Peripheral Lymphocyte Subpopulations Research Articles

Application of blood parameters for the early diagnosis of natural ascending placentitis in pregnant mares

Placental infection is an important cause of late-term pregnancy loss and neonatal diseases in horses. Detection of changes in blood parameters especially during early placentitis could improve the diagnostic accuracy, treatment decision, and potential outcomes. The objectives of this 2-part study were to identify differences in circulating immunological, inflammatory, and hormonal parameters between mares with natural ascending placentitis and control mares; evaluate each and combination of parameters as predictors of placentitis; and determine how these parameters indicate severity of placentitis. Reproductive examination and blood sampling were prospectively performed on pregnant mares in a natural setting. Study 1 enrolled mares diagnosed with early stage of ascending placentitis based on ultrasonographic findings (n = 12), and gestationally age-matched mares with healthy pregnancies as controls (n = 12). Blood samples were classified as pre-onset (before) and early onset (at the time of ultrasonographic changes) of placentitis. There were no detected statistically significant differences between groups and timepoints in immunological and inflammatory parameters, including peripheral lymphocyte subpopulations, cytokine, and serum amyloid A concentrations. The placentitis group showed a reduced (P = 0.01) DHP/20α-DHP ratio when compared to the control group at the early onset timepoint. Plasma estradiol-17β concentration <359 pg/mL predicted ascending placentitis with acceptable accuracy (area under the curve, AUC = 0.71). Combined albumin <3.7 g/dL, estradiol-17β < 499 ng/mL, and DHP <33 ng/mL predicted 100 % of cases of ascending placentitis. In study 2, samples were classified according to the presence and severity of the abnormal ultrasonographic findings as mild (n = 11) and moderate-severe (n = 23), and gestationally age-matched with samples from control mares (n = 34). Mares with moderate-severe ascending placentitis had increased (P = 0.03) plasma 20α-DHP concentration and reduced (P = 0.03) DHP/20α-DHP ratio when compared to control mares. Our results suggest that the early events of ascending placentitis detected by ultrasonographic findings include hormonal alterations of feto-placental metabolism measurable in the mare's circulation, yet without obvious systemic immunological and inflammatory changes. Additional studies are warranted to further assess how hormonal markers and cutoff values could guide decisions for timely therapeutic intervention.

CD3+/CD4+ cells combined with myosteatosis predict the prognosis in patients who underwent gastric cancer surgery.

This study aimed to investigate the predictive capacity of lymphocyte subpopulations, sarcopenia and myosteatosis for clinical outcomes in patients who underwent gastric cancer surgery. Additionally, the prognostic significance of CD3+/CD4+ cells in conjunction with myosteatosis was explored. A cohort of 190 patients with gastric cancer who underwent surgery and received computed tomography scans between July 2016 and December 2017 at our institution was examined. Complete clinical information and peripheral lymphocyte subpopulations were available for all patients. A comprehensive array of statistical methodologies was employed to scrutinize variances in both clinical and pathological characteristics among patients, with the aim of identifying autonomous prognostic determinants requisite for the development of a nomogram. Subsequent assessment of the predictive efficacy of the nomogram was conducted via calibration curve analysis. The study comprised a cohort of 190 participants, encompassing 126 males (66.32%) and 64 females (33.68%), with a mean age of 58.47 (±11.37) years. Patients were stratified into three groups based on CD3+/CD4+ cells and myosteatosis, with 24 in Group 1, 87 in Group 2 and 79 in Group 3. Notably, patients in the third group exhibited significantly shorter progression-free survival (PFS) (hazard ratio [HR]=0.208, P<0.001) and overall survival (OS) (HR=0.193, P<0.001). The subset of peripheral blood lymphocytes exhibited elevated levels of CD3+/CD4+ cells (HR=2.485, P<0.001) and heightened CD4+/CD8+ ratios (HR=1.705, P=0.038), whereas diminished CD19+ cell counts (HR=0.210, P=0.032) correlated with improved OS in patients. The individuals presenting with sarcopenia (HR=4.089, P=0.023) and myosteatosis (HR=2.857, P<0.001) displayed reduced OS. The multivariate Cox regression analysis showed that pathological tumour-node-metastasis stage, CD19+ cells, sarcopenia and CD3+/CD4+ cell-myosteatosis were identified as independent prognostic factors for PFS and OS in patients. The constructed nomograms for PFS and OS yielded C-index values of 0.839 (95% confidence interval [CI]: 0.798-0.880) and 0.836 (95% CI: 0.792-0.879), respectively. The calibration analysis demonstrated that the nomograms accurately predicted the 3- and 5-year survival rates of PFS and OS in patients. Lymphocyte subsets, including CD3+/CD4+ cells, CD4+/CD8+ ratio and CD19+ cells, are indicative of clinical prognosis in gastric cancer surgery patients. Body composition parameters, such as sarcopenia and myosteatosis, are also associated with the patient's prognosis. The combination of CD3+/CD4+ cells with myosteatosis demonstrates enhanced prognostic value, enabling the identification of patients at high risk of post-operative metastasis and recurrence.

Chicory root powder included as a prebiotic in different cereal-based diets for dogs: Influences on gut health, metabolic and immunological status

The present experiment was conducted to study the influence of chicory root powder as a prebiotic on gut health, metabolic, and immunological status of dogs fed with different cereal-based diets. Sixteen Labrador dogs, fed either a wheat (WT) or sorghum (SG) based diet, each without (C) or with (P) supplemental chicory root powder at 10 g/kg diet, were divided into four equal groups namely WT-C, WT-P, SG-C and SG-P. The digestibility of DM, OM, and total carbohydrates was significantly (p < 0.05) higher with the SG-C diet than with the other diets. Faecal ammonia was reduced (p = 0.002) in the WT-P group; however, there were no differences (p > 0.05) observed in the faecal pH and contents of lactate, acetate, propionate and total short chain fatty acids (SCFAs). The faecal lactobacilli population was higher (p = 0.001) in both the prebiotic-supplemented groups with a similar trend (p = 0.079) for bifidobacteria. Coliform counts were reduced (p = 0.002) in prebiotic-supplemented diets irrespective of cereals. There were significant (p < 0.05) variations in haemoglobin and haematocrit values among the dietary groups. The delayed-type hypersensitivity response to intradermal phytohemagglutinin (PHA-P) was higher (p<0.001) in both the prebiotic-supplemented groups; a similar trend was evident in the humoral immune response measured as antibody response to sheep erythrocytes. The peripheral lymphocyte subpopulations of CD4+ and CD8+ varied with prebiotic supplementation of the sorghum-based diet. Overall, the findings implies that tailoring the dietary composition to include specific cereals may optimize the beneficial effects of prebiotics in supporting canine health.

Lifelong exercise training promotes the remodelling of the immune system and prostate signalome in a rat model of prostate carcinogenesis

This work aimed to understand how lifelong exercise training promotes the remodelling of the immune system and prostate signalome in a rat model of PCa. Fifty-five male Wistar rats were divided into four groups: control sedentary, control exercised, induced PCa sedentary and induced PCa exercised. Exercised animals were trained in a treadmill for 53 weeks. Pca induction consisted on the sequential administration of flutamide, N-methyl-N-nitrosourea and testosterone propionate implants. Serum concentrations of C-reactive protein (CRP) and tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) were not different among groups. Peripheral levels of γδ T cells were higher in Pca exercised group than in the PCa sedentary group (p < 0.05). Exercise training also induced Oestrogen Receptor (ESR1) upregulation and Mitogen-activated Protein Kinase 13 (MAPK13) downregulation, changed the content of the phosphorylated (at Ser-104) form of this receptor (coded by the gene ESR1) and seemed to increase Erα phosphorylation and activity in exercised PCa rats when compared with sedentary PCa rats. Our data highlight the exercise-induced remodelling of peripheral lymphocyte subpopulations and lymphocyte infiltration in prostate tissue. Moreover, exercise training promotes the remodelling prostate signalome in this rat model of prostate carcinogenesis.

Lymphocyte Subpopulations Associated with Neutralizing Antibody Levels of SARS-CoV-2 for COVID-19 Vaccination.

The comprehensive knowledge regarding the immune response during coronavirus disease 2019 (COVID-19) vaccination is limited. The aim of this study was to longitudinally investigate not only the dynamic changes of peripheral lymphocyte subpopulations and cytokine levels but parallel changes of antibody levels against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Blood samples of 20 healthcare workers with two doses of COVID-19 vaccine were prospectively collected. The percentages of lymphocyte subpopulations from peripheral blood and cytokine production in lymphocytes with in vitro stimulation were assessed using eight-color flow cytometry. SARS-CoV-2 spike antibodies (anti-S Abs) and functional neutralizing antibodies (nAbs) were also measured. The relation between pre- and post-vaccination immunity was analyzed. There are 7 men and 13 women with a median age of 44.0 years (range: 25.7–59.5 years). The individuals had an increased percentage of lymphocytes at post-vaccination with statistical significance post first dose (p = 0.031). The levels of transitional cells (p = 0.001), such as plasmablasts (p < 0.001) and plasma cells (p = 0.031), were increased compared with pre-vaccination. Recent thymic emigrants of CD4+ T cells subsets were significantly higher at post-vaccination than those at pre-vaccination (p = 0.029). Intracellular levels of tumor necrosis factor-alpha, interferon-γ, interleukin (IL)-2, IL-21, transforming growth factor-beta and IL-17 produced by CD4+ T, CD8+ T, and natural killer cells were increased. All individual samples showed reactivity to anti-S Abs and the levels of nAbs were elevated after vaccination. The magnitude of adaptive immunity was associated with vaccine types and doses. Alterations of total memory B cells (p < 0.001), non-switched memory B cells (p = 0.016), and memory Treg cells (p < 0.001) were independent predictors for nAb levels. These findings might be helpful in elucidating the immune response of COVID-19 vaccination and in developing new strategies for immunization.

Immune Response in Vitamin D Deficient Metastatic Colorectal Cancer Patients: A Player That Should Be Considered for Targeted Vitamin D Supplementation.

Simple SummaryIn this study, we investigated the role of vitamin D levels and the impact on immune response in patients with mCRC and propose a vitamin D cut-off level. Among several hematological, biochemical and immunological variables, we identified the neutrophils-to-lymphocytes ratio, CD4+ T lymphocytes and B lymphocytes as being closely related to vitamin D status with an impact on survival.Background: Vitamin D deficiency is a poor prognostic factor in metastatic colorectal cancer (mCRC); however, targeted supplementation trials have so far yielded limited results. We investigated clinical-laboratory parameters influencing vitamin D deficiency, with a particular focus on immune response, and the effect on survival. These parameters could help optimize targeted supplementation therapy. Methods: Association of plasma 25-hydroxyvitamin D (25(OH])D) with overall survival (OS) was assessed with the Hazard Ratio Smoothed Curve with Restricted Cubic Splines (HRSC-RCS) and maximally selected rank statistics (MSRS) in mCRC patients who underwent first-line chemotherapy. Several hematobiochemical variables were evaluated as predictors of vitamin D deficiency by means of Least Absolute Shrinkage and Selection Operator (LASSO) analysis. In a patient subset, peripheral lymphocyte subpopulations were also analyzed. Results: One hundred thirty-three mCRC patients were included. The median(m) baseline 25(OH)D was 10.8 ng/mL (range 3–53.4). HRSC-RCS revealed a linear association between 25(OH)D and OS. MSRS found 10 ng/mL as the optimal 25(OH)D cut-off. The median OS for 25(OH)D < 10 (n = 60) vs. > 10 ng/mL (n = 73) was 12.3 and 24.5 months, respectively (p = 0.002). The LASSO analysis identified high neutrophil-to-lymphocyte ratio (NLR > 3.5) as the strongest predictor of vitamin D deficiency (Odds Ratio 3.35, p 0.0009). Moreover, patients with low 25(OH)D levels (< 10 ng/mL) and high NLR (>3.5) had the shortest survival and patients with 25(OH)D >10 ng/mL and NLR <3.5 had the longest: mOS 8.1 and 28.1 months, respectively, HR 3.40 (1.76–6.59), p 0.0004. Besides the significant difference in NLR between 25(OH)D < and > 10 ng/mL patients (mNLR 3.6 vs. 2.9, p 0.03), the lymphocyte subpopulation analysis revealed that vitamin D deficiency was associated with high T- CD4+ (p = 0.04) and low B (p = 0.03) lymphocyte frequency. Conclusions: NLR is a powerful predictor of Vitamin D deficiency and can further help in stratifying prognosis. Vitamin D deficiency was associated with significant variations in peripheral immune cells. We hypothesize that integrated targeted interventions to both vitamin D and immune system would improve the prognosis of mCRC patients.

Peripheral lymphocyte populations in ovarian cancer patients and correlations with clinicopathological features

BackgroundTo investigate the alterations of peripheral lymphocyte subpopulations in ovarian cancer patients compared to benign or borderline counterparts. The possible clinicopathological implications were also evaluated.MethodsWe enrolled 112 treatment-naive ovarian cancer patients, 14 borderline tumor patients and 44 benign tumor patients between 09/2016 and 01/2019. Flow cytometry was used to measure the peripheral lymphocyte subsets consisting of T cells (CD3+, CD3+CD4+, CD3+CD8+ and CD8+CD28+), regulatory T cells (Tregs, CD4+CD25+CD127−), natural killer cells (NK cells, CD3−CD56+) and B cells (CD19+).ResultsMost ovarian cancer patients were high-grade serous carcinoma (84.8%), followed by clear cell carcinoma (8.03%). Late-stage tumor (FIGO III + IV) accounted for 82.1%. The study showed that the proportions of peripheral lymphocyte subsets underwent apparent changes in ovarian cancer patients. We observed elevated levels of Treg cells in patients with both ovarian borderline and malignant tumor compared to those with benign tumors, which achieved statistic significance. In contrast, CD3+CD8+ T and CD8+CD28+ T cells were significantly lower in ovarian cancer patients. Interestingly, low level of B cells was correlated to clear cell carcinoma (P = 0.024), advanced tumor (P = 0.028) and platinum-resistant recurrence (P = 0.014). Regarding the changes of lymphocyte subsets after surgery, CD8+CD28+ T cells had a significant decreasing tendency (P = 0.007) while B cells were the opposite (P < 0.001).ConclusionsOvarian cancer patients have altered circulating lymphocyte profile (elevated Treg cell, depressed CD3+CD8+ T and CD8+CD28+ T cells). Low level of B cells might be related to disease aggressiveness, and it recovered after the removal of tumor, which merits further study.

Regulatory T cells in children with attention deficit hyperactivity disorder: A case-control study

ObjectiveThe pathophysiology of attention deficit hyperactivity disorder (ADHD) are still not fully elucidated. Immune system dysregulation has emerged as a major etiological focus as a result of the high comorbidity of allergic disease, inflammatory biomarkers, and genetic research. The present study aimed to evaluate peripheral lymphocyte subpopulations and regulatory T cells (Tregs) in children with ADHD. MethodsThis single-center cross-sectional case-control study assessed 49 children with ADHD and 35 age- and gender-matched healthy children aged 7–12 years (9.10 ± 2.37 and 9.45 ± 2.13, respectively). The participants were screened for psychopathology using the Kiddie Schedule for Affective Disorders and Schizophrenia for School-Age Children–Present and Lifetime Version, while the severity of ADHD symptoms was measured by means of the distracted-Continuous Performance Test. Peripheral lymphocyte subpopulations and Tregs were analyzed with flow-cytometry. ResultsThere is no significant difference in peripheral blood lymphocyte subsets between ADHD and control groups The children diagnosed with ADHD exhibited significantly higher levels of CD3+ CD4+ CD25+ Foxp3+ (Tregs) than the healthy control subjects (8.23 ± 2.09 vs. 6.61 ± 2.89; z = 2.965, p = .004). The Tregs cell (Exp(B) = 1.334; p = .042; CI = 1.011–1.761) levels were determined to be statistically significant according to regression analysis and were associated with an increased probability of ADHD. ConclusionElevated Treg levels were linked to an increased likelihood of ADHD. This study suggested that changes in immune regulatory cells represent an important part of research in treatment of ADHD.

Differences in immunophenotypes between myasthenia gravis patients with and without thyroid antibodies.

Immunophenotypes are related to the therapeutic efficacy of specific immunomodulating agents in patients with myasthenia gravis (MG), but the relationship of immunophenotype to the presence or absence of thyroid antibodies is unknown. This study aims to evaluate differences in the immunophenotypes between MG patients with and without thyroid antibody (TAb) positivity to provide insight for future targeted immunotherapies. This retrospective observational study included 48 MG patients with acetylcholine receptor antibody (AchR-Ab), of which 15 (31.25%) were TAb positive. Ocular MG (OMG) was defined as ocular-only manifestations for the duration for which records were available. Peripheral lymphocyte subpopulations were measured by flow cytometry. TAb positive patients appeared to have a higher prevalence of OMG than TAb negative patients (53.33% vs. 24.24%, p =.048). Percentages of B cells (mean difference (MD)=6.16, 95% confidence interval (CI): 1.91-10.40, p=.007) and CD8 + CD28+ cells (MD=15.14, 95%CI: 5.17-25.11, p =.013) were higher in TAb positive patients than those in TAb negative patients, while AChR-Ab titers (MD=-6.49 nmol/L, 95%CI: -9.29 to -3.70, p < .001), percentages of T cells (MD=-6.43, 95%CI: -11.92 to -0.94, p=.023), CD3 + HLA-DR+ cells (MD=-6.47, 95%CI: -12.31 to -0.63, p =.031) and CD8+ T cells (MD=-6.60, 95%CI: -9.86 to -3.34, p < .001) were lower. The immunophenotypes of MG patients with and without TAb positivity were significantly different, suggesting that their sensitivity to immunotherapy may be different. Further studies focused on differences between TAb positive and TAb negative MG patients in their responses to specific immunotherapies are needed to support our exploratory findings.

Absolute reduction in peripheral regulatory T cells in patients with Graves’ disease and post-treatment recovery

ObjectiveGraves’ disease (GD) is one of the most common autoimmune conditions, but the mechanisms underlying the associated induction of autoimmunity are not known. We explored the role of peripheral lymphocyte subpopulations in disease pathogenesis. MethodsIn total, 32 patients and 40 age- and sex-matched healthy controls were recruited in this study. Peripheral levels of T, B, NK, CD4+ T, CD8+ T, Th1, Th2, Th17, and Treg cells were measured using flow cytometry. For all patients, we compared all lymphocyte subpopulations between GD patients and healthy controls. Changes in patient lymphocyte subsets were compared before and after treatment. ResultsThe absolute numbers of circulating Th17 cells (0.45 ± 1.16, p > 0.05) between GD patients and healthy controls were not significantly different. However, the percentage of Th17 cells was significantly increased (0.25 ± 0.11, p < 0.05). The absolute numbers and percentages of circulating Tregs in GD patients were significantly decreased compared with those in healthy participants (11.61 ± 2.75, p < 0.05). There was a significant difference in Treg absolute numbers between the untreated and drug-treated groups. Furthermore, we found that the Treg percentage in untreated patients (mean=4.78) was not significantly different from that in the drug-treated group (mean=4.81). In addition, circulating Treg absolute numbers in GD patients with exophthalmos were significantly lower than those in GD patients without exophthalmos (9.96 ± 4.16, p < 0.05). A similar trend was observed in GD patients with weight loss (11.97 ± 3.28, p < 0.05). ConclusionGD pathogenesis was associated with a lower Treg population and an increased Th17/Treg ratio (T helper cell 17/ regulatory T cells). Th17 cells in this study were not related to the disease. Furthermore, anti-thyroid drug therapy improved immune-mediated system disorders. Finally, we found lower absolute numbers of circulating Tregs in GD patients with certain positive signs, such as exophthalmos and/or weight loss. Thus, immune changes are correlated with partial clinical manifestations.

Assessment of the Levels of Peripheral Lymphocytes Subpopulation in Healthy Women of Childbearing Age: a Prospective Cohort Study.

To evaluate changes in the levels of the peripheral lymphocyte subpopulation (PBLS) during the menstrual cycle in healthy women of childbearing age (HWOBA), for establishing a normal reference range. Flow cytometric analysis was performed for the assessment of the levels of PBLS during one menstrual cycle in HWOBA. Moreover, ovulation was monitored. The multivariable logistic regression models were used to evaluate the effects of age and body mass index (BMI) on PBLS. The levels of PBLS including CD3+, CD3+CD4+, CD3+CD8+, CD19+, CD16+CD56+ lymphocytes and ratio of Th1/Th2 cells, were relatively stable during the menstrual cycle. However, each increment in age (year) was proportional to a decrease (14%) in the level of CD8+ T cells and an increase (10%) in the level of CD19+ B cells (p < 0.05). Increased BMI was inversely proportional to the level of CD16+CD56+ natural killer (NK) cells. Thus, the normal reference intervals for PBLS in HWOBA were established. The levels of PBLS can be used for daily monitoring of immune status in HWOBA. Notably, age and BMI may affect PBLS level. The normal reference ranges for PBLS levels can provide a basis for the research and treatment of recurrent pregnancy loss.

Identifying Changes in Peripheral Lymphocyte Subpopulations in Adult Onset Type 1 Diabetes.

T- and B-lymphocytes play an important role in the pathogenesis of type 1 diabetes (T1D), a chronic disease caused by the autoimmune destruction of the insulin-producing cells in the pancreatic islets. Flow cytometry allows their characterization in peripheral blood, letting to investigate changes in cellular subpopulations that can provide insights in T1D pathophysiology. With this purpose, CD4+ and CD8+ T cells (including naïve, central memory, effector memory and terminally differentiated effector (TEMRA), Th17 and Tregs) and B cells subsets (naïve, unswitched memory, switched memory and transitional B cells) were analysed in peripheral blood of adult T1D patients at disease onset and after ≥2 years using multiparametric flow cytometry. Here we report changes in the percentage of early and late effector memory CD4+ and CD8+ T cells as well as of naïve subsets, regulatory T cells and transitional B cells in peripheral blood of adult patients at onset of T1D when compared with HD. After 2 years follow-up these changes were maintained. Also, we found a decrease in percentage of Th17 and numbers of T cells with baseline. In order to identify potential biomarkers of disease, ROC curves were performed being late EM CD4 T cell subset the most promising candidate. In conclusion, the observed changes in the percentage and/or absolute number of lymphocyte subpopulations of adult T1D patients support the hypothesis that effector cells migrate to the pancreas and this autoimmune process perseveres along the disease. Moreover, multiparametric flow allows to identify those subsets with potential to be considered biomarkers of disease.

Favorable outcomes of interferon-α and ribavirin treatment for a male with subacute sclerosing panencephalitis

Subacute sclerosing panencephalitis (SSPE) is a slow virus infection associated with mutant measles virus (MeV). The long-term outcome of antiviral treatments remains to be determined. We herein present a Japanese boy who was diagnosed with SSPE at 10 years of age. Intraventricular infusions of interferon-α effectively prevented the progress of symptoms during 14 years of follow-up period. Flow-cytometric analysis demonstrated higher proportion of T helper 17 cells (Th17, 18.2%) than healthy controls (4.8–14.5%) despite the normal subpopulation of peripheral lymphocytes. These data suggest that a group of patients with SSPE may show favorable responses to intraventricular infusions of interferon-α.

OP0307 GUT MICROBIOTA AND ITS RELEVANCE TO PERIPHERAL LYMPHOCYTE SUBPOPULATION IN PATIENTS WITH SYSTEMIC LUPUS ERYTHEMATOSUS

Background:Systemic lupus erythematosus (SLE) is an autoimmune disease with disturbance of lymphocyte subpopulations1. Growing experimental and clinical evidence suggests that chronic inflammatory response induced by gut microbiome critically contribute to the development of SLE2 3.Objectives:To investigate the characteristics of gut microbiome and the associations between flora and peripheral lymphocyte subpopulations in SLE patients.Methods:A total of 19 SLE patients who fulfilled the 2019 American college of Rheumatology (ACR) and European League Against Rheumatism (EULAR) classification criteria and 16 age- and sex- matched healthy controls (HC) were enrolled in this study. The peripheral T lymphocyte subsets of these participants were assessed by flow cytometry and the gut microbiota were investigated via 16s rRNA. Indicators of disease activity such as erythrocyte sedimentation rate (ESR), complement C3 and C4 were recorded at the same time. Mann-Whitney U test was applied to compare T lymphocyte subsets between SLE patients and HC. Spearman analysis was used for calculating correlation between T subsets and highly expressed differential flora as well as their correlation with disease activity indicators. All P-values reported herein were two-tailed and P-value&lt;0.05 was taken as statistically significant.Results:SLE patients had higher proportions of Th17 cells (P=0.020) and γδT cells (P=0.018) but lower levels of Treg cells (P=0.001), Tfh cells (P=0.018) and Naïve CD4+T cells (P=0.004) (Figure 1a-e). The diversity and relative abundance of intestinal flora in patients with SLE were significantly different from those in HCs. Detailly, the α-diversity was decreased in SLE (P&lt;0.05) (Figure 2a-c). Compared with HC, 11 species of flora were discovered to be distinctly different(P&lt;0.05) (Figure 2d-e). Moreover, there was a significant positive correlation between Treg levels and Ruminococcus2 (P=0.042), Th17 and Megamonas (P=0.009), γδT and Streptococcus (P=0.004) as well as Megamonas (P=0.003), Tfh and Bacteroides (P=0.040). Whereas Th1 levels and Bifidobacterium were negatively correlated in these participants (P=0.005). As for clinical disease measures, there were negative correlations not only between ESR and Treg cells (P=0.031) but also C4 and the amount of Unclassified Ruminococcaceae (P=0.032).Conclusion:Abnormality of T cell subsets, especially the level of Naïve CD4+T, γδT, Tfh, Treg, and Th17 cells contributes to the occurrence and progression of SLE, which may be related to the disturbance of gut microbiota. Therefore it is necessary to attach importance to the alteration of gut microbiota to prevent the outbreak of inflammation and maybe they can be identified as biomarkers predicting disease activity.

Utility of lymphocyte phenotype profile to differentiate primary Sjögren's syndrome from sicca syndrome.

To assess the potential diagnostic utility of advanced lymphocyte profiling to differentiate between primary Sjögren's Syndrome (pSS) and non-Sjögren Sicca syndrome. Distribution of peripheral lymphocyte subpopulations was analysed by flow cytometry in 68 patients with pSS, 26 patients with sicca syndrome and 23 healthy controls. The ability to discriminate between pSS and sicca syndrome was analysed using the area under the curve (AUC) of the receiver operating characteristic curve of the different lymphocyte subsets. The ratio between naïve/memory B cell proportions showed an AUC of 0.742 to differentiate pSS and sicca syndrome, with a sensitivity of 76.6% and a specificity of 72% for a cut-off value of 3.4. The ratio of non-switched memory B cells to activated CD4+ T cells percentage (BNSM/CD4ACT) presented the highest AUC (0.840) with a sensitivity of 83.3% and specificity of 81.7% for a cut-off value <4.1. To differentiate seronegative pSS patients from sicca patients, the BNSM/CD4ACT ratio exhibited an AUC of 0.742 (sensitivity 75%, specificity 66.7%, cut-off value <4.4), and the number of naïve CD4 T cells had an AUC of 0.821 (sensitivity 76.9%, specificity 88.9%, cut-off value <312/mm3). Patients with pSS show a profound imbalance in the distribution of circulating T and B lymphocyte subsets. The ratio BNSM/CD4ACT is useful to discriminate between pSS and sicca syndrome.

The Gut Microbiota and Its Relevance to Peripheral Lymphocyte Subpopulations and Cytokines in Patients with Rheumatoid Arthritis.

Growing experimental and clinical evidence suggests that a chronic inflammatory response induced by gut microbiome critically contribute to the development of rheumatoid arthritis (RA). Previous studies demonstrated the disturbance of lymphocyte subpopulations in RA patients. The purpose of this study was to explore the characteristics of gut microbiome and the associations between bacterium and lymphocyte subpopulations as well as cytokines in patients with RA. Fecal samples from 205 RA patients and 199 healthy controls (HCs) were collected for bacterial DNA extraction and 16S ribosomal RNA (rRNA) gene sequencing. The levels of peripheral lymphocyte subpopulation such as T, B, CD4+T, CD8+T, NK, T helper 1 (Th1), Th2, Th17, and regulatory T cells (Tregs) of these subjects were detected by flow cytometry combined with standard absolute counting beads. The serum levels of cytokines interleukin-2 (IL-2), IL-4, IL-6, IL-10, IL-17, tumour necrosis factor-α (TNF-α), and interferon-γ (INF-γ) were tested by flow cytometric bead array (CBA). Alpha and beta diversity of gut microbiome were explored by bioinformatics analysis. Spearman rank correlation test was used to explore the relationships between gut microbiome and lymphocyte subsets as well as serum cytokines. The diversity and relative abundance of intestinal microbiota in patients with RA were significantly different from those in HCs. Detailly, the abundant of phylum Proteobacteria in RA patients was more than that in HCs, while Firmicutes was less than in HCs. There was increased relative abundance of genus Clostridium_XlVa as well as genus Blautia, more abundance of Ruminococcus2 in patients with lower levels of T, B, CD4+T, and Tregs. In addition, the relative abundances of Pelagibacterium, Oxalobacter, ClostridiumXlVb, and ClostridiumXVIII were correlated with cytokines. Gut microbiome of RA patients was clearly different from that of HCs. Abnormal bacteria communities are associated with the altered levels of lymphocyte subpopulation and cytokines, which might be one of the pathogenesis of RA.

Circulating CD56+ NKG2D+ NK cells and postoperative fertility in ovarian endometrioma

The current biomarkers for postoperative fertility assessment caused by ovarian endometrioma (OE) are insufficient. The present study hypothesized that the peripheral lymphocyte subpopulation can be used as a candidate biomarker of postoperative infertility in OE. The association of the number of circulating CD4/CD8 T, NK, and γδ T cells with postoperative fertility was assessed in 33 OE patients aged 20 ~ 40 years between June 2018 and January 2019. Concomitantly, 68 healthy female subjects were recruited. The changes in the baseline immune characteristics between the two groups were compared. The data demonstrated significant differences in the ratio of CD4/CD8 T cells and the number of CD56+ NKG2D+ NK cells and γδ T cells between OE patients and control subjects. The patients were followed-up till December 2019 and the number of CD56+ NKG2D+ NK cells in the cases was a significant predictor for postoperative fertility as determined by different COX regression models (crude HR = 0.220, 95% CI = 0.059–0.822; adjusted HR = 0.127, 95% CI = 0.024–0.675). A significant delay to successful pregnancy was noted in OE patients (median time, 173 vs. 99 days, log-rank P = 0.013). The present findings suggested that CD56+ NKG2D+ NK cells are a candidate biomarker of postoperative fertility in OE patients. Larger population studies are warranted.

Increased Serum Interleukin-2 Levels Are Associated with Abnormal Peripheral Blood Natural Killer Cell Levels in Patients with Active Rheumatoid Arthritis.

Objective To investigate the relationship between serum interleukin-2 (IL-2) levels and disease activity, absolute numbers of peripheral lymphocyte subsets, autoantibodies, and associated cytokines in patients with rheumatoid arthritis (RA). Methods This study included 106 patients with RA, evaluated their disease activity (DAS28 score), and divided them into disease remission (DAS28 ≤ 2.6), low disease activity (DAS28 ≤ 3.2), and moderate-high disease activity (DAS28 > 3.2) groups. Flow cytometry was used to detect the absolute numbers of peripheral lymphocyte subpopulations and CD4+ T cell subsets in each group, and serum cytokine levels were measured using cytometric bead array. Results Serum IL-2 levels in RA patients were positively correlated with disease activity and rheumatoid factor titers (p < 0.001 and p = 0.045, respectively), and multiple regression analysis revealed that serum IL-2 levels were an independent factor affecting disease activity. Serum IL-2 levels were positively correlated with Th17/Treg ratios (p = 0.013). Compared with the remission group, peripheral lymphocyte and CD4+ T lymphocyte subsets in patients with active RA decreased to varying degrees; however, the numbers of peripheral natural killer (NK) cells were significantly higher in the moderate-high disease activity group than in the remission (p = 0.046) and low disease activity (p = 0.020) groups; the percentages of NK cells had the same trend. In addition, the number and percentage of NK cells were positively correlated with serum IL-2 levels (p = 0.018 and p = 0.006, respectively). Conclusions In RA patients, serum IL-2 levels were not only correlated with patients' disease activity and autoantibody levels but were also involved in their Th17/Treg immune imbalance. In addition, in patients with active RA, NK cell levels were abnormally elevated, possibly due to high serum levels of IL-2.

Abstract 2252: Significant impacts of IMRT on peripheral blood lymphocyte subpopulation and immune globulin levels in head and neck cancer patients

Abstract Purpose This study aims at investigate the regulation of peripheral lymphocyte subpopulations and immune globulins in head and neck cancer patients during intensity-modulated radiation therapy (IMRT). Patients and Methods Patients with non-metastatic head and neck cancers consecutively treated in our institute by conventional IMRT with or without induction or concomitant chemotherapy from January 2019 to September 2019 were enrolled in this study. Radiation doses were given 68-70Gy to gross tumor volumes, 60-66Gy to high-risk cervical lymphatic draining areas, 54Gy to low-risk lymphatic draining areas. Induction chemotherapy was composed of two to three cycles of triweekly taxanes and platinum salts, and concomitant chemotherapy was composed of weekly or triweekly platinum salts. Data of lymphocyte subpopulation counts and immune globulin levels were collected before, in the middle of and at the end of IMRT. Friedman's two-way ANOVA was used to compare values of indicated time points. Generalized estimating equations (GEE) were used to examine the impacts of induction and concomitant chemotherapy. SPSS (IBM version 23.0) software package was used to perform statistical analysis and P&amp;lt;0.05 was considered statistically significant. Results A total of 126 patients were enrolled in this study. There were 86 male and 40 female patients with the median age being 54 (range: 24-82) years. There were 47 patients with nasopharyngeal carcinoma and 79 patients with other head and neck cancers. 44 patients received induction chemotherapy, and 56 received concurrent chemotherapy. Total CD3+ T cells (from 1118.69/μL to 335.57/μL, P&amp;lt;0.001), CD3+CD4+ T cells (from 596.0/μL to 119.5/μL, P&amp;lt;0.001) and CD3+CD8+ T cells (from 408.0/μL to 107.5/μL, P&amp;lt;0.001), CD16+CD56+ NK cells (from 337.74/μL to 150.11/μL, P&amp;lt;0.013), CD19+ B cells (from 147.01/μL to 19.27/μL, P&amp;lt;0.001) counts declined significantly during IMRT. However, the percentage of CD8+ T cells and NK cells increased from 26.34% to 28.06% (P&amp;lt;0.001), and from 20.93% to 29.58% (P&amp;lt;0.001), respectively. Complement C4 also increased from 0.2688g/L to 0.3171g/L (P&amp;lt;0.001). Other items including C3, IgA, IgE, IgG, IgM were not altered significantly during IMRT. GEE analysis indicated that induction chemotherapy impacted significantly on B cell count (P=0.018), IgA (P=0.034) and IgM (P=0.017), while concomitant chemotherapy impacted significantly on CD3+ T cell (P=0.010), CD3+CD4+ T cell (P=0.050) and CD3+CD8+ T cell (P=0.017) counts and IgG (P=0.002), and did not impact significantly on other lymphocyte subpopulations or immune globulins. Conclusion IMRT induced a significant alteration in peripheral lymphocyte subpopulations and immune globulins in head and neck patients. Induction and concomitant chemotherapy also impacted on selected components. The clinical relevance of these findings warrants further investigations. Keywords Head and neck cancer, IMRT, lymphocyte subpopulation, immune globulin Citation Format: Qiuji WU, Shuyuan Zhang, Zheng LI, Yahua Zhong. Significant impacts of IMRT on peripheral blood lymphocyte subpopulation and immune globulin levels in head and neck cancer patients [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2252.

Absolute reduction of peripheral regulatory T cell in patients with relapsing polychondritis

Although relapsing polychondritis (RP) is considered as an immune-mediated systemic disease, the levels of peripheral lymphocyte subpopulations are rarely studied in patients with RP. In this study, we focused on changes of peripheral CD4+T cell subsets in patients with RP. Absolute numbers and percentages of CD4+T cell subsets including helper T(Th)1, Th2, Th17 cells and regulatory T (Treg) cells in peripheral blood (PB) from 19 RP patients, healthy controls and RA patients respectively were assessed by flow cytometry combined a microbead-based single-platform method. We compared the CD4+T cell levels in all RP patients and healthy controls. In addition, we analysed the difference of the absolute number and percentage of Treg cells between RP and RA patients. Compared with healthy controls, all RP patients had significantly both lower absolute number and proportion of Treg cells (absolute number, 45.10/μl vs. 22.48/μl, p<0.001; proportion, 5.19% vs. 3.78%, p<0.001) no matter whether they had received treatment or not. Similarly, the absolute number of Th2 cells in all RP patients was decreased (10.19/μl vs. 7.44/μl, p=0.030). However, there were no significant differences in percentages and absolute numbers of Th1 and Th17 cells between RP patients and healthy controls. The above results led to increased ratios of Th1/Treg (3.68 vs. 2.06, p=0.020), Th2/Treg (0.29 vs. 0.21, p=0.037) and Th17/Treg (0.25 vs. 0.14, p<0.001) in RP patients, and untreated RP patients were mainly characterised by the imbalance of Th17/Treg (0.25 vs. 0.14, p<0.01). There was no significant difference in Treg cells between RP and RA patients (p>0.05). Our data suggest that the reduction of Treg cells and its imbalance with Th cells play an important role in the pathogenesis of RP.