Immune cell kinetics and antibody response in COVID-19 patients with low-count monoclonal B-cell lymphocytosis.

Low-count monoclonal B-cell lymphocytosis (MBLlo ) has been associated with an underlying immunodeficiency and has recently emerged as a new risk factor for severe COVID-19. Here, we investigated the kinetics of immune cell and antibody responses in blood during COVID-19 of MBLlo versus non-MBL patients. For this study, we analyzed the kinetics of immune cells in blood of 336 COVID-19 patients (74 MBLlo and 262 non-MBL), who had not been vaccinated against SARS-CoV-2, over a period of 43 weeks since the onset of infection, using high-sensitivity flow cytometry. Plasma levels of anti-SARS-CoV-2 antibodies were measured in parallel by ELISA. Overall, early after the onset of symptoms, MBLlo COVID-19 patients showed increased neutrophil, monocyte, and particularly, plasma cell (PC) counts, whereas eosinophil, dendritic cell, basophil, and lymphocyte counts were markedly decreased in blood of a variable percentage of samples, and with a tendency toward normal levels from week +5 of infection onward. Compared with non-MBL patients, MBLlo COVID-19 patients presented higher neutrophil counts, together with decreased pre-GC B-cell, dendritic cell, and innate-like T-cell counts. Higher PC levels, together with a delayed PC peak and greater plasma levels of anti-SARS-CoV-2-specific antibodies (at week +2 to week +4) were also observed in MBLlo patients. In summary, MBLlo COVID-19 patients share immune profiles previously described for patients with severe SARS-CoV-2 infection, associated with a delayed but more pronounced PC and antibody humoral response once compared with non-MBL patients.

Single-Cell DNA Sequencing and Immunophenotypic Profiling to Track Clonal Evolution in an Acute Myeloid Leukemia Patient.

Single-cell DNA sequencing can address the sequence of somatic genetic events during myeloid transformation in relapsed acute myeloid leukemia (AML). We present an NPM1-mutated AML patient with an initial low ratio of FLT3-ITD (low-risk ELN-2017), treated with midostaurin combined with standard chemotherapy as front-line treatment, and with salvage therapy plus gilteritinib following allogenic stem cell transplantation after relapse. Simultaneous single-cell DNA sequencing and cell-surface immunophenotyping was used in diagnostic and relapse samples to understand the clinical scenario of this patient and to reconstruct the clonal composition of both tumors. Four independent clones were present before treatment: DNMT3A/DNMT3A/NPM1 (63.9%), DNMT3A/DNMT3A (13.9%), DNMT3A/DNMT3A/NPM1/FLT3 (13.8%), as well as a wild-type clone (8.3%), but only the minor clone with FLT3-ITD survived and expanded after therapy, being the most represented one (58.6%) at relapse. FLT3-ITD was subclonal and was found only in the myeloid blast population (CD38/CD117/CD123). Our study shows the usefulness of this approach to reveal the clonal architecture of the leukemia and the identification of small subclones at diagnosis and relapse that may explain how the neoplastic cells can escape from the activity of different treatments in a stepwise process that impedes the disease cure despite different stages of complete remission.

Siglec-6 is a novel target for CAR T-cell therapy in acute myeloid leukemia.

Acute myeloid leukemia (AML) is an attractive entity for the development of chimeric antigen receptor (CAR) T-cell immunotherapy because AML blasts are susceptible to T-cell-mediated elimination. Here, we introduce sialic acid-binding immunoglobulin-like lectin 6 (Siglec-6) as a novel target for CAR T cells in AML. We designed a Siglec-6-specific CAR with a targeting domain derived from the human monoclonal antibody JML-1. We found that Siglec-6 is commonly expressed on AML cell lines and primary AML blasts, including the subpopulation of AML stem cells. Treatment with Siglec-6 CAR T cells confers specific antileukemia reactivity that correlates with Siglec-6 expression in preclinical models, including induction of complete remission in a xenograft AML model in immunodeficient mice (NSG/U937). In addition, we confirmed Siglec-6 expression on transformed B cells in chronic lymphocytic leukemia (CLL), and specific anti-CLL reactivity of Siglec-6 CAR T cells in vitro. Of particular interest, we found that Siglec-6 is not detectable on normal hematopoietic stem and progenitor cells (HSPCs) and that treatment with Siglec-6 CAR T cells does not affect their viability and lineage differentiation in colony-formation assays. These data suggest that Siglec-6 CAR T-cell therapy may be used to effectively treat AML without the need for subsequent allogeneic hematopoietic stem cell transplantation. In mature normal hematopoietic cells, we detected Siglec-6 in a proportion of memory (and naïve) B cells and basophilic granulocytes, suggesting the potential for limited on-target/off-tumor reactivity. The lack of expression of Siglec-6 on normal HSPCs is a key to differentiating it from other Siglec family members (eg, Siglec-3 [CD33]) and other CAR target antigens (eg, CD123) that are under investigation in AML, and it warrants the clinical investigation of Siglec-6 CAR T-cell therapy.