Association between memory B-cells and clinical and immunological features of primary Sjögren's syndrome and Sicca patients.

B-cells play a pivotal role in primary Sjögren's syndrome (pSS) pathogenesis. We aim to (1) evaluate the distribution of B-lymphocyte subpopulations in pSS and Sicca patients, (2) establish cut-off points that discriminate pSS from controls, (3) evaluate the association between memory B-cells and phenotypic features in pSS. We included 57 pSS patients, 68 Sicca and 24 healthy controls. Circulating B-cells were characterized by flow cytometry as naïve and memory subsets and classified from Bm1 to Bm5. Compared to controls, pSS patients had lower percentages (29.5 vs 44.4%) and absolute numbers (47 vs 106 cells/µl) of memory B-cells. Through ROC curves, a cut-off of ≤ 58 total memory B-cells/µl yielded a specificity of 0.88 and a sensitivity of 0.60 for pSS, and was met by 59.6% of pSS patients, 38.8% of Sicca and 12.5% of controls. A cut-off of < 23.5 Switched-memory B-cells/µl yielded a specificity of 0.88 and a sensitivity of 0.54 and was met by 54.4% of pSS patients, 37.3% of Sicca and 12.5% of controls. In pSS, lower total memory B-cells count was associated with longer disease duration (14.3 vs 8.1 years, p = 0.006) and more active disease profile, as evaluated by the European League Against Rheumatism (EULAR) Sjögren's Syndrome Disease Activity Index (ESSDAI) (3.1 vs 1.4, p = 0.043). Decreased numbers of memory B-cells clearly discriminated pSS from controls and can also have prognostic value. It remains to be clarified whether Sicca patients with decreased memory B-cells represent pSS and if B-cell profiling could help in the diagnosis of pSS.

Regulatory T Cells Show Dynamic Behavior During Late Pregnancy, Delivery, and the Postpartum Period.

Regulatory T cells (Tregs) are critical immunomodulators during early pregnancy by preventing maternal T-cell activation against fetal cells. However, how populations of maternal Tregs vary during and after pregnancy in humans is still unclear. Therefore, we investigated Treg subsets in the peripheral blood of pregnant women from late pregnancy through the postpartum period. To accomplish this, the following circulating Treg subsets were analyzed in 43 healthy pregnant women and 35 nonpregnant women by flow cytometry during the third trimester, on the day of delivery, and postpartum: CD4DimCD25Hi, CD4+CD25HiFoxp3+, and CD4+CD25HiCD127-/dim. Additionally, the expression levels of the transcription factor Foxp3 in CD4DimCD25Hi Treg were analyzed. We have found that CD4DimCD25Hi Treg subset significantly decreased in the pregnant women on the day of delivery relative to the third trimester ( P < .05), and that all Treg subsets significantly increased postpartum compared to the third trimester and the day of delivery ( P < .05). Moreover, the Foxp3 expression ratios within the CD4DimCD25Hi Treg subset decreased during pregnancy and until delivery compared to those measured in the nonpregnant women and significantly increased postpartum compared to the third trimester and the day of delivery ( P < .05). Thus, despite their established role in offering immunoprotection to the fetus in early pregnancy, the number of circulating Tregs also varies from late pregnancy to the postpartum period. Our results offer an explanation for the possible effects of pregnancy on the clinical outcomes of some autoimmune diseases during the postpartum period.

Impact of Labor on Peripheral Blood Maternal T-Cell Subsets and on Regulatory T and B Cells.

PURPOSE: Labor is thought to positively influence immune system development in the offspring, but studies investigating the impact of different modes of delivery on maternal immune system cells are scarce. Therefore, the aim of this study was to investigate the effect of labor on maternal peripheral blood T-cell subsets and on the recently described regulatory T and B cells. METHODS: Cross-sectional study comparing the absolute counts and percentages of peripheral blood T-cell subsets (maturation and activation profiles) and regulatory T and B cells between healthy pregnant women who delivered their newborns via elective cesarean (no labor; n = 14) and those who had a spontaneous vaginal delivery (after labor; n = 18). The cells were characterized using flow cytometry. RESULTS: We found that compared to the women who had elective cesareans, those who had spontaneous vaginal deliveries had significantly ( P < .05) lower absolute counts of B cells (median [cells/µL]: 146 [interquartile range, IQR = 49] vs 192 [IQR = 65]) and natural killer-like T (NKT-like) cells (median [cells/µL]: 154 [IQR = 125] vs 224 [IQR = 117]) in the peripheral blood. No further significant differences, particularly in regulatory T and B cells, were identified between the study groups. CONCLUSION: Labor does not seem to have a major impact on maternal peripheral blood T-cell subsets or regulatory T and B cells.

Pregnancy alters the circulating B cell compartment in atopic asthmatic women, and transitional B cells are positively associated with the development of allergy manifestations in their progeny.

PROBLEM: Maternal atopy is a risk factor for allergy. B cells are poorly studied in reproduction and atopy. We aimed to assess how pregnancy affects B cells in atopic women and whether B cells relate to allergic manifestations in offspring. METHOD OF STUDY: Women with and without atopic asthma, pregnant and non-pregnant were enrolled for the study, and circulating B cells were evaluated by flow cytometry, using CD19, CD27, CD38, IgD, and IgM. RESULTS: Compared to healthy non-pregnant, atopic asthmatic non-pregnant (ANP) women presented increased B cell counts, enlarged memory subsets, less transitional cells, and plasmablasts. Atopic asthmatic pregnant (AP) and healthy pregnant (HP) women showed similarities: reduced B cell counts and percentages, fewer memory cells, especially switched, and higher plasmablast percentages. Transitional B cell percentages were increased in AP women with allergic manifestations in their progeny. CONCLUSION: Atopic asthmatic non-pregnant women have a distinctive B cell compartment. B cells change in pregnancy, similarly in AP and HP women. The recognition that AP women with allergy in their progeny have a typical immune profile may help, in the future, the adoption of preventive measures to avoid the manifestation of allergic diseases in their newborns.

Characterization of B cells in healthy pregnant women from late pregnancy to post-partum: a prospective observational study.

BACKGROUND: B cells play a role in pregnancy due to their humoral and regulatory activities. To our knowledge, different maturational stages (from transitional to memory) of circulating B cell subsets have not yet been characterized (cell quantification and phenotype identification) in healthy pregnant women. Thus, the objective of our study was to characterize these subsets (as well as regulatory B cells) from late pregnancy to post-partum and to compare them with the circulating B cells of non-pregnant women. METHODS: In all of the enrolled women, flow cytometry was used to characterize the circulating B cell subsets according to the expression of IgD and CD38 (Bm1-Bm5 classification system). Regulatory B cells were characterized based on the expression of surface antigens (CD24, CD27, and CD38) and the production of IL-10 after lipopolysaccharide stimulation. RESULTS: Compared to the absolute counts of B cells in the non-pregnant women (n = 35), those in the pregnant women (n = 43) were significantly lower (p < 0.05) during the 3rd trimester of pregnancy and on delivery day (immediately after delivery). The percentages of these cells on delivery day and at post-partum were significantly lower than those in the non-pregnant women. In general, the absolute counts and percentages of the majority of the B cell subsets were significantly lower in the 3rd trimester of pregnancy and on delivery day than in the non-pregnant women. However, these counts and percentages did not differ significantly between the post-partum and the non-pregnant women. The most notable exceptions to the above were the percentages of naïve B cells (which were significantly higher in the 3rd trimester and on delivery day than in the non-pregnant women) and of CD24(hi)CD38(hi) regulatory B cells (which were significantly higher in the post-partum than in the non-pregnant women). CONCLUSION: According to our study, the peripheral B cell compartment undergoes quantitative changes during normal late pregnancy and post-partum. Such findings may allow us to better understand immunomodulation during human pregnancy and provide evidence that could aid in the development of new strategies to diagnose and treat pregnancy-associated disturbances. Our findings could also be useful for studies of the mechanisms of maternal responses to vaccination and infection.

Immature myeloid cells and tolerogenic cytokine profile in lung adenocarcinoma metastatic lymph nodes assessed by endobronchial ultrasound.

In lung cancer, the immune cell compartment of tumor-draining lymph nodes (TDLNs) dictate the response against tumors. This response is predominantly triggered by myeloid antigen-presenting cells (mAPCs) that capture antigens and, if matured, prime anti-tumor-specific T cell populations. However, the clinical role of mAPCs infiltrated in TDLN from lung cancer patients is poorly understood. The purpose of this study was to study mAPCs in TDLN from lung adenocarcinoma patients, in comparison to individuals with non-malignant diseases, using minimally invasive sampling methods. Mediastinal lymph nodes were assessed by endobronchial ultrasound-transbronchial needle aspiration (EBUS-TBNA). mAPCs were characterized by flow cytometry and cytokine expression by quantitative polymerase chain reaction. The association with tumor burden, overall survival, and response to treatment was assessed. TDLN from lung adenocarcinoma patients (n = 24) showed a reduced immune cell compartment, but a higher level of infiltrating mAPCs, when compared with control lymph nodes (n = 17). A decreased expression of co-stimulatory molecules CD80/CD86 by TDLN and blood mAPC was observed. TDLN showed lower levels of TNF-α and IL-12 and increased levels of immunosuppressive cytokines TGF-ß and IL-10. The IL-12 expression was inversely correlated with the percentage of infiltrated tumor cells, while IL-10 was directly correlated. Patients with lower expression of IL-12 in TDLN or lower expression of CD80/86 in blood mAPCs had worse overall survival and response to therapy. mAPCs of lung adenocarcinoma patients express less co-stimulatory molecules, and within TDLN, the cytokine profile is biased towards a tolerance-inducing phenotype. Patients with enhanced immune parameters have better survival and response to treatment. EBUS-TBNA allows the collection of viable specimens from TDLN that may provide further insight on relevant immunological mechanisms.

Cytokeratin 19, carcinoembryonic antigen, and epithelial cell adhesion molecule detect lung cancer lymph node metastasis in endobronchial ultrasound-guided transbronchial aspiration samples.

INTRODUCTION: Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) holds promise for accurate examination of mediastinal lymph nodes in NSCLC patients. However, it is not always possible to achieve a definitive diagnosis or subtype all cases. We aimed to evaluate the role of EBUS-TBNA combined with quantitative reverse transcription polymerase chain reaction (qRT-PCR) and flow cytometry (FCM) to assess tumor-associated antigens and immune responses to identify metastases and pathological patterns in lymph node aspirates. PATIENTS AND METHODS: EBUS-TBNA samples from patients with NSCLC (n = 33) and nonmalignant diseases (n = 17) were prospectively collected. Cytokeratin 19 (CK-19), carcinoembryonic antigen (CEA), epithelial cell adhesion molecule (EPCAM), sialyl-Lewis(x), CD44, and the immune compartment were analyzed using qRT-PCR and FCM. RESULTS: In the NSCLC patients, the epithelial cell compartment was significantly increased (30.8% vs. 12% CD45⁻ CK-19⁺ cells) and showed brighter CK-19 staining than controls (P = .039) using FCM. Carcinoembryonic antigen was exclusively expressed by the NSCLC epithelial compartment (35% of the cases) and absent in controls. The NSCLC immune compartment showed an increased monocyte population (P = .04), and decreased lymphocyte subpopulations, anticipating a disruption in the distribution of myeloid and lymphoid immune cells. Quantitative reverse transcription polymerase chain reaction showed that CK-19, CEA, and EPCAM transcripts were significantly higher in NSCLC. A positive correlation between the primary tumor lesion size and EPCAM (ρ = 0.476; P = .005), CK-19 (ρ = 0.594; P = .001), and CEA (ρ = 0.394; P = .023) was also found. CONCLUSION: The identification of CK-19, CEA, and EPCAM in EBUS-TBNA samples using FCM and qRT-PCR is feasible and might further aid in the detection of NSCLC lymph node metastasis.