Impaired adaptive immune response in COVID-19 convalescent patients with hematological malignancies.

OBJECTIVES: The immune dysregulation during SARS-CoV-2 has the potential to worsen immune homeostasis after recovery. Patients with hematological malignancies with COVID-19 have changes both in the innate and adaptive immune responses. Little is known about the severity of immune dysfunction following recovery from COVID-19 in hematological patients. METHODS: Here, we performed a comprehensive analysis of the lymphocyte subsets in peripheral blood mononuclear cells by FACS Canto II in 55 patients, including 42 with hematological malignancies 4-6 weeks after COVID-19. RESULTS: Hematological COVID-19 convalescents had deep reduction in CD3+ T cells, including helper T cells (CD3 + CD4+), naïve helper T cells (CD3 + CD4 + CD45RA+), and memory CD4+ T cells among with extremely low levels of Treg cells and decreased expression of both TCRα/ß and TCRγ/δ. Severe immune dysregulation in hematological convalescents was expressed by increased activation of T lymphocytes, both as elevated levels of activated T cells (CD3 + HLA-DR+) and activated cytotoxic T cells (CD3 + CD8 + HLA-DR+). CONCLUSIONS: Our findings showed a profound impairment of the adaptive immune response in hematological convalescents which might be a result of persistent activation of T cells. Convalescents with lymphoid malignancies showed more pronounced depletion of key T lymphocytes subpopulations in creating an effective adaptive response and immune memory.

Small RNA-Seq Reveals Similar miRNA Transcriptome in Children and Young Adults with T-ALL and Indicates miR-143-3p as Novel Candidate Tumor Suppressor in This Leukemia.

We aimed to identify miRNAs and pathways specifically deregulated in adolescent and young adult (AYA) T-ALL patients. Small RNA-seq showed no major differences between AYA and pediatric T-ALL, but it revealed downregulation of miR-143-3p in T-ALL patients. Prediction algorithms identified several known and putative oncogenes targeted by this miRNA, including KRAS, FGF1, and FGF9. Pathway analysis indicated signaling pathways related to cell growth and proliferation, including FGFR signaling and PI3K-AKT signaling, with the majority of genes overrepresented in these pathways being predicted targets of hsa-miR-143-3p. By luciferase reporter assays, we validated direct interactions of this miRNA with KRAS, FGF1 and FGF9. In cell proliferation assays, we showed reduction of cell growth upon miR-143-3p overexpression in two T-ALL cell lines. Our study is the first description of the miRNA transcriptome in AYA T-ALL patients and the first report on tumor suppressor potential of miR-143-3p in T-ALL. Downregulation of this miRNA in T-ALL patients might contribute to enhanced growth and viability of leukemic cells. We also discuss the potential role of miR-143-3p in FGFR signaling. Although this requires more extensive validation, it might be an interesting direction, since FGFR inhibition proved promising in preclinical studies in various cancers.

Pattern of human monocyte subpopulations in health and disease.

Monocytes are important cells of the innate system. They are a heterogeneous type of cells consisting of phenotypically and functionally distinct subpopulations, which play a specific role in the control, development and escalation of the immunological processes. Based on the expression of superficial CD14 and CD16 in flow cytometry, they can be divided into three subsets: classical, intermediate and non-classical. Variation in the levels of human monocyte subsets in the blood can be observed in patients in numerous pathological states, such as infections, cardiovascular and inflammatory diseases, cancer and autoimmune diseases. The aim of this review is to summarize current knowledge of human monocyte subsets and their significance in homeostasis and in pathological conditions.

The comparison of effectiveness and safety between different biosimilars of G-CSF in the mobilization of peripheral blood stem cells (PBSCs) for autologous transplantation (autologous peripheral blood stem cell transplantation, auto-PBSCT).

BACKGROUND: Autologous peripheral blood marrow stem cell transplantation (auto-PBSCT) preceded by high-dose chemotherapy is a well-known method of treatment for patients with hematological cancers. Performing the procedure entails obtaining from the patient their own stem cells from peripheral blood using G-CSF. Currently, various filgrastim biosimilars are widely used. AIM OF THE STUDY: The purpose of this study is to compare the efficacy and safety of three different biosimilars of filgrastim in PBSC mobilization in patients with hematological malignancies. MATERIALS AND METHODS: This is a retrospective analysis of 282 patients (118 women and 164 men) who underwent stem cells mobilization for auto-PBSCT in the Department of Hematology in Wroclaw in 2012-2014. Three filgrastim biosimilars were used: Tevagrastim (95), Nivestim (92), and Zarzio (95). Ninety patients (32%) were diagnosed with multiple myeloma, 55 (19%) with Hodgkin's lymphoma, 90 (32%) with NHLs, 20 (7%) with acute myeloid leukemia, and 27 (10%) with another hematological cancer. RESULTS: The mean number of CD34+ cells collected during the first leukapheresis was 5.95 × 106 /kg for Tevagrastim, 7.08 × 106 /kg for Nivestim, and 6.8 × 106 /kg for Zarzio (P > .05). The necessary number of leukapheresis for patients receiving Zarzio, Nivestim, and Tevagrastim was 1.32, 1.37, and 1.66, respectively (P > .05). The percentage of effective mobilizations was 88.2% for Zarzio, 86.2% for Nivestim, and 84.9% for Tevagrastim. The side effects included bone pain and headache. CONCLUSION: All tested biosimilars demonstrated similar effectiveness and safety profiles in patients with hematological tumors undergoing PBSC mobilization; therefore, they can be used interchangeably.

Higher efficacy of intermediate dose cytarabine + G-CSF compared to cyclophosphamide + G-CSF in hematopoietic stem cell mobilization in patients with multiple myeloma.

BACKGROUND: There are several regimens used in hematopoietic stem cell (HSC) mobilization in multiple myeloma (MM). Cyclophosphamide (Cy) is one of the most commonly used agents, although it does not always result in collecting adequate number of CD34+ cells. Recently, cytarabine (Ara-C) has been proposed as potentially efficient and safe option. AIMS: Since the data regarding Ara-C in HSC mobilization is limited, the aim of our study was to compare retrospectively the efficiency and toxicity of G-CSF combined with either Ara-C or Cy in MM patients. MATERIALS & METHODS: Of a total of 89 patients, 43 received low or intermediate doses of Cy, and 46 were treated with 800 mg/m2 /day of Ara-C administered for two days. RESULTS: The mean peak of CD34+ cells/ul in peripheral blood was 132 (range, 84-202) in Ara-C and 51 (range, 29-69) in Cy cohort (p < 0.001). The median number of collected CD34+ cells (×106/kg) was 10.3 (range, 4.2-17.9) vs 4.5 (range, 2.7-8.9), respectively (p < 0.001). Mobilization failure was observed in one patient in Ara-C cohort (2%) and in 8 patients treated with Cy (19%) (p = 0.013). In the Ara-C group 98% of patients obtained more than 4×106 CD34+ cells/kg required for tandem transplantation. Moreover, we observed a trend toward increased paraprotein levels measured at transplant compared to before HSC mobilization in Ara-C cohort and significantly higher transfusion rates in that group. CONCLUSION: Our findings confirm higher HSC mobilization efficacy of Ara-C compared to Cy in MM patients. However, lower transfusions rate and better disease control of Cy may justify its use in some cases.

Soluble MICA concentrations and genetic variability of MICA and its NKG2D receptor as factors affecting Graft-versus-Host Disease development after allogeneic haematopoietic stem cell transplantation.

Despite new treatment strategies, graft-versus-host disease (GvHD) remains a formidable complication after allogeneic hematopoietic stem cell transplantation (HSCT). This study aimed to investigate the impact of polymorphisms and expression of MICA and NKG2D receptor on the development of GvHD in allogeneic HSCT recipients. Soluble MICA (sMICA) concentration was measured in serum collected 30 days after transplantation and the genetic variability of MICA and NKG2D genes was evaluated. The frequency of NKG2D+NK cells was determined by flow cytometry before and (21, 30, 60 and 90 days) after transplantation. Recipients with acute GvHD grades II-IV carried the NKG2D rs1049174 C allele more frequently than controls or patients with no or mild disease. Patients with chronic GvHD had higher frequency of NKG2D expressing NK cells posttransplant, reflecting increased activity of their NK cells. Although no direct relationship between MICA SNPs and GvHD were observed, the presence of MICA rs1051792 GG genotype correlated with elevated sMICA levels and increased serum level of sMICA was associated with higher risk of chronic GvHD. Our findings suggest that sMICA concentration may serve as a potential biomarker for chronic GvHD and emphasize the impact of genetic variability of NKG2D and its surface expression on the HSCT outcome.

Significance of HLA-E and its two NKG2 receptors in development of complications after allogeneic transplantation of hematopoietic stem cells.

Transplantation of hematopoietic stem cells (HSCT) is a procedure commonly used in treatment of various haematological disorders which is associated with significantly improved survival rates. However, one of its drawbacks is the possibility of development of post-transplant complications, including acute and chronic graft-versus-host disease (GvHD) or CMV infection. Various studies suggested that NK cells and their receptors may affect the transplant outcome. In the present study, patients and donors were found to significantly differ in the distribution of the NKG2A rs7301582 genetic variants - recipients carried the C allele more often than their donors (0.975 vs 0.865, p<0.0001). Increased soluble HLA-E (sHLA-E) levels detected in recipients' serum 30 days after transplantation seemed to play a prognostic and protective role. It was observed that recipients with higher sHLA-E levels were less prone to chronic GvHD (11.65 vs 6.33 pg/mL, p=0.033) or more severe acute GvHD grades II-IV (11.07 vs 8.04 pg/mL, p=0.081). Our results also showed an unfavourable role of HLA-E donor-recipient genetic incompatibility in CMV infection development after transplantation (OR=5.92, p=0.014). Frequencies of NK cells (both CD56dim and CD56bright) expressing NKG2C were elevated in recipients who developed CMV, especially 30 and 90 days post-transplantation (p<0.03). Percentages of NKG2C+ NK cells lacking NKG2A expression were also increased in these patients. Moreover, recipients carrying a NKG2C deletion characterized with decreased frequency of NKG2C+ NK cells (p<0.05). Our study confirms the importance of NK cells in the development of post-transplant complications and highlights the effect of HLA-E and NKG2C genetic variants, sHLA-E serum concentration, as well as NKG2C surface expression on transplant outcome.

Immunophenotypic Characteristics of Bone Marrow Microenvironment Cellular Composition at the Biochemical Progression of Multiple Myeloma.

Multiple myeloma (MM) relapses are inevitable in the majority of patients, and in addition to genetic changes in the MM clone, the immune profile of the bone marrow (BM) plays a key role in this process. Biochemical progression or relapse (BR) precedes clinical relapse in a significant proportion of patients with MM. In the present study, we used flow cytometry to assess the cellular composition of the BM microenvironment in MM patients with confirmed BR. Fifteen distinct cells subsets in the BM were evaluated with the panel of antibodies used routinely for MRD monitoring in MM in 52 patients with MM (MRD-negative n = 20, BR n = 20, and clinically relapsed MM, RMM n = 12). The median percentage of MM cells detected in BR patients was 0.90% versus not detectable in MRD-negative patients and of 3.0% in RMM cohort. Compared to the MRD-negative group, BR status was associated with an increase in the percentage of lymphoid subpopulations, including memory B cells (p = 0.003), CD27+T cells (p = 0.002), and NK/NKT cells (p < 0.001). Moreover, a decrease in B-cell precursors (p < 0.001) and neutrophils (p = 0.006) was observed. There were no significant differences in the composition of the BM cell subpopulations between the BR and RMM groups. Our results indicate the involvement of B-, T-, and NK cells in the process of losing immune surveillance over the MM clone that leads to relapse. It can be speculated that similar studies of a larger cohort of BR patients can potentially identify a group of patients for which an early treatment intervention would be beneficial.

Two Doses of BNT162b2 mRNA Vaccine in Patients after Hematopoietic Stem Cell Transplantation: Humoral Response and Serological Conversion Predictors.

Vaccination against SARS-CoV-2 is currently the best tool in the fight against the COVID-19 pandemic. However, there are limited data on its efficacy and safety after hematopoietic stem cell transplantation (HCT). We present the results of a prospective analysis of the humoral response to two doses of BNT162b2 mRNA vaccine in 93 adult patients, including 29 after autologous HCT (autoHCT) and 64 after allogeneic HCT (alloHCT). Positive anti-SARS-CoV-2 antibodies were detected before vaccination in 25% of patients despite a negative medical history of COVID-19. Seroconversion after vaccination was achieved in 89% of patients after alloHCT and in 96% after autoHCT, without grade 3/4 adverse events. Post-vaccination anti-SARS-CoV-2 antibody level correlated with the time from transplant and absolute B-cell count at the vaccination. In univariate analysis restricted to the alloHCT group, short time since transplantation, low B-cell count, low intensity conditioning, GvHD, and immunosuppressive treatment at the vaccination were associated with lack of seroconversion. In the multivariate model, the only negative predictor of seroconversion remained treatment with calcineurin inhibitor (CNI). In conclusion, the BNT162b2 mRNA vaccine is highly immunogenic in patients after HCT, but treatment with CNI at the time of vaccination has a strong negative impact on the humoral response.

Immunosuppression as a Hallmark of Critical COVID-19: Prospective Study.

The dysregulation of both the innate and adaptive responses to SARS-CoV-2 have an impact on the course of COVID-19, and play a role in the clinical outcome of the disease. Here, we performed a comprehensive analysis of peripheral blood lymphocyte subpopulations in 82 patients with COVID-19, including 31 patients with a critical course of the disease. In COVID-19 patients who required hospitalization we analyzed T cell subsets, including Treg cells, as well as TCRα/ß and γ/δ, NK cells, and B cells, during the first two weeks after admission to hospital due to the SARS-CoV-2 infection, with marked reductions in leukocytes subpopulations, especially in critically ill COVID-19 patients. We showed decreased levels of Th, Ts cells, Treg cells (both naïve and induced), TCRα/ß and γ/δ cells, as well as CD16+CD56+NK cells in ICU compared to non-ICU COVID-19 patients. We observed impaired function of T and NK cells in critically ill COVID-19 patients with extremely low levels of secreted cytokines. We found that the IL-2/INFγ ratio was the strongest indicator of a critical course of COVID-19, and was associated with fatal outcomes. Our findings showed markedly impaired innate and adaptive responses in critically ill COVID-19 patients, and suggest that the immunosuppressive state in the case of a critical course of SARS-CoV-2 infection might reflect subsequent clinical deterioration and predict a fatal outcome.

Harmonization of Flow Cytometric Minimal Residual Disease Assessment in Multiple Myeloma in Centers of Polish Myeloma Consortium.

Minimal residual disease (MRD) status is now considered as one of the most relevant prognostic factors in multiple myeloma (MM) while MRD negativity became an important endpoint in clinical trials. Here, we report the results of the first study evaluating the reproducibility of high-sensitivity flow cytometry MM MRD assessment in four laboratories in Poland. EuroFlow protocols for instrument setting standardization and sample preparation in MM MRD assessment were implemented in each laboratory. In the inter-laboratory reproducibility study, 12 bone marrow samples from MM patients were distributed and processed in participant laboratories. In the inter-operator concordance study, 13 raw data files from MM MRD measurements were analyzed by five independent operators. The inter-laboratory study showed high 95% overall concordance of results among laboratories. In the inter-operator study, 89% of MRD results reported were concordant, and the highest immunophenotype interpretation differences with regard to expression of CD27, CD45, CD81 were noticed. We confirmed the applicability and feasibility of the EuroFlow protocol as a highly sensitive method of MRD evaluation in MM. Results of our inter-center comparison study demonstrate that the standardization of MM MRD assessment protocols is highly desirable to improve quality and comparability of results within and between different clinical trials.

Lymphocyte subsets in haematological patients with COVID-19: Multicentre prospective study.

The role of immune dysregulation in the course and prognosis of COVID-19 is not clearly established. In particular, immune status in specific populations such as haematological patients, who have an impaired immunological system, has not been described so far. Here, we performed a comprehensive analysis of peripheral blood lymphocyte subsets in 27 SARS-CoV-2-infected patients, including 16 patients with haematological malignancies. We identified T cell subpopulations, B cells, NK cells and TCR α/ß and É£/ƍ-expressing T cells during COVID-19 infection, with significant changes observed in immune profiles during the course of disease, especially in haematological patients. We observed an increase in activated T lymphocytes (CD3+HLA-DR+ and CD3+CD8+HLA-DR+) in the early stages of SARS-CoV-2 infection with a concomitant decrease in the CD4/CD8 ratio in haematological patients compared to non-haematological patients affected by COVID-19. We also found a decrease in É£/ƍ T cells in both studied groups of patients, with lower numbers of CD25+ T cells and CD16+CD56+ NK cells in haematological patients compared to non-haematological patients with COVID-19. Our findings demonstrate, for the first time, impaired adaptive immunity in patients with haematological malignancies infected with COVID-19, resulting in impaired cellular immune responses to SARS-CoV-2. This warrants further investigation of this disease group in COVID-19 patient cohorts.

Successful Salvage Haploidentical Alpha-Beta T Cell-Depleted Stem Cell Transplantation After Busulfan-Based Myeloablation in a Patient With IPEX Syndrome: A Case Report.

BACKGROUND: X-linked immunodysregulation syndrome with polyendocrinopathy and enteropathy (IPEX) is caused by FOXP3 gene mutations that block the generation of regulatory T lymphocytes. We report an 18-month-old boy with classic IPEX who underwent 2 hematopoietic stem cell transplantations (HSCTs). METHODS: The first HSCT from an unrelated 8/10 HLA-matched umbilical cord blood donor (UCB) was performed after a conditioning regimen consisting of treosulfan, fludarabine, thiotepa, and thymoglobulin. Due to complete rejection of the UCB transplant, a second transplantation from a 6/10 HLA-matched mother was performed after alpha-beta T-cell depletion. The second conditioning regimen consisted of busulfan, fludarabine, a single dose of cyclophosphamide 1 g/m2, and Grafalon (Neovii Pharmaceuticals, Rapperswil, Switzerland). The T-cell depletion product contained 15.06 x 106 CD34+ cells per kilogram body weight (BW) and 4.19 x 105 alpha-beta T lymphocytes per kilogram BW. Due to acute graft rejection, the boy was treated with thymoglobulin, and full donor chimerism in both T lymphocytes and mononuclear cells was achieved. The immunosuppressive therapy was stopped 1 year after transplantation. To date, the patient remains free from graft-vs-host disease (GVHD) and immunosuppression. CONCLUSIONS: HSCT after busulfan-based reduced-toxicity conditioning in patients with IPEX syndrome is feasible and well tolerated and can result in full donor engraftment. Monitoring of chimerism and aggressive therapy in cases of graft rejection are warranted due to the high reactivity of residual autologous T lymphocytes. T-cell depletion reduces the risk of GVHD and the need for steroid therapy, which is especially challenging in patients with diabetes.

The role of hypoxia-inducible factors in leukemias.

Hypoxia, understood as low partial oxygen pressure, has become one of the most explored fields in recent years. Cellular response to hypoxia is mediated by hypoxia-inducible factors (HIFs) - potent transcription regulators, and their downstream pathways. In general, HIFs modify energy metabolism, inflammation and immune response, enhance cancer invasion, metastasis, resistance to treatment, and relapse. The influence of HIFs on the progression of leukemia is still under investigation in various studies, but in mice and some human models HIFs have been recognized as leukemia immortalizers by promoting leukemic stem cell quiescence and inhibiting their cell cycle. This makes leukemic stem cells resistant to most known treatment approaches. The role of HIFs in solid tumors and leukemia makes them almost ideal targets for an anticancer treatment. Although the first attempts with new molecules are encouraging, there is a need to investigate the ambiguous role of HIFs to develop a modern antileukemic treatment.