OMIP-106: A 30-color panel for analysis of check-point inhibitory networks in the bone marrow of acute myeloid leukemia patients.

Acute myeloid leukemia (AML) is the most common form of acute leukemia diagnosed in adults. Despite advances in medical care, the treatment of AML still faces many challenges, such as treatment-related toxicities, that limit the use of high-intensity chemotherapy, especially in elderly patients. Currently, various immunotherapeutic approaches, that is, CAR-T cells, BiTEs, and immune checkpoint inhibitors, are being tested in clinical trials to prolong remission and improve the overall survival of AML patients. However, early reports show only limited benefits of these interventions and only in a subset of patients, showing the need for better patient stratification based on immunological markers. We have therefore developed and optimized a 30-color panel for evaluation of effector immune cell (NK cells, γδ T cells, NKT-like T cells, and classical T cells) infiltration into the bone marrow and analysis of their phenotype with regard to their differentiation, expression of inhibitory (PD-1, TIGIT, Tim3, NKG2A) and activating receptors (DNAM-1, NKG2D). We also evaluate the immune evasive phenotype of CD33+ myeloid cells, CD34+CD38-, and CD34+CD38+ hematopoietic stem and progenitor cells by analyzing the expression of inhibitory ligands such as PD-L1, CD112, CD155, and CD200. Our panel can be a valuable tool for patient stratification in clinical trials and can also be used to broaden our understanding of check-point inhibitory networks in AML.

OMIP-080: 29-Color flow cytometry panel for comprehensive evaluation of NK and T cells reconstitution after hematopoietic stem cells transplantation.

This 29-color panel was developed and optimized for the monitoring of NK cell and T cell reconstitution in peripheral blood of patients after HSCT. We considered major post-HSCT complications during the design, such as relapses, viral infections, and GvHD and identification of lymphocyte populations relevant to their resolution. The panel includes markers for all major NK cell and T cell subsets and analysis of their development and qualitative properties. In the NK cell compartment, we focus mainly on CD57 + NKG2C+ cells and the expression of activating (NKG2D, DNAM-1) and inhibitory receptors (NKG2A, TIGIT). Another priority is the characterization of T cell reconstitution; therefore, we included detection of CD4+ RTEs based on CD45RA, CD62L, CD95, and CD31 as a marker of thymus function. Besides that, we also analyze the emergence and properties of major T cell populations with a particular interest in CD8, Th1, ThCTL, and Treg subsets. Overall, the panel allows for comprehensive analysis of the reconstituting immune system and identification of potential markers of immune cell dysfunction.

Multiparametric analysis of the specific immune response against SARS-CoV-2.

BACKGROUND: SARS-CoV-2, which causes COVID-19, has killed more than 7 million people worldwide. Understanding the development of postinfectious and postvaccination immune responses is necessary for effective treatment and the introduction of appropriate antipandemic measures. OBJECTIVES: We analysed humoral and cell-mediated anti-SARS-CoV-2 immune responses to spike (S), nucleocapsid (N), membrane (M), and open reading frame (O) proteins in individuals collected up to 1.5 years after COVID-19 onset and evaluated immune memory. METHODS: Peripheral blood mononuclear cells and serum were collected from patients after COVID-19. Sampling was performed in two rounds: 3-6 months after infection and after another year. Most of the patients were vaccinated between samplings. SARS-CoV-2-seronegative donors served as controls. ELISpot assays were used to detect SARS-CoV-2-specific T and B cells using peptide pools (S, NMO) or recombinant proteins (rS, rN), respectively. A CEF peptide pool consisting of selected viral epitopes was applied to assess the antiviral T-cell response. SARS-CoV-2-specific antibodies were detected via ELISA and a surrogate virus neutralisation assay. RESULTS: We confirmed that SARS-CoV-2 infection induces the establishment of long-term memory IgG+ B cells and memory T cells. We also found that vaccination enhanced the levels of anti-S memory B and T cells. Multivariate comparison also revealed the benefit of repeated vaccination. Interestingly, the T-cell response to CEF was lower in patients than in controls. CONCLUSION: This study supports the importance of repeated vaccination for enhancing immunity and suggests a possible long-term perturbation of the overall antiviral immune response caused by SARS-CoV-2 infection.

Specific immune response to mRNA vaccines against COVID-19 in patients receiving allogeneic stem cell transplantation for myeloid malignancy was altered by immunosuppressive therapy.

BACKGROUND: Allogeneic hematopoietic stem cell transplant (allo-HSCT) recipients are at high risk of complications associated with COVID-19 infection due to dysfunction of their immune system. Vaccination can protect from the adverse consequences of COVID-19. However, studies on the efficacy of COVID-19 vaccines in HSCT recipients with insufficient post-HSCT immune reconstitution are still scarce. In our study, we determined how immunosuppressive medication and the reconstitution of the cellular immune system influenced T cell responses specific for the surface glycoprotein of SARS-CoV-2 virus (S antigen) after two doses of mRNA vaccine against COVID-19 in patients with myeloid malignancies treated with HSCT. METHODS: Vaccination outcomes were followed in 18 (allo-HSCT) recipients and 8 healthy volunteers. The IgG antibodies against SARS-CoV-2 spike (S) and nucleocapsid (NCP) protein were determined in ELISA and S-specific T cells were detected using a sensitive ELISPOT-IFNγ based on in vitro expansion and restimulation of T cells in pre- and post-vaccination blood samples. Multiparametric flow cytometry analysis of peripheral blood leukocyte differentiation markers was employed for determination of reconstitution of the main subpopulations of T cells and NK cells at month 6 after HSCT. RESULTS: S- specific IgG antibody response detected in 72% of the patients was lower than in healthy vaccinees (100%). Vaccine-induced T-cell responses to S1 or S2 antigen were significantly reduced in HSCT recipients, which were treated with corticosteroids in dose 5 mg of prednisone- equivalents or higher during the vaccination period or in preceeding 100 days in comparison with recipients un-affected with corticosteroids. A significant positive correlation was found between the level of anti-SARS-Cov-2 spike protein IgG antibodies and the number of functional S antigen-specific T cells. Further analysis also showed that the specific response to vaccination was significantly influenced by the interval between administration of vaccine and transplantation. Vaccination outcomes were not related to age, sex, type of mRNA vaccine used, basic diagnosis, HLA match between HSC donor and recipient, and blood counts of lymphocytes, neutrophils, and monocytes at the time of vaccination. Multiparametric flow cytometry analysis of peripheral blood leukocyte differentiation markers showed that good humoral and cellular S-specific immune responses induced by vaccination were associated with well-reconstituted CD4+ T cells, mainly CD4+ effector memory subpopulation at six months after HSCT. CONCLUSIONS: The results showed that both humoral and cellular adaptive immune responses of HSCT recipients to the SARS-CoV-2 vaccine were significantly suppressed by corticosteroid therapy. Specific response to the vaccine was significantly affected by the length of the interval between HSCT and vaccination. Vaccination as early as 5 months after HSCT can lead to a good response. Immune response to the vaccine is not related to age, gender, HLA match between HSC donor and recipient, or type of myeloid malignancy. Vaccine efficacy was dependent on well-reconstituted CD4+ T cells, at six months after HSCT.

Expression of circular RNAs in myelodysplastic neoplasms and their association with mutations in the splicing factor gene SF3B1.

Mutations in the splicing factor 3b subunit 1 (SF3B1) gene are frequent in myelodysplastic neoplasms (MDS). Because the splicing process is involved in the production of circular RNAs (circRNAs), we investigated the impact of SF3B1 mutations on circRNA processing. Using RNA sequencing, we measured circRNA expression in CD34+ bone marrow MDS cells. We defined circRNAs deregulated in a heterogeneous group of MDS patients and described increased circRNA formation in higher-risk MDS. We showed that the presence of SF3B1 mutations did not affect the global production of circRNAs; however, deregulation of specific circRNAs was observed. Particularly, we demonstrated that strong upregulation of circRNAs processed from the zinc finger E-box binding homeobox 1 (ZEB1) transcription factor; this upregulation was exclusive to SF3B1-mutated patients and was not observed in those with mutations in other splicing factors or other recurrently mutated genes, or with other clinical variables. Furthermore, we focused on the most upregulated ZEB1-circRNA, hsa_circ_0000228, and, by its knockdown, we demonstrated that its expression is related to mitochondrial activity. Using microRNA analyses, we proposed miR-1248 as a direct target of hsa_circ_0000228. To conclude, we demonstrated that mutated SF3B1 leads to deregulation of ZEB1-circRNAs, potentially contributing to the defects in mitochondrial metabolism observed in SF3B1-mutated MDS.