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Simple Summary: The current prognostic index for chronic lymphocytic leukemia does not account for the dynamic changes in the B-cell clone over time. This study aims to investigate the association between the tumor microenvironment and the kinetics of clonal B-cells in early-stage CLL patients. By categorizing patients based on the rate of clonal B-cell increase, we identified significant differences in immune cell profiles and clinical outcomes. Patients with rapidly increasing clones exhibited poorer prognosis and shorter time to treatment, but notably, lower Tαβ CD4+CD8lo cell counts, altered B-cell subsets, and higher plasma cell counts were associated with highly proliferative clones. Multivariate analysis confirmed that the number of clonal B-cells, Tαβ CD4+CD8lo cells, and the IGHV mutational status were independent predictors of clonal expansion. These findings suggest that the interplay between CLL cells and the immune microenvironment might play a relevant role in disease progression, potentially leading to the development of novel prognostic markers. Background/Objectives: Despite the current international prognostic index for chronic lymphocytic leukemia (CLL) being widely accepted and broadly used, it does not consider the kinetics of the B-cell clone over time. Here, we investigated the potential association between distinct features of leukemic cells and other immune cells in blood and the kinetics of clonal B-cells in CLL stage Binet A/Rai 0 (A/0) patients; Methods: Based on the leukemia cell kinetics, 69 CLL A/0 cases followed for a median of 105 months were classified as carrying stable (n = 53) vs. rapidly increasing in size (n = 16) CLL clones; Results: Patients with increasing CLL clones had a significantly higher risk of disease progression and shortened time to first therapy vs. those carrying stable B-cell clones (p ≤ 0.001). Strikingly, the distribution of various immune-cell populations in blood at diagnosis also differed significantly between the two groups, with lower Tαβ CD4+CD8lo cell counts (p = 0.03), a greater switched/unswitched memory B-cell ratio (p = 0.01), and higher plasma cell counts (p = 0.05) in CLL with increasing vs. stable clones. Multivariate analysis revealed that the number of circulating clonal B-cells (≥15 × 109/L) and Tαβ CD4+CD8lo cells (≤35 cells/µL), together with an IGHV unmutated gene status at diagnosis, were independent predictors of an increasing CLL clone; Conclusions: Altogether, these data suggest that the expansion of the CLL clone in stage A/0 patients may depend on both the intrinsic characteristics of CLL cells and the surrounding immune microenvironment. [ABSTRACT FROM AUTHOR]

The early immune kinetics after SARS-CoV-2 vaccination remain poorly understood, particularly among individuals with low-count monoclonal B-cell lymphocytosis (MBLlo). We investigated the cellular and humoral kinetics in the blood of 50 non-MBL healthy donors (HD) vs. 16 MBLlo subjects after SARS-CoV-2 vaccination, who were subclassified according to their history of previous exposure to SARS-CoV-2 into SARS-CoV-2 naïve and previously infected subjects. Overall, we found decreased neutrophil and lymphocyte counts at day +4 following each dose in non-MBL HD, together with an earlier and higher increase in plasma cell (PC) counts and SARS-CoV-2-specific antibody levels after the first vaccine in previously infected non-MBL HD. MBLlo subjects showed a similar profile, except for lower B-cell and higher PC counts after vaccination, and a trend towards a higher (but delayed) antibody response. In summary, we found different cell-kinetic profiles following vaccination in SARS-CoV-2 naïve vs. previously infected non-MBL HD (earlier PC and antibody responses in the latter group); additionally, MBLlo subjects had significantly lower B-cell and higher PC counts after vaccination, and a delayed SARS-CoV-2-specific antibody response. [ABSTRACT FROM AUTHOR]

Excessive alcohol consumption impairs the immune system, induces oxidative stress, and triggers the activation of peripheral blood (PB) monocytes, thereby contributing to alcoholic liver disease (ALD). We analyzed the M1/M2 phenotypes of circulating classical monocytes and macrophage-derived monocytes (MDMs) in excessive alcohol drinkers (EADs). PB samples from 20 EADs and 22 healthy controls were collected for isolation of CD14+ monocytes and short-term culture with LPS/IFNγ, IL4/IL13, or without stimulation. These conditions were also used to polarize MDMs into M1, M2, or M0 phenotypes. Cytokine production was assessed in the blood and culture supernatants. M1/M2-related markers were analyzed using mRNA expression and surface marker detection. Additionally, the miRNA profile of CD14+ monocytes was analyzed. PB samples from EADs exhibited increased levels of pro-inflammatory cytokines. Following short-term culture, unstimulated blood samples from EADs showed higher levels of soluble TNF-α and IL-8, whereas monocytes expressed increased levels of surface TNF-α and elevated mRNA expression of pro-inflammatory cytokines and inducible nitric oxide synthase. MDMs from EADs showed higher levels of TNF-α and CD206 surface markers and increased IL-10 production. LPS/IFNγ induced higher mRNA expression of Nrf2 only in the controls. miRNA analysis revealed a distinctive miRNA profile that is potentially associated with liver carcinogenesis and ALD through inflammation and oxidative stress. This study confirms the predominantly pro-inflammatory profile of PB monocytes among EADs and suggests immune exhaustion features in MDMs. [ABSTRACT FROM AUTHOR]

Simple Summary: Assessment of the status of the immune system in both health and disease requires robust and reliable reference ranges for the different blood leukocyte (sub)populations that take into consideration factors that might influence their distribution, such as age, sex, ethnicity and the presence vs. absence of low-count monoclonal B-cell lymphocytosis with a chronic-lymphocytic-leukemia-like phenotype (MBLlo). It should be noted that despite MBLlo being highly prevalent in the general population and being associated with immune impairment, MBLlo individuals have not been previously excluded in the definition of normal leukocyte ranges. Here, we provide reference cell-count ranges for the major leukocyte populations identified in blood using an optimized and fully validated 8-color flow-cytometry antibody combination based on the largest (n = 706) cohort reported to date of Caucasian adult donors from the general population, grouped by age and sex, and highlight the altered immune profiles associated with MBLlo (622 non-MBL and 84 MBLlo subjects). Reference ranges of blood-circulating leukocyte populations by, e.g., age and sex, are required for monitoring immune-cell kinetics. Most previous reports in which flow cytometry has been used to define the reference ranges for leukocyte counts included a limited number of donors and/or cell populations and/or did not consider age and sex simultaneously. Moreover, other factors not previously considered in the definition of normal ranges, such as the presence of chronic-lymphocytic-leukemia (CLL)-like low-count monoclonal B-cell lymphocytosis (MBLlo), might also be associated with an altered distribution of leukocytes in blood in association with an immunodeficiency and increased risk of infection and cancer. Here, we established reference cell-count ranges for the major populations of leukocytes in blood of non-MBL and MBLlo adult Caucasians matched by age and sex using the EuroFlow Lymphocyte Screening Tube (LST). A total of 706 Caucasian adult donors—622 non-MBL and 84 MBLlo—were recruited from the general population. Among non-MBL donors, the total leukocyte, neutrophil, basophil dendritic cell and monocyte counts remained stable through adulthood, while the absolute numbers of T- and B-cell populations and plasma cells decreased with age. The number of eosinophils and NK-cell increased over time, with clear differences according to sex for certain age ranges. In MBLlo subjects, few differences in the absolute cell counts by age (vs. non-MBL) were observed, and MBLlo men and women showed similar trends to non-MBL subjects except for the B-cell count drop observed in >70 y-men, which was more pronounced in MBLlo vs. non-MBL controls. Building robust age- and sex-matched reference ranges for the most relevant immune-cell populations in the blood of non-MBL donors is essential to appropriately identify an altered immune status in different clinical settings and highlight the altered immune-cell profiles of MBLlo subjects. [ABSTRACT FROM AUTHOR]

Simple Summary: TRBC1 expression analysis by flow cytometry (FCM) has been recently proved to be a useful, simple and fast approach to assessing Tαβ-cell clonality. The aim of this study was to validate the utility of this assay specifically for the diagnosis of T-cell clonality of T-large granular lymphocytic leukemias (T-LGLL), as more mature polyclonal Tαβ large granular lymphocytes (Tαβ-LGL) show broader TRBC1+/TRBC1− ratios vs. total Tαβ cells. Our results showed that a TRBC1-FCM assay is also a fast and easy method for detecting T-cell clonality in T-LGLL based on altered (increased or decreased) percentages of TRBC1+ Tαβ cells of LGL expansions (i.e., with lymphocytosis) suspected of T-LGLL, whereas in the absence of lymphocytosis (or in TαβCD4-LGLL), the detection of increased absolute cell-counts of more precisely defined subpopulations of T-LGL expressing individual TCRVβ families is required. Flow cytometric (FCM) analysis of the constant region 1 of the T-cell receptor β chain (TRBC1) expression for assessing Tαβ-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 Tαβ cells (i.e., T-LGL normal-counterpart) show broader TRBC1+/TRBC1− ratios vs. total Tαβ cells. We compared the distribution and absolute counts of TRBC1+ and TRBC1− Tαβ-LGL in blood containing polyclonal (n = 25) vs. clonal (n = 29) LGL. Overall, polyclonal TRBC1+ or TRBC1− Tαβ-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 TCRVβ families, the CD28− effector-memory and terminal-effector polyclonal Tαβ 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 Tαβ-LGL suspected of T-LGLL based on altered percentages of TRBC1+ Tαβ cells. However, in the absence of lymphocytosis or in the case of TαβCD4-LGL expansion, the detection of increased absolute cell counts by the TRBC1-FCM assay for more accurately defined subpopulations of Tαβ-LGL-expressing individual TCRVβ families, allows the detection of T-cell clonality, even in the absence of phenotypic aberrations. [ABSTRACT FROM AUTHOR]

Simple Summary: The anti-TRBC1 antibody JOVI-1 has recently been identified as a flow cytometry marker potentially useful for assessment of T-cell clonality. The aim of this study was to optimize a flow cytometric method for routine use of anti-TRBC1 to assess T-cell clonality and validate it in a large series of normal and pathological samples. Our results showed that the best resolution to accurately identify TRBC1+ cells was achieved by adding the CD3 antibody either simultaneously or after TRBC1. In addition, TRBC1+/TRBC1− ratios within different Tαβ-cell subsets are provided as expected reference ranges for polyclonal T-cells. Based on the optimized approach here proposed, we detected monoclonal Tαβ-cell populations with high specificity (96%) and a high analytical sensitivity/level of detection (≤10−4), when clonal T-cells exhibited immunophenotypic aberrancies. These findings further support and extend previous observations about the utility of TRBC1 for the diagnostic screening and monitoring of clonal Tαβ-cell populations. A single antibody (anti-TRBC1; JOVI-1 antibody clone) against one of the two mutually exclusive T-cell receptor β-chain constant domains was identified as a potentially useful flow-cytometry (FCM) marker to assess Tαβ-cell clonality. We optimized the TRBC1-FCM approach for detecting clonal Tαβ-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 Tαβ-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 Tαβ-cell subsets are provided as reference for polyclonal cells, among which a bimodal pattern of TRBC1-expression profile was found for all TCRVβ families, whereas highly-variable TRBC1+/TRBC1− ratios were observed in more mature vs. naïve Tαβ-cell subsets (vs. total T-cells). In 112/117 (96%) samples containing clonal Tαβ-cells in which the approach was validated, monotypic expression of TRBC1 was confirmed. Dilutional experiments showed a level of detection for detecting clonal Tαβ-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 Tαβ+ leukemia/lymphoma patients. [ABSTRACT FROM AUTHOR]

Simple Summary: We investigated the distribution of different subsets of monocytes (Mo) in blood and bone marrow (BM) of newly-diagnosed untreated monoclonal gammopathy of undetermined significance (MGUS), smoldering (SMM) and active multiple myeloma (MM), and its relationship with immune/bone serum-marker profiles. Our results showed decreased production of BM Mo with decreased counts of classical Mo (cMo) in BM and blood of SMM and MM, but not MGUS. Conversely, intermediate and non-classical Mo were significantly increased in MGUS, SMM and MM BM. In parallel, increased levels of interleukin (IL)1β were observed in a fraction of MGUS and SMM, while increased serum IL8 was characteristic of SMM and MM, and higher serum IL6, RANKL and bone alkaline phosphatase concentrations, together with decreased counts of FcεRI+cMo, were restricted to MM presenting with bone lesions. These results provide new insights in the pathogenesis of plasma cell neoplasms and the potential role of FcεRI+cMo in normal bone homeostasis. Background. Monocyte/macrophages have been shown to be altered in monoclonal gammopathy of undetermined significance (MGUS), smoldering (SMM) and active multiple myeloma (MM), with an impact on the disruption of the homeostasis of the normal bone marrow (BM) microenvironment. Methods: We investigated the distribution of different subsets of monocytes (Mo) in blood and BM of newly-diagnosed untreated MGUS (n = 23), SMM (n = 14) and MM (n = 99) patients vs. healthy donors (HD; n = 107), in parallel to a large panel of cytokines and bone-associated serum biomarkers. Results: Our results showed normal production of monocyte precursors and classical Mo (cMo) in MGUS, while decreased in SMM and MM (p ≤ 0.02), in association with lower blood counts of recently-produced CD62L+ cMo in SMM (p = 0.004) and of all subsets of (CD62L+, CD62L− and FcεRI+) cMo in MM (p ≤ 0.02). In contrast, intermediate and end-stage non-classical Mo were increased in BM of MGUS (p ≤ 0.03), SMM (p ≤ 0.03) and MM (p ≤ 0.002), while normal (MGUS and SMM) or decreased (MM; p = 0.01) in blood. In parallel, increased serum levels of interleukin (IL)1β were observed in MGUS (p = 0.007) and SMM (p = 0.01), higher concentrations of serum IL8 were found in SMM (p = 0.01) and MM (p = 0.002), and higher serum IL6 (p = 0.002), RANKL (p = 0.01) and bone alkaline phosphatase (BALP) levels (p = 0.01) with decreased counts of FcεRI+ cMo, were restricted to MM presenting with osteolytic lesions. This translated into three distinct immune/bone profiles: (1) normal (typical of HD and most MGUS cases); (2) senescent-like (increased IL1β and/or IL8, found in a minority of MGUS, most SMM and few MM cases with no bone lesions); and (3) pro-inflammatory-high serum IL6, RANKL and BALP with significantly (p = 0.01) decreased blood counts of immunomodulatory FcεRI+ cMo-, typical of MM presenting with bone lesions. Conclusions: These results provide new insight into the pathogenesis of plasma cell neoplasms and the potential role of FcεRI+ cMo in normal bone homeostasis. [ABSTRACT FROM AUTHOR]

Simple Summary: STAT3 and STAT5B mutations have been identified in a subset of T and NK large granular lymphocytic leukemia (T/NK-LGLL). The aim of our study was to evaluate the frequency and type of these mutations in all different subtypes of T/NK-LGL expansions (n = 100 patients), as well as to analyze its association with biological and clinical features of the disease. We show for the first time that STAT3/5B mutations were present in all different T/NK-cell LGLL categories here studied; further, STAT3 mutations were associated with overall reduced counts of almost all normal residual populations of immune cells in blood, together with a shorter time-to-therapy vs. wild type T/NK-LGLL. These findings contribute to support the utility of the STAT3 mutation analysis for diagnostic and prognostic purposes in LGLL. STAT3 and STAT5B (STAT3/STAT5B) mutations are the most common mutations in T-cell large granular lymphocytic leukemia (T-LGLL) and chronic lymphoproliferative disorders of NK cells (CLPD-NK), but their clinical impact remains unknown. We investigated the frequency and type of STAT3/STAT5B mutations in FACS-sorted populations of expanded T/NK-LGL from 100 (82 clonal; 6 oligoclonal; 12 polyclonal) patients, and its relationship with disease features. Seventeen non-LGL T-CLPD patients and 628 age-matched healthy donors were analyzed as controls. STAT3 (n = 30) and STAT5B (n = 1) mutations were detected in 28/82 clonal T/NK-LGLL patients (34%), while absent (0/18, 0%) among oligoclonal/polyclonal LGL-lymphocytosis. Mutations were found across all diagnostic subgroups: TCD8+-LGLL, 36%; CLPD-NK, 38%; TCD4+-LGLL, 7%; Tαβ+DP-LGLL, 100%; Tαβ+DN-LGLL, 50%; Tγδ+-LGLL, 44%. STAT3-mutated T-LGLL/CLPD-NK showed overall reduced (p < 0.05) blood counts of most normal leukocyte subsets, with a higher rate (vs. nonmutated LGLL) of neutropenia (p = 0.04), severe neutropenia (p = 0.02), and cases requiring treatment (p = 0.0001), together with a shorter time-to-therapy (p = 0.0001), particularly in non-Y640F STAT3-mutated patients. These findings confirm and extend on previous observations about the high prevalence of STAT3 mutations across different subtypes of LGLL, and its association with a more marked decrease of all major blood-cell subsets and a shortened time-to-therapy. [ABSTRACT FROM AUTHOR]