Cell Subpopulations In Peripheral Blood Research Articles

Monocyte-to-platelets ratio (MPR) at diagnosis is associated with inferior progression-free survival in patients with mantle cell lymphoma: a multi-center real-life survey.

Mantle cell lymphoma (MCL) pathogenesis is strongly related to the role of the tumor immune microenvironment (TIME) in which MCL cells proliferate. TIME cells can produce growth signals influencing MCL cells' survival and exert an antitumoral immune response suppression. The activity of TIME cells might be mirrored by some ratios of peripheral blood cell subpopulations, such as the monocyte-to-platelet ratio (MPR). We reviewed the clinical features of 165 consecutive MCL patients newly diagnosed and not eligible for autologous stem cell transplantation (both for age or comorbidities) who accessed two Italian Centers between 2006 and 2020. MPR was calculated using data obtained from the complete blood cell count at diagnosis before any cytotoxic treatment and correlated with PFS. Univariate analysis showed that MPR ≥ 3 was associated with inferior PFS (p = 0.02). Multivariate analysis confirmed that MPR ≥ 3, LDH > 2.5 ULN, and bone marrow involvement were significant independent variables in predicting PFS. For these reasons, MPR ≥ 3 seems the most promising prognostic factor in patients with MCL, and it could be considered a variable in new predictive models.

Peripheral Blood Mononuclear Cells and Serum Cytokines in Patients with Lupus Nephritis after COVID-19.

Systemic lupus erythematosus (SLE) patients have an increased risk of infections and infection-related mortality. Therefore, during the global SARS-CoV-2 pandemic, SLE patients were particularly vulnerable to SARS-CoV-2 infections. Also, compared to other patients, SLE patients seem to develop more severe manifestations of coronavirus disease 2019 (COVID-19), with higher rates of hospitalization, invasive ventilation requirements, or death. This study evaluated the immune parameters after SARS-CoV-2 infection in SLE patients. We analyzed subpopulations of peripheral blood cells collected from patients with renal manifestation of SLE (lupus nephritis, LN). LN patients were divided into two subgroups: those unexposed to SARS-CoV-2 (LN CoV-2(-)) and those who had confirmed COVID-19 (LN-CoV-2(+)) six months earlier. We analyzed basic subpopulations of T cells, B cells, monocytes, dendritic cells (DCs), and serum cytokines using flow cytometry. All collected data were compared to a healthy control group without SARS-CoV-2 infection in medical history. LN patients were characterized by a decreased percentage of helper T (Th) cells and an increased percentage of cytotoxic T (Tc) cells regardless of SARS-CoV-2 infection. LN CoV-2(+) patients had a higher percentage of regulatory T cells (Tregs) and plasmablasts (PBs) and a lower percentage of non-switched memory (NSM) B cells compared to LN CoV-2(-) patients or healthy controls (HC CoV-2(-)). LN patients had a higher percentage of total monocytes compared with HC CoV-2(-). LN CoV-2(+) patients had a higher percentage of classical and intermediate monocytes than LN CoV-2(-) patients and HC CoV-2(-). LN CoV-2(+) patients had higher serum IL-6 levels than HC CoV-2(-), while LN CoV-2(-) patients had higher levels of serum IL-10. LN patients are characterized by disturbances in the blood's basic immunological parameters. However, SARS-CoV-2 infection influences B-cell and monocyte compartments.

Correlation of cTfh cells and memory B cells with AMR after renal transplantation

Renal transplantation is the preferred treatment option for patients with end-stage renal disease (ESRD) in a clinical setting. Antibody mediated rejection (AMR) is one of the leading causes of graft dysfunction. To address the current shortcomings in the early diagnosis and treatment of AMR in clinical practice, this article analyzes the distribution of different circulating T follicular helper (cTfh) cell subtypes and B cell subpopulations in peripheral blood and detects the cytokine levels of chemokine ligand 13 (CXCL13), interleukin-21 (IL-21), and interleukin-4 (IL-4) related to cTfh cells in peripheral blood of kidney transplant recipients. Moreover, we also explore the correlation between cTfh cells, peripheral blood memory B cells, and AMR, their value as early predictive indicators of AMR, and explore potential therapeutic targets for AMR patients. Our results indicate that the proportion of cTfh cells increased at the onset of AMR, which plays an important role in antigen-specific B-cell immune regulation. Activation of cTfh cells in AMR patients correlates with phenotypes of memory B cells and plasma blasts. cTfh cells and memory B cells have promising diagnostic efficacies and predictive values for AMR. The proportion of cTfh cells to CD4+ T cells and the proportion of memory B cells to CD19+ B cells are correlated with serum creatinine levels, indicating that cTfh cells and memory B cells may be involved in the progression of AMR. In addition, the CXCL13, IL-21, and IL-4, which were associated with cTfh cells, may be involved in the onset of AMR.

Expression of Molecules Characterizing Metabolic and Cytotoxic Activity of Different Natural Killer Cell Subpopulations in Peripheral Blood during Pregnancy

Expression of Molecules Characterizing Metabolic and Cytotoxic Activity of Different Natural Killer Cell Subpopulations in Peripheral Blood during Pregnancy

Asociación entre HLA-DR4 y prurigo actínico: una revisión exploratoria

IntroductionDifferent studies on actinic prurigo suggest that this pathology may have an autoimmune component. Alterations have been found in peripheral blood cell subpopulations, increased lymphocytes and the expression of adhesion molecules in dermal lesions, in addition to a strong association with HLA class II. ObjectiveDescribe the current status of the association of HLA class II and actinic prurigo in the scientific literature published. Material and methodsScoping review that included PubMed, Scopus y LILACS. Empirical and theoretical publications, without a time limit written in English and Spanish were included. Result26 documents were included there are original research articles (n = 11), congress presentation (n = 1), narrative reviews (n = 6), letters to the editor (n = 4), comment (n = 1), case report (1), and case series (n = 2). ConclusionA strong association between actinic prurigo and HLA-DR4 and subtype DR4 DRB1*0407 is described. It is necessary to conduct a major number of studies for typification in other populations in order to establish the presence of unknown alleles associated with actinic prurigo. Simultaneously, it is important to increase the sample of patients with the disease and control the patient's sample to explore a possible influence of an environmental factor that modulates presence of the actinic prurigo.

Contents of CD4⁺ and CD8⁺ memory cell subpopulations and circulating cytokine levels in psoriasis in children

In psoriasis, memory T cells form in the immune system and contribute to the development of disease recurrence. In the presence of secondary antigen exposure, CD4⁺ and CD8⁺ memory cells begin to proliferate intensively and develop a more sustained and stronger immune response. There is an imbalance of cytokines in the immunopathogenesis of psoriasis. The aim was to study the content of CD4⁺ and CD8⁺ memory cell subpopulations in peripheral blood and circulating cytokines in children with psoriasis. Materials and methods — 114 children with vulgar psoriasis on different pathogenetic therapies between 5 and 18 years of age were examined. The results were obtained by flow cytofluorimetry and multiplex analysis using xMAP technology. Analysis of the data obtained was performed using Statistica 10.0. Results. We found that the content of naïve and central memory T-cells was significantly higher than similar CD8+ memory cells, and the number of effector CD8⁺ cells was significantly higher than CD4⁺ in children with psoriasis (p<0.05). The dependence of memory cells on the age and duration of the disease was found to be significant. The number of effector and terminally differentiated memory cells was shown to depend on the type of therapy. Elevated serum levels of IL-12p70, IL-22, IL-28A, GM-CSF, IL-13, IL-4, IL-23, IL-17A, IL-17F, IL-27, TNFα and their association with memory cell count, and psoriasis severity according to PASI were found. Conclusion. The study of immunological memory will allow a more detailed understanding of the immunopathogenesis of psoriasis, finding new effective treatments and improving patients’ quality of life.

The relationship between myeloid-derived supressor cells and clinico-laboratory parameters in patients with liver cyrrosis

In the present study, we used multicolor flow cytometry assay to measure the numbers of various myeloid suppressor cell subpopulations (Lin-HLA-DR-CD33+, Lin-HLA-DR-CD33+CD66b+, and CD14+HLA-DRlow/-) in peripheral blood of 51 participants, including 33 patients with viral- and 1 8 patients with «nonviral» (alcoholic or biliary/autoimmune) liver cirrhosis. Patients in both groups had increased proportions of Lin-HLA-DR-CD33+, Lin-HLA-DR-CD33+CD66b+, and CD 14+HLA-DRlow/-cells which levels did not depend on the type and replication of the virus in viral liver cirrhosis. In viral liver cirrhosis, the relative numbers of Lin-HLA-DR-CD33+cells directly correlated with the albumin levels (Rs=0.45; p=0.029). In «nonviral» group an inverse relationship was found between these indicators (Rs = -0.56; p=0.02), in addition the proportion of Lin-HLA-DR-CD33+and CD14+HLA-DRlow/-cells directly correlated with disease severity scores (Child-Pugh and MELD) direct correlation of the proportion of Lin-HLA-DR-CD33+and CD14+HLA-DRlow/-cells with the disease severity scores (Child-Pugh and MELD). Comparison of the initial content of myeloid suppressors with the response to complex therapy (that included autologous bone marrow-derived cell transplantation), showed that in viral liver cirrhosis, the proportion of Lin-HLA-DR-CD33+cells was characterized by a prognostic significance and, at values

Regulatory T Cells as Predictors of Clinical Course in Hospitalised COVID-19 Patients.

BackgroundThe host immune response has a prominent role in the progression and outcome of SARS-CoV-2 infection. Lymphopenia has been described as an important feature of SARS-CoV-2 infection and has been associated with severe disease manifestation. Lymphocyte dysregulation and hyper-inflammation have been shown to be associated with a more severe clinical course; however, a T cell subpopulation whose dysfunction correlate with disease progression has yet to be identify.MethodsWe performed an immuno-phenotypic analysis of T cell sub-populations in peripheral blood from patients affected by different severity of COVID-19 (n=60) and undergoing a different clinical evolution. Clinical severity was established based on a modified WHO score considering both ventilation support and respiratory capacity (PaO2/FiO2 ratio). The ability of circulating cells at baseline to predict the probability of clinical aggravation was explored through multivariate regression analyses.ResultsThe immuno-phenotypic analysis performed by multi-colour flow cytometry confirmed that patients suffering from severe COVID-19 harboured significantly reduced circulating T cell subsets, especially for CD4+ T, Th1, and regulatory T cells. Peripheral T cells also correlated with parameters associated with disease severity, i.e., PaO2/FiO2 ratio and inflammation markers. CD4+ T cell subsets showed an important significant association with clinical evolution, with patients presenting markedly decreased regulatory T cells at baseline having a significantly higher risk of aggravation. Importantly, the combination of gender and regulatory T cells allowed distinguishing between improved and worsened patients with an area under the ROC curve (AUC) of 82%.ConclusionsThe present study demonstrates the association between CD4+ T cell dysregulation and COVID-19 severity and progression. Our results support the importance of analysing baseline regulatory T cell levels, since they were revealed able to predict the clinical worsening during hospitalization. Regulatory T cells assessment soon after hospital admission could thus allow a better clinical stratification and patient management.

Changes in B Cell Pool of Patients With Multibacillary Leprosy: Diminished Memory B Cell and Enhanced Mature B in Peripheral Blood.

Despite being treatable, leprosy still represents a major public health problem, and many mechanisms that drive leprosy immunopathogenesis still need to be elucidated. B cells play important roles in immune defense, being classified in different subgroups that present distinct roles in the immune response. Here, the profile of B cell subpopulations in peripheral blood of patients with paucibacillary (TT/BT), multibacillary (LL/BL) and erythema nodosum leprosum was analyzed. B cell subpopulations (memory, transition, plasmablasts, and mature B cells) and levels of IgG were analyzed by flow cytometry and ELISA, respectively. It was observed that Mycobacterium leprae infection can alter the proportions of B cell subpopulations (increase of mature and decrease of memory B cells) in patients affected by leprosy. This modulation is associated with an increase in total IgG and the patient’s clinical condition. Circulating B cells may be acting in the modulation of the immune response in patients with various forms of leprosy, which may reflect the patient’s ability to respond to M. leprae.

Peripheral Blood Biomarkers Associated With Improved Functional Outcome in Patients With Chronic Left Ventricular Dysfunction: A Biorepository Evaluation of the FOCUS-CCTRN Trial.

Cell therapy trials for heart failure (HF) have shown modest improvement; however, the mechanisms underlying improvement in some patients but not others are not well understood. Although immune cells are important in the course of HF, our understanding of the immune processes in HF is limited. The objective of this study was to evaluate associations between temporal changes in peripheral blood (PB) cell subpopulations and improved outcome in patients with chronic ischemic cardiomyopathy after bone marrow-derived mononuclear cell therapy or placebo in the FOCUS-CCTRN trial. Peripheral blood was collected at days 0, 1, 30, 90, and 180 from consented participants. We used flow cytometry to compare PB populations in patients with the best (cohort 1) or worst functional outcome (cohort 2) in three primary endpoints: left ventricular (LV) ejection fraction, LV end-systolic volume, and maximal oxygen consumption (VO2 max). A linear mixed model was used to assess changes over time in 32 cell populations. The difference between each time point and baseline was calculated as linear contrast. Compared with cohort 2, patients who improved (cohort 1) had a higher frequency of CD45+CD19+ B cells at days 0, 1, 90, and 180. CD11B+ cells increased over baseline at day 1 in both cohorts and remained higher in cohort 2 until day 30. CD45+CD133+ progenitor cells decreased over baseline at day 30 in cohort 1. We identified specific cell subpopulations associated with improved cardiac function in patients with chronic LV dysfunction. These findings may improve patient selection and prediction of outcomes in cell therapy trials.

Low pan-immune-inflammation-value predicts better chemotherapy response and survival in breast cancer patients treated with neoadjuvant chemotherapy

Blood-based biomarkers reflect systemic inflammation status and have prognostic and predictive value in solid malignancies. As a recently defined biomarker, Pan-Immune-Inflammation-Value (PIV) integrates different peripheral blood cell subpopulations. This retrospective study of collected data aimed to assess whether PIV may predict the pathological complete response (pCR) to neoadjuvant chemotherapy (NAC) in Turkish women with breast cancer. The study consisted of 743 patients with breast cancer who were scheduled to undergo NAC before attempting cytoreductive surgery. A pre-treatment complete blood count was obtained in the two weeks preceding NAC, and blood-based biomarkers were calculated from absolute counts of relevant cell populations. The pCR was defined as the absence of tumor cells in both the mastectomy specimen and lymph nodes. Secondary outcome measures included disease-free survival (DFS) and overall survival (OS). One hundred seven patients (14.4%) had pCR. In receiver operating characteristic analysis, optimal cut-off values for the neutrophile-to-lymphocyte ratio (NLR), monocyte-to-lymphocyte ratio (MLR), platelet-to-lymphocyte (PLR), PIV, and Ki-67 index were determined as ≥ 2.34, ≥ 0.22, ≥ 131.8, ≥ 306.4, and ≥ 27, respectively. The clinical tumor (T) stage, NLR, MLR, PLR, PIV, estrogen receptor (ER) status, human epidermal growth factor receptor-2 (HER-2) status, and Ki-67 index were significantly associated with NAC response in univariate analyses. However, multivariate analysis revealed that the clinical T stage, PIV, ER status, HER-2 status, and Ki-67 index were independent predictors for pCR. Moreover, the low PIV group patients had significantly better DFS and OS than those in the high PIV group (p = 0.034, p = 0.028, respectively). Based on our results, pre-treatment PIV seems as a predictor for pCR and survival, outperforming NLR, MLR, PLR in predicting pCR in Turkish women with breast cancer who received NAC. However, further studies are needed to confirm our findings.

Low Circulating B Lymphocytes in Newly Diagnosed Follicular and Diffuse Large B Cell Lymphoma: Differences Compared to Normal Subjects and Possible Prognostic Role

Introduction: Reduction of the lymphocyte to monocyte ratio (LMR) has an unfavorable impact on overall survival in Diffuse Large B Cell Lymphoma (DLBCL), and on treatment response in Follicular Lymphoma (FL). Abnormal LMR seems primarily due to a reduction in absolute value of lymphocytes. Based on these observations, we have prospectively investigated peripheral blood (PB) and Bone Marrow (BM) lymphocyte subpopulations in a series of previously untreated DLBCL and FL. Aim of the study was: i. to assess whether LMR alteration is linked to a distinct lymphocyte subset; ii. to correlate PB with BM cell subpopulations; iii. to compared DLBCL and FL with regards to PB and BM lymphocyte subpopulations. Methods: Overall, PB cells from 58 DLBCL and 27 FL at diagnosis were investigated; BM cell populations were analyzed in 77 DLBCL and 54 FL. None of the studied patients had BM involvement nor circulating lymphoma cells detectable by cytometry; in addition, none of the patients had been previously treated with immunosuppressive drugs. PB from 40 healthy subjects and BM samples from 69 subjects without primary BM dysfunctions were used as control. Multicolor flow cytometry (MFC) was used to evaluate percentages and absolute values of the following cell subsets: CD3+ T cell, CD3-CD56+ NK cells, CD19+CD20+ B cells. Among B cells, t he analysis included transitional (CD38+high/CD24+high/IgD+/CD27-), naive (CD38+/CD24+/IgD+/CD27-), memory IgD+ (CD38-/+CD24+/IgD+/CD27+), and memory IgD- (CD38-/CD24+high/IgD-/CD27+) subsets; B cell precursors were investigated as CD19+CD20-CD10+ and mature B cells as CD19+CD20+CD10-. Results: A significant reduction in median absolute lymphocyte count was seen in PB from lymphoma patients (median: 1,526/µL) compared to controls (median: 2,195/ µL). Moreover, absolute lymphocytes were more markedly reduced in DLBCL (median: 1,386 /µL) compared to FL (median: 1883/µl) (p<0.001). This resulted in a significantly lower median PB-LMR in DLBCL (median LMR: 3) compared to FL patients (median LMR: 5) (p=0.001). Among PB cell subpopulations, a marked reduction in circulating B cells was observed in lymphoma patients (median 96/µL) compared to controls (median 224/µL). Again, loss of circulating B-cells was more markedly pronounced in DLBCL (median: 81.5 cell/µL) compared to FL (median: 150 cell/µL) (p=0.013). Differences in circulating B-cells among DLBCL, FL and controls are shown in Figure 1A. At BM analysis, a reduction of B cell precursor (CD19+/CD20-/CD10+) could be documented only in DLBCL. A reduction in circulating T-lymphocytes in lymphoma patients compared to controls was detected as well, although at a lower extent compared to B-cells. No significant abnormalities in the amount of circulating NK-cells was seen in both DLBCL and FL patients. When these data were matched with treatment outcome, the reduction of circulating B cells proved to be associated with an unfavorable outcome in younger patients with DLBCL. Indeed, among DLBCL patients aged < 60 yrs, a poorer progression free survival was seen in those having less than 96 B-cells/µL compared to those with PB B-cells equal or above 96 B-cells/µl (Figure 1B). Conclusions: The study indicates that circulating B lymphocytes are significantly reduced in both DLBCL and FL compared to age-matched healthy controls; however, their loss is much more pronounced in DLBCL compared to FL. Indeed, abnormalities in B-cell progenitors are present in BM from DLCBL but not from FL. In addition, B-cell reduction may have an unfavorable impact in the long-term outcome of DLBCL, at least in younger patients. Lastly, both B and T-cell subsets are decreased in DLBCL and FL whereas circulating NK subset does not seem to be affected. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Distribution of Myeloid and Plasmacytoid Dendritic Cell Subpopulations in Peripheral Blood of Hyperprolactinemic Women

Dendritic cells (DCs) play key roles in regulating the immune response using the specialized function of processing and presenting antigens. Prolactin (PRL), a hormone produced by the pituitary gland, participates in DC maturation and function. The present study was aimed to determine the frequencies of peripheral blood DC subpopulations of myeloid DC (MDC) and plasmacytoid DC (PDC) in hyperprolactinemic (HPRL) women compared to normal healthy volunteers. This study was conducted on 70 women, including 35 HPRL patients and 35 matched healthy controls, whose PRL serum levels were in the normal range (lower than 25 ng/mL). Serum thyroid-stimulating hormone (TSH) levels were measured in both groups as an indicator of normal thyroid function. The electrochemiluminescence immunoassay method was applied to measure the serum levels of TSH and PRL. The frequencies of MDC and PDC in the peripheral blood samples of both groups were determined by flow cytometry. The mean serum PRL levels in the HPRL patients and healthy individuals were 46.41±21.96 and 13.75±11.19, respectively (p<0.0001); however TSH levels in both groups were similar and within the normal range (0.4-4.5 mIU/mL) (p=0.2). The frequencies of both MDC and PDC subpopulations in the peripheral blood of HPRL patients were significantly lower than they were in the healthy controls. However, the ratio of MDCs/PDCs in HPRL patients was not significantly different between the two groups (p=0.8). Our study revealed that an increased level of serum PRL may lead to a reduction in the number of MDC and PDC subpopulations. These results could help clarify the complex relationship between the immune system and the neuroendocrine axis and may be of potential use in understanding the pathogenesis of endocrine and immune disorders.

Development of a method for the identification of receptor activator of nuclear factor-κB+ populations in vivo

ObjectivesOsteoclasts are induced by macrophage colony-stimulating factor-1 (CSF-1) and receptor activator of nuclear factor-κB (RANK) ligand (RANKL). Monocyte/macrophage lineages are thought to be osteoclast precursors; however, such cells have not been fully characterized owing to a lack of tools for their identification. Osteoclast precursors express colony-stimulating factor-1 receptor (CSF-1R) and RANK. However, the capacity of conventional methods using anti-RANK antibodies to detect RANK+ cells by flow cytometry is insufficient. Here, we developed a high-sensitivity method for detecting RANK+ cells using biotinylated recombinant glutathione S-transferase-RANKL (GST-RANKL-biotin). MethodsWe sorted sub-populations of mouse bone marrow (BM) or peripheral blood (PB) cells using GST-RANKL-biotin, anti-CSF1R, and anti-B220 antibodies and induced osteoclastogenesis in vitro. ResultsThe frequency of the RANK+ population in BM detected by GST-RANKL-biotin was significantly higher than that detected by anti-RANK antibodies. Although RANK+ cells were detected in both the B220+ and B220− populations, the macrophage lineage was present only in B220−. Unexpectedly, a significantly higher number of osteoclasts was induced in RANK−CSF-1R+ cells than in RANK+CSF-1R+ cells contained in the B220− population. In contrast, the PB-derived B220−RANK+CSF-1R+ population contained a significantly higher frequency of osteoclast precursors than the B220−RANK−CSF-1R+ population. ConclusionsThese results suggest that GST-RANKL-biotin is useful for the detection of RANK+ cells and that RANK and CSF-1R may be helpful indicators of osteoclast precursors in PB.

P-257 Deep polychromatic flow cytometry characterization of circulating endothelial cells in metastatic colorectal cancer patients

Circulating endothelial cells (CEC) and their progenitors (EPC) are restricted subpopulations of peripheral blood (PB), cord blood (CB), and bone marrow (BM) cells, involved in the endothelial remodeling and turnover. Both CEC and EPC have been studied as potential biomarkers in colon cancer, but a lack of specificity has limited their clinical application. In this work, a highly sensitive polychromatic flow cytometry method has been applied for CEC characterization, exploring the role of different circulating endothelial cell subpopulations as potential and predictive biomarkers in colorectal cancer patients.

Phosphoflow Protocol for Signaling Studies in Human and Murine B Cell Subpopulations.

BCR signaling, involving phosphorylation of various downstream molecules, including kinases, lipases, and linkers, is crucial for B cell selection, survival, proliferation, and differentiation. Phosphoflow cytometry (phosphoflow) is a single-cell-based technique to measure phosphorylated intracellular proteins, providing a more quantitative read-out than Western blotting. Recent advances in phosphoflow basically allow simultaneous analysis of protein phosphorylation in B cell (sub)populations, without prior cell sorting. However, fixation and permeabilization procedures required for phosphoflow often affect cell surface epitopes or mAb conjugates, precluding the evaluation of the phosphorylation status of signaling proteins across different B cell subpopulations present in a single sample. In this study, we report a versatile phosphoflow protocol allowing extensive staining of B cell subpopulations in human peripheral blood or various anatomical compartments in the mouse, starting from freshly isolated or frozen cell suspensions. Both human and mouse B cell subpopulations showed different basal and BCR stimulation-induced phosphorylation levels of downstream signaling proteins. For example, peritoneal B-1 cells and splenic marginal zone B cells exhibited significantly increased basal (ex vivo) signaling and increased responsiveness to in vitro BCR stimulation compared with peritoneal B-2 cells and splenic follicular B cells, respectively. In addition, whereas stimulation with anti-IgM or anti-Igκ L chain Abs resulted in strong pCD79a and pPLCγ2 signals, IgD stimulation only induced CD79a but not pPLCγ2 phosphorylation. In summary, the protocol is user friendly and quantifies BCR-mediated phosphorylation with high sensitivity at the single-cell level, in combination with extensive staining to identify individual B cell development and differentiation stages.

Pattern of Immunocompetent Peripheral Blood Cell Subpopulations in B-Cell Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma

Background. In the WHO classification small lymphocytic lymphoma (SLL) and B-cell chronic lymphocytic leukemia (В-CLL) are combined into one nosological entity of lymphoid tumors due to their similar tumor cell immunophenotype. Up to now, there is no consensus on either their similarities or the differences between them. Distinction between В-CLL and SLL is drawn with respect to clinical and hematological manifestations of tumors. The reason for the differences that determine tumor spreading in a patient may lie in specific states of some immune system components. Comparison of immune system parameters within the CLL/SLL model provides a unique opportunity to trace the behavior of immunity indicators in local und disseminated pathogenetically similar neoplastic processes and to identify possible prognostic factors. Aim. To compare quantitative representations of peripheral blood lymphocyte subpopulations in SLL and В-CLL. Materials &amp; Methods. Immunocompetent cells (relative and absolute Т- and NK-cell counts), immunophenotype, and tumor clone volume were assessed using multicolor flow cytometry based on the expression of СD3, CD4, CD8, CD16, CD19, CD20, CD23, CD5, CD79b, FMC7, CD22, CD43, CD38 antigens, and immunoglobulins light chain IgK and IgA. Before chemotherapy onset, the data of 17 SLL and 81 CLL patients (22 of them with B-lymphocyte count of 35-79 % and 59 with 80-99 %) were compared. As a control, peripheral blood lymphocyte subpopulations in 50 relatively healthy individuals (blood donors) were analyzed. Results. The analysis of NK-cells and Т-lymphocyte subpopulations in SLL showed the preserved number of killer/cytotoxic cells of innate and adaptive immunity (CD16+, CD8+), the reduction of CD4+ Т-cell count, and CD4/CD8 ratio. In CLL a considerable increase of main subpopulations of residual normal lymphocytes was detected. However, the extent of their increase proved to be considerably lower than increase in the volume of tumor B-cell clone, which signifies a rising exhaustion of immune system effector components. Conclusion. The present study yielded characteristic features of residual normal lymphocyte subpopulations in SLL and CLL with different leukocytosis grades. SLL patients demonstrated the reduction of relative and absolute Т-cell counts with Т-helper (CD3+, СD4+) phenotype, and the increase of cytotoxic CD8+ Т-cells and NK-cells. Lymphocytosis (35-79 %) in the CLL-I group was due not only to tumor В-cells but also to Т-killer (CD16+, CD8+) and Т-helper (CD4+) absolute counts, which were 1.7-2.5 times higher than in SLL and the control group. Residual lymphocyte subpopulation pattern (80-99 %) in the CLL-II group compared with the control group was characterized by a significantly higher absolute count of CD8+ T-cells and CD16+ NK-cells, as well as higher Т-regulatory index compared with SLL and CLL-I groups. These data point to the necessity for further and more detailed study of residual lymphocyte subpopulation pattern within the CLL/SLL model in order to identify additional predisposing factors.

Identification of Autoreactive B Cell Subpopulations in Peripheral Blood of Autoimmune Patients With Pemphigus Vulgaris.

Pemphigus vulgaris (PV) is a rare blistering disease caused by IgG autoantibodies against the epidermal adhesion molecules desmoglein (Dsg)3 and Dsg1 providing a well-characterized paradigm of an antibody-mediated organ-specific autoimmune disease. In PV patients who have achieved clinical remission after B cell-depleting therapy, relapses often coincide with a reoccurrence of B cells and Dsg-specific autoantibodies. Here, we analyzed Dsg3-specific B cell subpopulations (i.e., total CD19+ B cells, CD19+CD27−B cells, CD19+CD27+ memory B cells, and CD19+CD27hiCD38hi plasmablasts) in peripheral blood of both PV patients (n = 14) at different stages of disease and healthy individuals (n = 14) by flow cytometry using fluorescently labeled recombinant human Dsg3 protein. Applying this approach, Dsg3-specific B cells could be detected at low frequencies (0.11–0.53% of CD19+ B cells) and numbers of Dsg3-specific memory B cells were significantly increased in PV patients in clinical remission receiving minimal immunosuppressive therapy. Finally, we confirmed in vitro that Dsg3-reactive memory B cells were able to produce anti-Dsg3 IgG autoantibodies upon ex vivo activation. Thus, monitoring of Dsg3-specific B cells in PV is of particular interest to further characterize the immunopathogenesis of PV.

Correlation of the tumor mutational burden with the composition of the immune cell subpopulations in peripheral blood of triple-negative breast cancer patients undergoing neoadjuvant therapy with durvalumab: Results from the prospectively randomized GeparNuevo trial.

588 Background: The GeparNuevo trial is a randomized, double-blind, multi-center phase II trial of neoadjuvant therapy in patients with early-stage triple negative breast cancer (TNBC) investigating the role of durvalumab, an anti-PD-L1 antibody, which blocks PD-L1 binding to PD1 and CD80, in addition to standard chemotherapy with nab-Paclitaxel (nab-Pac) followed by Epirubicin plus Cyclophosphamid (EC; Loibl S et al. ASCO 2018). Since the tumor mutational burden (TMB) has been suggested to be associated with a better outcome of patients undergoing immunotherapy and an increased T cell response, we determined whether there exists a link between TMB and immune cell composition, frequency and function in patients of the GeparNuevo trial. Methods: In order to determine possible predictive and / or prognostic biomarkers, tumor biopsies taken at recruitment from 149 patients out of the 174 enrolled patients underwent deep sequencing in order to determine the TMB. In addition, for 120 patients blood samples were taken at recruitment and during different time points of treatment (after durvalumab pre-treatment, after Nab-Pac and at surgery after EC) and evaluated using multicolor flow cytometry by monitoring the absolute cell counts of T cells, B cells and NK cells as well as the frequency, composition and functionality of different immune cell populations. Results: The TMB of the GeparNuevo cohort was in line with published data with a mean of 1.8 mutations/MB (range 0.02 – 7.65), respectively. Preliminary evaluation demonstrated a significant correlation of TMB with blood parameters, in particular with subsets of CD8+ T cells. Interestingly, the data suggest a negative correlation of TMB with the frequency of effector cells while a positive correlation exists with the effector memory cells at recruitment. In depth analyses of a correlation with treatment arm and clinical responses are currently performed. Conclusions: Using this approach we hope to identify biomarkers, which will allow a better selection of TNBC patients undergoing specific immunotherapies. Clinical trial information: NCT02685059.

Characterization and functional analysis of novel circulating NK cell sub-populations.

Natural killer (NK) cells are innate lymphoid cells having potent cytolytic function that provide host defense against microbial infections and tumors. Using our generated monoclonal antibody (mAb), named FE-1H10, new NK cell sub-populations in peripheral blood were identified. The molecules recognized by mAb FE-1H10 were expressed on a sub-population of CD3-CD56dim NK cells. The epitope recognized by mAb FE-1H10 was demonstrated to be N-glycan and proven to be different from CD57. Upon K562 stimulation, the CD56dimFE-1H10+ NK cell sub-population exhibited significantly lower cytolytic function with low ability to degranulate and release cytolytic granules compared to the CD56dimFE-1H10- NK cell sub-population. Moreover, the CD56dimFE-1H10+ NK cells produced less IFN-γ and TNF-α than the CD56dimFE-1H10- NK cells. We demonstrated here that mAb FE-1H10 could identify two sub-populations of circulating CD56dim NK cells with different functions. Our discovery of new sub-populations of NK cells improves our understanding of NK cell biology and may lead to the development of new approaches for NK cell therapy.