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.

High-Sensitive TRBC1-Based Flow Cytometric Assessment of T-Cell Clonality in Tαß-Large Granular Lymphocytic Leukemia.

Flow cytometric (FCM) analysis of the constant region 1 of the T-cell receptor ß chain (TRBC1) expression for assessing Tαß-cell clonality has been recently validated. However, its utility for the diagnosis of clonality of T-large granular lymphocytic leukemia (T-LGLL) needs to be confirmed, since more mature Tαß cells (i.e., T-LGL normal-counterpart) show broader TRBC1+/TRBC1- ratios vs. total Tαß cells. We compared the distribution and absolute counts of TRBC1+ and TRBC1- Tαß-LGL in blood containing polyclonal (n = 25) vs. clonal (n = 29) LGL. Overall, polyclonal TRBC1+ or TRBC1- Tαß-LGL ranged between 0.36 and 571 cells/µL (3.2-91% TRBC1+ cells), whereas the clonal LGL cases showed between 51 and 11,678 cells/µL (<0.9% or >96% TRBC1+ cells). Among the distinct TCRVß families, the CD28- effector-memory and terminal-effector polyclonal Tαß cells ranged between 0 and 25 TRBC1+ or TRBC1- cells/µL and between 0 and 100% TRBC1+ cells, while clonal LGL ranged between 32 and 5515 TRBC1+ or TRBC1- cells/µL, representing <1.6% or >98% TRBC1+ cells. Our data support the utility of the TRBC1-FCM assay for detecting T-cell clonality in expansions of Tαß-LGL suspected of T-LGLL based on altered percentages of TRBC1+ Tαß cells. However, in the absence of lymphocytosis or in the case of TαßCD4-LGL expansion, the detection of increased absolute cell counts by the TRBC1-FCM assay for more accurately defined subpopulations of Tαß-LGL-expressing individual TCRVß families, allows the detection of T-cell clonality, even in the absence of phenotypic aberrations.

Anti-TRBC1 Antibody-Based Flow Cytometric Detection of T-Cell Clonality: Standardization of Sample Preparation and Diagnostic Implementation.

A single antibody (anti-TRBC1; JOVI-1 antibody clone) against one of the two mutually exclusive T-cell receptor ß-chain constant domains was identified as a potentially useful flow-cytometry (FCM) marker to assess Tαß-cell clonality. We optimized the TRBC1-FCM approach for detecting clonal Tαß-cells and validated the method in 211 normal, reactive and pathological samples. TRBC1 labeling significantly improved in the presence of CD3. Purified TRBC1+ and TRBC1- monoclonal and polyclonal Tαß-cells rearranged TRBJ1 in 44/47 (94%) and TRBJ1+TRBJ2 in 48 of 48 (100%) populations, respectively, which confirmed the high specificity of this assay. Additionally, TRBC1+/TRBC1- ratios within different Tαß-cell subsets are provided as reference for polyclonal cells, among which a bimodal pattern of TRBC1-expression profile was found for all TCRVß families, whereas highly-variable TRBC1+/TRBC1- ratios were observed in more mature vs. naïve Tαß-cell subsets (vs. total T-cells). In 112/117 (96%) samples containing clonal Tαß-cells in which the approach was validated, monotypic expression of TRBC1 was confirmed. Dilutional experiments showed a level of detection for detecting clonal Tαß-cells of ≤10-4 in seven out of eight pathological samples. These results support implementation of the optimized TRBC1-FCM approach as a fast, specific and accurate method for assessing T-cell clonality in diagnostic-FCM panels, and for minimal (residual) disease detection in mature Tαß+ leukemia/lymphoma patients.