Lymphocyte Subtypes Research Articles

Abstract 15847: Mass Cytometry Reveals Immune Cell Subtypes Responsive to Il-1b and Il-6 Stimulation in CAD Subjects and Controls

Introduction: Inflammation plays a key role in the development of atherosclerosis, yet targeted therapies are lacking. Previous trials have demonstrated that IL-1b is a potential target, albeit with adverse effects on immune suppression. IL-6 has emerged as another mediator of coronary artery disease. It clear that a strategy to identify a biomarker customized for inflammatory inhibition is needed. Mass cytometry shows promise as a technology for customized biomarker identification. Hypothesis: A mass cytometry panel including markers to identify circulating immune subtypes in humans and phosphorylation of key transcription factors driving downstream inflammation can identify cell subtypes most responsive to cytokine stimulation. Methods: Peripheral blood mononuclear cells (PBMCs) were isolated from patients undergoing coronary computed tomography angiography (CCTA). Using PBMCs from 12 pairs of matched subjects, we stimulated cells with vehicle, IL-6, and IL-1b. These samples were analyzed using a custom mass cytometry panel. Results: Mass cytometry analysis of PMBCs after 15 minute cytokine stimulation identified specific lymphocyte and monocyte subtypes that were particularly responsive to IL-1b and IL-6. Notably, there was clear heterogeneity in this response suggesting this approach may identify those most impacted by cytokine inhibition or those with most severe disease. Conclusions: High dimensional mass cytometry has the potential to better identify individuals at risk and those that may respond to cytokine inhibition. We are in the process of performing advanced bioinformatic analysis including machine learning to leverage this novel technology for precision medicine.

FLASH Modulation of Immune Microenvironment in Diffuse Midline Glioma (DMG) at Single-Cell Resolution

<h3>Purpose/Objective(s)</h3> Diffuse Midline Glioma with H3K27M mutation (DMG) is a universally fatal tumor primarily treated with radiotherapy (RT) and typically unresponsive to immune-based therapies. FLASH is a novel RT technique associated with decreased toxicity and effective tumor control in glioma models as well as immune-sparing properties in other cancer models. However, the effect of FLASH on the DMG tumor immune microenvironment (TIME) has not yet been explored. <h3>Materials/Methods</h3> Using a syngeneic murine brainstem DMG, we performed single-cell RNA sequencing on CD45+ cells isolated from tumors 4 days after posterior fossa irradiation (15Gy x 1, 9MeV), utilizing either ultra-high dose rate (90Gy/sec, FLASH) or conventional (2Gy/min, CONV) RT, and compared to unirradiated tumor (SHAM). For differential gene expression analysis, we compared CONV to SHAM (CvS) and FLASH to SHAM (FvS). We also performed single cell gene set enrichment scores (ssGSEA) for evaluation of immune signatures from gene ontology (GO); ssGSEA scores were deemed significant by a p<0.05 using Mann-Whitney U test. We validated our most robust immune markers with dual immunofluorescence. <h3>Results</h3> Unsupervised clustering of 23,165 CD45+ cells revealed 16 immune clusters, including subtypes of resident microglia (MG, 78.2% of total cells), lymphocytes (6.4%), macrophages (MPs, 5.2%), monocytes (1.8%), and dendritic cells (DCs, 1.7%). MG consisted of four distinct subtypes representing a spectrum of homeostatic to activated cellular states. In the most activated MG subtype, both CONV and FLASH showed an upregulation of interferon (IFN) genes such as Isg15 (Fold Change (FC) 1.03 CvS, 0.94 FvS) and Ifit3 (FC 0.77 CvS, 0.66 FvS) when compared to SHAM. Furthermore, ssGSEA of MG found IFN scores increased in both FvS (p < 0.0001) and CvS (p < 0.0001) while TGFbR signaling score was increased in FvS (p < 0.0001) and decreased in CvS (p < 0.0001). In non-resident myeloid clusters, the most differential response was found in MPs and DCs with robust upregulation of IFN genes for CvS (Isg15 FC 2.30 MPs, 2.67 DCs; Ifit3 FC 2.02 MPs, 2.06 DCs), while this response was not seen with FvS. This was confirmed with ssGSEA with increased IFN scores in both MPs (CvS p<0.0001, p=0.04) and DCs (CvS p<0.0001, FvS p=0.69). FLASH showed an increase in anti-inflammatory MP markers such as Mrc1, Cd163, and Maf (FC 1.40, 1.11, 1.19) and an upregulated myeloid-derived suppressor cell signature in monocytes (FvS, p < 0.0001), not seen in CvS (p = 0.09). <h3>Conclusion</h3> Our work is the first to map CONV and FLASH immune alterations with single-cell resolution in the DMG TIME. FLASH induces IFN differentially across microglia and non-resident myeloid subsets. Further exploration is warranted to understand how FLASH impacts tumor growth through differential cytokine signaling, inflammatory responses and immune checkpoint expression within the DMG TIME.

Treatment Induced Changes in Systemic Immune Environment in Patients with Unresectable Hepatocellular Carcinoma

<h3>Purpose/Objective(s)</h3> Host immune status plays an essential role in cancer patients. However, it is unknown whether therapy can change or remodel this environment which can influence treatment response to the remaining treatment. It is unknown however whether such change varies with treatment modality. This study aimed to exam the systemic immune status, explore the changes after arterial chemotherapy embolization (TACE), stereotactic body radiation therapy (SBRT) and immunotherapy (IO) as well as their correlation with treatment responses in patients with hepatocellular carcinoma (HCC). <h3>Materials/Methods</h3> This is a pilot study of correlative research for a prospective phase II trial. Patients with unresectable HCC were eligible. The protocol treatment included sequential TACE, SBRT (27.5–40 Gy/5 fractions) and IO. TACE and SBRT were per standard of care. Treatment response was assessed according to mRECIST by CT or MRI around 6 months from treatment commencement. Peripheral blood samples were collected at baseline and after TACE, SBRT and IO for lymphocyte subtypes of our interest by flow cytometry, including NKT cell, T, CD4+T, CD8+T, CD25+CD8+T, CD127+CD8+T, Th1, Th2, Th17, Treg, CD4/CD8 T cell, Th2/Th1, Th1/Th17 and Th17/Treg. Generalized estimation equation was employed for longitudinal analyses, and Bonferroni adjustment for multiple testing was performed for statistical significance at a p < 0.0083 (0.05/6) level. <h3>Results</h3> A pilot of 12 patients were enrolled between July 2020 and February 2021, with Child Pugh score of A, mean tumor greatest dimension of 8.4 ± 3.7 cm, and lesion number of less than 4. The circulating immune cells and lymphocyte subtypes were quite heterogeneous among patients at baseline, e.g., the median percent of CD8+T cell was 27% (95% CI, 19% - 43%). The treatment induced changes of circulating immune components were heterogeneous from patient to patient but most significant after SBRT. On average, CD8+T cell (27% to 51%, p = 0.01) increased after sequential treatment, while its non-effector component CD127+CD8+T cell (51% to 18%, p < 0.001) decreased significantly. SBRT reduced the counts of lymphocyte (1.3 to 0.7, p < 0.001) and platelet (226 to 156, p=0.001), while T cell increased significantly (45% to 54%, p = 0.031). Our pilot study of 12 patients demonstrated 58.3% tumor control (7 complete response, 5 progression disease). After grouping by treatment response, the baseline CD4+T (65% vs. 53%, p = 0.01), Th1 cells (21% vs. 10%, p = 0.007) and Th1/Th17 (10% vs. 1%, p = 0.001) were significantly higher in responders, while CD25+CD8+T (4% vs. 12%, p < 0.001), CD127+CD8+T (42% vs. 60%, p = 0.003), Th17 (2% vs. 8%, p = 0.047) and Treg cells (p = 0.002) and ratio of Th2/Th1 (3% vs. 9%, p = 0.028) were significantly higher in non-responders. <h3>Conclusion</h3> This pilot study revealed heterogeneous components of systemic immunities at baseline and changes after different treatments, with most significant changes from SBRT. Future scale verification trials are warranted.

The Recovery of the Absolute Lymphocyte Subpopulation Count in Cervical Cancer Patients after Radiotherapy

<h3>Purpose/Objective(s)</h3> Radiotherapy (RT) results in varying degrees of lymphopenia. However, to date, there is still no consensus on the persistence of lymphopenia after RT treatment for cervical cancer. Therefore, this study aimed to evaluate the dynamic changes of absolute lymphocyte subset count (ALSC) in cervical cancer patients after receiving RT. In addition, the study also explored the diagnostic value of the lymphocyte count (LC) in patients with declining ALSC <h3>Materials/Methods</h3> The peripheral blood ALSC was tested by flow cytometry before RT (baseline) and at regular follow-up intervals after completion of RT. The blood cell counts were also collected on the same day. Patient characteristics, tumor variables, and treatment factors were gathered for risk assessment. The primary endpoint was the degree of reduction in the lymphocyte subpopulation and the secondary endpoint was the predictive value of LC for declining ALSC. Logistic regression was used to identify the risk factors of lymphopenia, and the receiver operating characteristic (ROC) curve was used to assess the predictive value of LC. <h3>Results</h3> A total of 260 consecutive cervical cancer patients met the study criteria, among which 228 (87.7%) patients had a reduction in either one or some of the lymphocyte subpopulations. The median percentage decrease in ASLC ranged from 22.52% to 58.32%. When compared with the baseline the lymphocyte subpopulations dropped immediately after initiation of RT, to eventually reach their minimum levels at 3 to 6 months after completion of treatment. The B-lymphocyte and CD4+ to CD8+ ratios recovered within the first 6 months and the CD8+ T cell count recovered to normal levels within one year after completion of RT. In comparison, the CD3+ and CD4+ T cell counts persisted at low levels for one year and recovered five years after RT. The natural killer (NK) cells rarely decreased after RT. Multivariate logistic regression analysis identified advanced tumor stage and age above 60 years as independent risk factors affecting the count of lymphocyte cell subsets after RT. The ROC analysis confirmed that the LC obtained from a routine blood examination could be used to predict the count for each lymphocyte subtype except for the NK cell subtype. <h3>Conclusion</h3> The lymphocyte subpopulation counts decreased immediately after initiation of RT, to eventually reach their minimum levels after 3 to 6 months following completion of treatment, and it took several months or years to recover. Advanced tumor stage and age above 60 were identified as independent risk factors for lymphopenia in cervical cancer patients. The LC was a good predictor of ALSC decline. Therefore, this test could be used as a cheaper alternative to complex ALSC monitoring with flow cytometry.

Plasma Extracellular Vesicles Play a Role in Immune System Modulation in Minimal Hepatic Encephalopathy.

Minimal hepatic encephalopathy (MHE) is associated with changes in the immune system including an increased pro-inflammatory environment and altered differentiation of CD4+ T lymphocytes. The mechanisms remain unknown. Changes in extracellular vesicle (EV) cargo including proteins and miRNAs could play a main role as mediators of immune system changes associated with MHE. The aim was to assess whether plasma EVs from MHE patients played a role in inducing the pro-inflammatory environment and altered differentiation of CD4+ T lymphocyte subtypes in MHE patients. We characterized the miRNA and protein cargo of plasma EVs from 50 cirrhotic patients (27 without and 23 with MHE) and 24 controls. CD4+ T cells from the controls were cultured with plasma EVs from the three groups of study, and the cytokine release and differentiation to CD4+ T-cell subtypes were assessed. Plasma EVs from MHE patients had altered miRNA and protein contents, and were enriched in inflammatory factors compared to the controls and patients without MHE. EVs from MHE patients modulated the expression of pro-inflammatory IL-17, IL-21, and TNF-α and anti-inflammatory TGF-β in cultured CD4+ T lymphocytes, and increased the proportion of Th follicular and Treg cells and the activation of Th17 cells. In conclusion, plasma EVs could play an important role in the induction of immune changes observed in MHE.

T-lymphocyte subtyping: an early warning and a potential prognostic indicator of active cytomegalovirus infection in patients with sepsis.

Cytomegalovirus (CMV) infection is very common in patients suffering from sepsis and may cause poor prognosis. To explore the relationship between immune status of patients with sepsis and CMV infection, we assessed T lymphocyte subtyping and other commonly used clinical parameters in patients with sepsis upon admission to the intensive care unit (ICU) and evaluated their potential impact on diagnosis and outcomes of active CMV infection. In our study, 82 of 599 patients with sepsis were diagnosed with active CMV infection. The 28-day mortality was higher in active CMV-infected than nonactive CMV-infected patients (20.7% versus 9.9%); 51of 82 active CMV-infected patients with sepsis were assessed to have CMV-DNA-negative conversion, while 31 were persistently positive for CMV DNA. Higher CD8+ CD28+ T-cell counts at presentation were associated with CMV-DNA-negative conversion and lower 28-day mortality. The CMV-DNA-negative conversion and 28-day mortality of active CMV-infected patients with sepsis could be predicted using cutoff values of 151 (74.5% sensitivity and 87.1% specificity) and 64.5 (52.9% sensitivity and 92.3% specificity) CD8+ CD28+ T cells mL-1 at ICU admission, respectively. Higher CD8+ CD28+ T-cell count was significantly associated with active CMV infection, higher CMV-DNA-negative conversion and lower 28-day mortality, which may be a potential marker for early warning of active CMV infection and outcome prediction.

Th17 cells in primary Sjögren's syndrome negatively correlate with increased Roseburia and Coprococcus.

BackgroundDysbiosis of the gut microbiota is closely related to chronic systemic inflammation and autoimmunity, playing an essential role in the pathogenesis of primary Sjögren’s syndrome (pSS). Abnormalities in the proportions of blood T lymphocyte subtype, that is Th17/Treg, were detected in pSS patients. We aimed to determine the associations between gut microbiota and Th17/Treg in pSS.Method98 pSS patients and 105 healthy controls (NC) were enrolled between Dec 1, 2018, and Aug 31, 2019. The baseline information and clinical parameters on pSS patients and healthy controls were collected. 16S rRNA sequencing was performed to characterize the gut microbiome and identify gut microbes that are differentially abundant between patients and healthy controls. Lastly, associations between relative abundances of specific bacterial taxa in the gut and clinical outcome parameters were evaluated.ResultsPatients with pSS show decreased gut microbial diversity and richness, decreased abundance of butyrate producing bacteria, such as Roseburia and Coprococcus, and increased abundance of other taxa, such as Eubacterium rectale and Roseburia inulinivorans. These bacteria are enriched with functions related to glycolytic and lipogenic, energy, substance, galactose, pentose metabolism pathways and glucuronate interconversions, decreased with functions related to peptidoglycan biosynthesis, pyrimidine metabolism pathways. An integrative analysis identified pSS-related specific bacterial taxa in the gut, for which the abundance of Eubacterium rectale is negatively correlated with Th17/Treg. Furthermore, the pathways of biosynthesis of secondary metabolites, biosynthesis of amino acids, peptidoglycan biosynthesis and pyrimidine, galactose, pentose, microbial metabolism in diverse environments, glyoxylate and dicarboxylate metabolism are associated with Treg or Th17/Treg.ConclusionsPrimary Sjögren’s syndrome could lead to decreased gut microbial diversity and richness of intestinal flora in patients. The proportions of Th17 and Treg cells induced by microbiota were predictive pSS manifestations and accounted for the pSS severity.

Investigation of the Relationship Between the Immune Responses due to COVID-19 Vaccine and Peripheral Bloodlymphocyte Subtypes of Healthcare Workers

Worldwide vaccination is the best strategy to limit the Coronavirus disease-2019 (COVID-19) pandemic. The aim of this study was to investigate the antibody levels and hemogram parameters and the prediction of T-helper, T-cytotoxic and B lymphocyte subtypes in the peripheral blood for the development of these antibodies in the selected group of healthcare workers who were vaccinated with CoronaVac® (Sinovac, China). In a previous study with the same researchers, blood samples were taken from healthcare workers one month after the second vaccination, and anti-RBD anti-severe acute respiratory syndrome coronavirus-2 (anti-SARS-CoV-2) IgG antibodies were measured by enzyme linked immunosorbent assay (ELISA) method. Test results were classified as binding antibody <25.6 BAU/mL unresponsive, 25.6-35.1 BAU/mL weak response, <35.2 BAU/mL strong response, according to the assay manufacturer's recommendations. According to these criteria, it was determined that 22 (8.3%) people had no response, 25 (9.5%) people had a moderate response, and 217 (82.2%) people had a strong response. According to these results, 30 people from each group were invited. The results of three individuals were excluded from the evaluation due to chronic illness. A total of 87 health workers were included in the study. Twenty-two of these people had no response, 25 had a moderate response, and 40 had a good response. Flow cytometry was used to examine the peripheral blood lymphocyte percentage distribution. Statistical analyzes were performed with SPSS 26 software. The conformity of the variables to the normal distribution was examined using analytical methods (Kolmogorov-Smirnov/ Shapiro-Wilk tests). Pearson's Chi-square test was used to compare categorical variables. The correlation between the antibody level and the measurement results of hemogram parameters and lymphocyte indicators was evaluated by Pearson's correlation analysis. The model was established by performing linear regression analysis with the variables correlated with the antibody level. Cases with a p value below 0.05 were considered statistically significant. The age range of the participants in the study was 22-69. The mean age was 45.5 ± 10.4 years. Antibodies against SARS-CoV-2 virus were detected by ELISA method in 65 (74.7%) of the healthcare professionals, while no antibodies were detected in 22 (25.3%) of them. Lymphocyte count (p= 0.002), CD3 count (p= 0.0004), CD4 count (p= 0.0001), CD3/CD19 ratio (p= 0.011), CD4 percentage (p= 0.004) in the antibody positive group was found to be significant. According to the level of correlation between antibody level and lymphocyte indicators, a statistically significant negative correlation was found in CD3, CD4, CD8 and CD19 positive lymphocytes, respectively, and the best correlation was observed in CD3 positive lymphocytes. The formula f (Antibody level)= y= 358002 -0.176 x CD3 + 0.469 x CD19 was created using the linear regression model. In the light of the data of this study, the central role of T and B cells in COVID-19 immunization emerges. The vaccine-related antibody level-related formula may be useful for healthcare professionals in patient follow-up. It is thought that conducting vaccine related immune response studies involving larger populations will contribute more to the literature.

Real-world effectiveness and protection of SARS-CoV-2 vaccine among patients hospitalized for COVID-19 in Xi'an, China, December 8, 2021, to January 20, 2022: A retrospective study.

IntroductionIn December 2021, a large-scale epidemic broke out in Xi’an, China, due to SARS-CoV-2 infection. This study reports the effect of vaccination on COVID-19 and evaluates the impact of different vaccine doses on routine laboratory markers.MethodsThe laboratory data upon admission, of 231 cases with COVID-19 hospitalized from December 8, 2021 to January 20, 2022 in Xi’an, including blood routine, lymphocyte subtypes, coagulative function tests, virus specific antibodies and blood biochemical tests were collected and analyzed.ResultsOf the 231 patients, 21 were not vaccinated, 158 were vaccinated with two doses and 52 with three doses. Unvaccinated patients had a higher proportion of moderate and severe symptoms than vaccinated patients, while two-dose vaccinated patients had a higher proportion than three-dose vaccinated patients. SARS-CoV-2 specific IgG levels were significantly elevated in vaccinated patients compared with unvaccinated patients. Particularly, unvaccinated patients had lower counts and percentages of lymphocytes, eosinophils and CD8+ T-lymphocytes, and elevated coagulation-related markers. In addition, vaccination had no effect on liver and kidney function.ConclusionsVaccination against SARS-CoV-2, inducing high IgG level and increased CD8+ T cells and eosinophils, and regulating coagulation function, can significantly attenuate symptoms of COVID-19, suggesting that the vaccine remains protective against SARS-CoV-2.

Altered distribution and function of NK-cell subsets lead to impaired tumor surveillance in JAK2V617F myeloproliferative neoplasms.

In cancer, tumor cells and their neoplastic microenvironment can sculpt the immunogenic phenotype of a developing tumor. In this context, natural killer (NK) cells are subtypes of lymphocytes of the innate immune system recognized for their potential to eliminate neoplastic cells, not only through direct cytolytic activity but also by favoring the development of an adaptive antitumor immune response. Even though the protective effect against leukemia due to NK-cell alloreactivity mediated by the absence of the KIR-ligand has already been shown, and some data on the role of NK cells in myeloproliferative neoplasms (MPN) has been explored, their mechanisms of immune escape have not been fully investigated. It is still unclear whether NK cells can affect the biology of BCR-ABL1-negative MPN and which mechanisms are involved in the control of leukemic stem cell expansion. Aiming to investigate the potential contribution of NK cells to the pathogenesis of MPN, we characterized the frequency, receptor expression, maturation profile, and function of NK cells from a conditional Jak2V617F murine transgenic model, which faithfully resembles the main clinical and laboratory characteristics of human polycythemia vera, and MPN patients. Immunophenotypic analysis was performed to characterize NK frequency, their subtypes, and receptor expression in both mutated and wild-type samples. We observed a higher frequency of total NK cells in JAK2V617F mutated MPN and a maturation arrest that resulted in low-numbered mature CD11b+ NK cells and increased immature secretory CD27+ cells in both human and murine mutated samples. In agreement, inhibitory receptors were more expressed in MPN. NK cells from Jak2V617F mice presented a lower potential for proliferation and activation than wild-type NK cells. Colonies generated by murine hematopoietic stem cells (HSC) after mutated or wild-type NK co-culture exposure demonstrated that NK cells from Jak2V617F mice were deficient in regulating differentiation and clonogenic capacity. In conclusion, our findings suggest that NK cells have an immature profile with deficient cytotoxicity that may lead to impaired tumor surveillance in MPN. These data provide a new perspective on the behavior of NK cells in the context of myeloid malignancies and can contribute to the development of new therapeutic strategies, targeting onco-inflammatory pathways that can potentially control transformed HSCs.

Relationship between onco-immunological and morphologic characteristics of lymphoepithelioma-like carcinoma and lymphocyte subtypes of peripheral blood

Objective: To study the relationship between the onco-immunological and morphologic characteristics of lymphoepithelioma-like carcinoma (LELC) and peripheral blood lymphocyte subtypes and its clinical significance. Methods: The pathologic and clinical data of 117 LELC patients who were admitted to the Tumor Hospital of the University of Chinese Academy of Sciences from 2006 to 2018 were collected. The histological classification was based on previously reported morphological classification method. The onco-immunological and morphologic characteristics of the tumors such as lymphoid follicle formation and interstitial fibrous hyperplasia, patient's peripheral blood lymphocyte subtypes and prognosis data were collected. The relationship between various factors and their impact on prognosis were analyzed. Results: There were 117 patients, including 61 females and 56 males. The male to female ratio was 0.9∶1.0. The age of onset was 24-89 years (median 52 years). Primary sites included head and neck (68 cases), lungs (26 cases), stomach (15 cases), and others (eight cases). Morphologically, 54 cases were type Ⅰ, 62 cases were type Ⅱ, and one case could not be classified. The onco-immunological and morphologic features of the LELC tumors showed a continuous spectrum. Interstitial TILs were noted from focally to diffuse, and the interstitial fibrous tissues were from hardly visible to obvious sclerotic. Formation of lymphoid follicles was seen in 42 patients; obvious fibrosis was seen in 31 cases. Data of peripheral blood lymphocyte subtyping by flow cytometry were available in 73 cases. These data included CD3+total T cells, CD3+CD4+helper T cells, CD3+CD8+cytotoxic T cells, CD3-CD56+natural killer (NK) cells, CD3-CD19+B cells, CD4+CD45RA-T helper induction subgroup, CD4+CD45RA+ T suppression induction subgroup, CD4+CD45RO+memory T cell subgroup, CD45RA+CD45RO+activated T cell subgroup, CD8+CD38+activated cytotoxic T cell, and CD25+lymphocytes and CD44+lymphocyte. The proportion of lymphocytes of each subtype was normal in most patients, but the proportion of CD44+lymphocytes in 61 cases (83.6%) was increased; the proportion of T cell suppression induced subgroups was decreased in 53 cases (72.6%). Correlation analysis found a significant correlation between clinical stage and NK cells (P=0.023); tumor histologic type and cytotoxic T cells were significantly positively correlated (P=0.012); while tumor cell morphologic differentiation was significantly related to total T cells (P=0.003) and NK cells (P=0.026); Formation of interstitial lymphoid follicles was positively correlated with memory T cell subsets (P=0.025); Tumor interstitial fibrosis was significantly positively correlated with T suppression-induced subpopulations (P=0.004), and was significantly negatively correlated with total T cells (P=0.023) and with the expression of CD44 adhesion molecules (P=0.003). Survival analysis found that lymphoid follicle formation was a favorable prognostic factor for LELC (P=0.001). Conclusions: The onco-immunological and morphologic features in LELC show a continuous spectrum; the tumor clinicopathological characteristics and onco-immunological morphology are closely related to peripheral blood T lymphocyte subtypes, and the formation of interstitial lymphoid follicles is a favorable prognostic factor for LELC.

Rotenone aggravates PD-like pathology in A53T mutant human α-synuclein transgenic mice in an age-dependent manner.

Multiple factors such as genes, environment, and age are involved in developing Parkinson’s disease (PD) pathology. However, how various factors interact to cause PD remains unclear. Here, 3-month and 9-month-old hα-syn+⁣/− mice were treated with low-dose rotenone for 2 months to explore the mechanisms that underline the environment–gene–age interaction in the occurrence of PD. We have examined the behavior of mice and the PD-like pathologies of the brain and gut. The present results showed that impairments of the motor function and olfactory function were more serious in old hα-syn+/– mice with rotenone than that in young mice. The dopaminergic neuron loss in the SNc is more in old hα-syn+/– mice with rotenone than in young mice. Expression of hα-syn+/– is increased in the SNc of hα-syn+/– mice following rotenone treatment for 2 months. Furthermore, the number of activated microglia cells increased in SNc and accompanied the high expression of inflammatory cytokines, namely, TNF-α and IL-18 in the midbrain of old hα-syn+/– mice treated with rotenone. Meanwhile, we found that after treatment with rotenone, hα-syn positive particles deposited in the intestinal wall, intestinal microflora, and T lymphocyte subtypes of Peyer’s patches changed, and intestinal mucosal permeability increased. Moreover, these phenomena were age-dependent. These findings suggested that rotenone aggravated the PD-like pathologies and affected the brain and gut of human α-syn+/– transgenic mice in an age-dependent manner.

Primary CNS vasculitis (PCNSV): a cohort study

Primary CNS Vasculitis (PCNSV) is a rare inflammatory disorder affecting the blood vessels of the central nervous system. Patients present with a combination of headaches, seizures, and focal neurological deficits. There is usually a diagnostic delay. Treatment is based on observational studies and expert opinion. Our objective was to identify clinical, laboratory, neuroimaging, pathologic or management-related associations with 2 year outcome in patients with primary CNS vasculitis. We conducted a cohort study at a single tertiary care referral centre of prospectively (2018-2019) and retrospectively (2010-2018) identified individuals with primary CNS vasculitis (diagnosis was proven by either brain biopsy or cerebral digital subtraction angiography). Clinical, imaging and histopathologic findings, treatment, and functional outcomes were recorded. Univariate and stepwise multiple logistic regression were applied. P-value<0.05 was considered statistically significant. The main outcome measures were documentation of clinical improvement or worsening (defined by mRS scores) and identification of independent predictors of good functional outcome (mRS 0-2) at 2 years. We enrolled eighty-two biopsy and/or angiographically proven PCNSV cases. The median age at presentation was 34 years with a male predilection and a median diagnostic delay of 23 months. Most patients presented with seizures (70.7%). All patients had haemorrhages on MRI. Histologically lymphocytic subtype was the commonest. Corticosteroids with cyclophosphamide was the commonest medication used. The median mRS at follow-up of 2 years was 2 (0-3), and 65.2% of patients achieved a good functional outcome. Myelitis and longer duration of illness before diagnosis were associated with poorer outcomes. The presence of hemorrhages on SWI sequence of MRI might be a sensitive imaging marker. Treatment with steroids and another immunosuppressant probably reduced relapse rates in our cohort. We have described the third largest PCNSV cohort and multi-centre randomised controlled trials are required to study the relative efficacy of various immunosuppressants.Study registration: CTRI/2018/03/012721.

Flowcytometric Ratios of Immune Cells in the Prognosis and Staging of Acute Biliary Pancreatitis.

To evaluate several lymphocyte subtypes with various parameters that can be applied easily and give fast results for their roles in evaluating the stage and prognosis of acute biliary pancreatitis. Case-control study. The Emergency Department of Istanbul Training and Research Hospital, and Gaziosmanpasa Research and Training Hospital, Turkey, between June 2020 and April 2021. Patients, who admitted to the Emergency Department with acute pancreatitis and treated after hospitalisation, were included in the study. The patients were divided into three groups; mild, moderately severe and severe, according to the 2012 revised Atlanta classification. Hematocrit, creatinine, potassium, sodium values, and flow cytometry ratios of lymphocyte, monocyte, CD4+, CD8+, and regulatory T cells were measured and the difference between the groups were evaluated. Their results were compared with healthy volunteers. A total of 53 persons including 40 with acute pancreatitis (14 mild, 14 moderately severe, and 12 in the severe pancreatitis groups) and 13 healthy volunteers, were included in the study. The average age of the studied participants was 50.9 ±13.42 years, 43.3% males and 56.7% females. Leukocyte values, lymphocyte rates, hematocrit rates, age, hospital staying duration, creatinine, potassium values, CD4+, and CD3+ lymphocyte rates were found to be different at a statistically significant level between the groups. High leukocyte, low lymphocyte, high hematocrit, advanced age, elevated creatinine, elevated potassium, low CD4+ T lymphocyte, low CD3+ T lymphocyte, and low lymphocyte/monocyte ratio were identified as poor prognostic indicators. Acute pancreatitis, Flow cytometry, Regulatory T cell, Atlanta.

Revisiting regulatory T cells for stroke therapy.

Stroke is a leading cause of death and long-term disability. T cells have been extensively studied for their dual role in regulating immunity and inflammation following stroke. In this issue of the JCI, Cai, Shi, et al. demonstrated that CD8+ regulatory-like T cells (CD8+ TRLs) are one of the earliest lymphocyte subtypes to enter the brain after experimental ischemic stroke. Using a mouse model of stroke and comprehensive experimental approaches, the authors found that CD8+ TRLs reduced both brain damage and functional deficits in both young and aged mice. These unique early responding regulatory T cells may also play a role in a wide array of other T cell–mediated neurological disorders.

T-Cell Assay after COVID-19 Vaccination Could Be a Useful Tool? A Pilot Study on Interferon-Gamma Release Assay in Healthcare Workers.

Background: SARS-CoV-2 T-cells are crucial for long-term protection against reinfection. The aim was to demonstrate the Interferon-gamma Release Assay (IGRA) test could be useful for vaccination monitoring. Methods: In a prospective cohort of 98 vaccinated healthcare workers for SARS-CoV-2, we selected 23 people in low-antibodies (Group 1, N = 8), high-antibodies (Group 2, N = 9), and negative control groups (Group 3, N = 6). SARS-CoV-2-specific humoral and cellular responses were analyzed at 8 months after two doses of Pfizer BioNTech, evaluating anti-RBD (Receptor Binding Domain) and RBD-ACE2 (Angiotensin Converting Enzyme-2) blocking antibodies in sera through a Chemiluminescence Immunoassay (CLIA) and T-cells through the IGRA test in heparinized plasma. Moreover, lymphocyte subtyping was executed by a flow cytometer. Statistical analysis was performed. Results: The data confirmed that RBD and RBD-ACE2 blocking ACE2 antibody levels of Group 1 were significantly lower than Group 2; p < 0.001. However, T-cells showed no significant difference between Group 1 and Group 2. Conclusions: This work suggests the need for new strategies for booster doses administration.

Conditioned media of pancreatic cancer cells and pancreatic stellate cells induce myeloid-derived suppressor cells differentiation and lymphocytes suppression

As pancreatic cancer cells (PCCs) and pancreatic stellate cells (PSCs) are the two major cell types that comprise the immunosuppressive tumor microenvironment of pancreatic cancer, we aimed to investigate the role of conditioned medium derived from PCCs and PSCs co-culture on the viability of lymphocytes. The conditioned medium (CM) collected from PCCs and/or PSCs was used to treat peripheral blood mononuclear cells (PBMCs) to determine CM ability in reducing lymphocytes population. A proteomic analysis has been done on the CM to investigate the differentially expressed protein (DEP) expressed by two PCC lines established from different stages of tumor. Subsequently, we investigated if the reduction of lymphocytes was directly caused by CM or indirectly via CM-induced MDSCs. This was achieved by isolating lymphocyte subtypes and treating them with CM and CM-induced MDSCs. Both PCCs and PSCs were important in suppressing lymphocytes, and the PCCs derived from a metastatic tumor appeared to have a stronger suppressive effect than the PCCs derived from a primary tumor. According to the proteomic profiles of CM, 416 secreted proteins were detected, and 13 DEPs were identified between PANC10.05 and SW1990. However, CM was found unable to reduce lymphocytes viability through a direct pathway. In contrast, CM that contains proteins secreted by PCC and/or PSC appear immunogenic as they increase the viability of lymphocytes subtypes. Lymphocyte subtype treated with CM-induced MDSCs showed reduced viability in T helper 1 (Th1), T helper 2 (Th2), and T regulatory (Treg) cells, but not in CD8+ T cells, and B cells. As a conclusion, the interplay between PCCs and PSCs is important as their co-culture displays a different trend in lymphocytes suppression, hence, their co-culture should be included in future studies to better mimic the tumor microenvironment.

Immune reconstitution after allogenic stem cell transplantation: An observational study in pediatric patients

IntroductionThe immune reconstitution (IR) after the allogenic hematopoietic stem cell transplantation (allo-HSCT) is a progressive process intrinsically correlated to the therapeutic success. It is essential to understand the interfering factors in IR to prevent the HSCT-related mortality. MethodsWe retrospectively evaluated the clinical outcomes, absolute lymphocyte counts (ALCs) and lymphocyte subtypes at different time-points of 111 pediatric patients with allogeneic HSCT for malignant and non-malignant diseases from 2013 to 2018. ResultsThe ALCs gradually increased on D+30, D+100, and D+180 (medians 634/μL, 1022/μL and 1541/μL, respectively). On D+100, the CD3+CD8+ achieved the highest recovery rate (68%), followed by the CD16+CD56+ (47%), CD3+CD4+ (39%) and CD19+ (8%). The adequate ALC recovery was associated with age < 8 years, bone marrow grafts, myeloablative conditioning, non-use of serotherapy and non-haploidentical donors. The ALC and CD3+CD8+ on D+100 counts were higher in patients with the cytomegalovirus infection. The CD3+CD4+ recovery was associated with an age < 8 years, a non-malignant disease and a lower incidence of acute graft-versus-host disease ≥ grade 2. Furthermore, the ALC recovery on D+100 resulted in a higher overall survival, regardless of the disease type (HR 3.65, 1.05 - 12.71, p = 0.04). ConclusionSeveral factors influenced the IR after the allo-HSCT. The ALC ≥ 500/μL on D+100 was a simple IR predictor of survival, easily available to resource-limited centers.

Role of Inflammation in Cardiac Remodeling After Acute Myocardial Infarction.

Atherosclerosis is defined as an inflammatory disease. Low-grade inflammation is present in all phases of the cardiovascular continuum, since the establishment of cardiovascular risk factors and ischemic heart disease until cardiovascular events, such as myocardial infarction, heart failure and death. Not all inflammatory pathways are linked to cardiovascular outcomes, and thus, not all anti-inflammatory approaches decrease cardiovascular events. The most common cause of ventricular remodeling and heart failure is ischemic heart disease. Biomarkers such as high-sensitivity C-reactive protein can identify individuals at risk of major cardiovascular complications, but this biomarker has no causal effect on cardiovascular disease. On the other hand, interleukin 6 appears to be causally associated with cardiovascular disease. CANTOS was the first proof of concept study showing that anti-inflammatory therapy reduces major cardiovascular outcomes. Based on many anti-inflammatory trials, only therapies acting on the NLRP3 inflammasome, or interleukin 1beta, showed benefits on cardiovascular disease. Ventricular remodeling, particularly after myocardial infarction seems also influenced by the intensity of inflammatory responses, suggesting that anti-inflammatory therapies may reduce the residual cardiovascular risk. Inflammasome (NLRP3) activation, subtypes of lymphocytes, interleukin 6, and some inflammatory biomarkers, are associated with larger infarct size and impaired ventricular function after myocardial infarction. Cardiovascular risk factors commonly present in patients with myocardial infarction, and advanced age are associated with higher inflammatory activity.

Appendectomy and Future Risk of Microscopic Colitis: A Population-Based Case-Control Study in Sweden

Background and AimsMicroscopic colitis (MC) is an inflammatory bowel disease and a common cause of chronic diarrhea. Appendectomy has been suggested to have immunomodulating effects in the colon, influencing the risk of gastrointestinal disease. The relationship between appendectomy and MC has only been sparsely studied. MethodsThis was a case-control study based on the nationwide ESPRESSO (Epidemiology Strengthened by histoPathology Reports in Sweden) cohort, consisting of histopathological examinations in Sweden, linked to national registers. Patients with MC were matched to population controls by age, sex, calendar year of biopsy, and county of residence. Data on antecedent appendectomy and comorbidities were retrieved from the Patient Register. Unconditional logistic regression models were conducted presenting odds ratios (ORs) and 95% confidence intervals (CIs) adjusted for country of birth and matching factors. Further subanalyses were made based on MC subtypes (lymphocytic colitis and collagenous colitis), follow-up time postappendectomy and severity of appendicitis. ResultsThe study included 14,520 cases of MC and 69,491 controls, among these 7.6% (n = 1103) and 5.1% (n = 3510), respectively, had a previous appendectomy ≥1 year prior to MC or matching date. Patients with a previous appendectomy had an increased risk of MC in total (OR, 1.50; 95% CI, 1.40–1.61) and per the collagenous colitis subtype (OR, 1.67; 95% CI, 1.48–1.88) or lymphocytic colitis subtype (OR, 1.42; 95% CI, 1.30–1.55). The risk remained elevated throughout follow-up, and the highest risk was observed in noncomplicated appendicitis. ConclusionsThis nationwide case-control study found a modestly increased risk of developing MC following appendectomy.