Tregs In Peripheral Blood Research Articles

Liangxue Tongyu Prescription Alleviates Brain Damage in Acute Intracerebral Hemorrhage Rats by Regulating Intestinal Mucosal Barrier Function.

Liangxue Tongyu prescription (LTP) is a commonly used formula for acute intracerebral hemorrhage (AICH) in clinical practice that has significant ameliorative effects on neurological deficits and gastrointestinal dysfunction, yet the mechanism remains elusive. The aim of this study was to investigate the pathway by which LTP alleviates brain damage in AICH rats. The AICH rat models were established by autologous caudal arterial blood injection. The neurological function scores were evaluated before and after treatment. The water content and the volume of Evans blue staining in the brain were measured to reflect the degree of brain damage. RT-PCR was used to detect the inflammatory factors of the brain. Western blotting was used to detect the expression of the tight junction proteins zonula occludens 1 (ZO-1), occludin (OCLN), and claudin (CLDN) in the brain and colon, followed by mucin 2 (MUC2), secretory immunoglobulin A (SIgA), and G protein-coupled receptor 43 (GPR43) in the colon. Flow cytometry was used to detect the ratios of helper T cells 17 (Th17) and regulatory T cells (Treg) in peripheral blood, and the vagus nerve (VN) discharge signals were collected. LTP reduced the brain damage of the AICH rats. Compared with the model group, LTP significantly improved the permeability of the colonic mucosa, promoted the secretion of MUC2, SigA, and GPR43 in the colon, and regulated the immune balance of peripheral T cells. The AICH rats had significantly faster VN discharge rates and lower amplitudes than normal rats, and these abnormalities were corrected in the LTP and probiotics groups. LTP can effectively reduce the degree of brain damage in AICH rats, and the mechanism may be that it can play a neuroprotective role by regulating the function of the intestinal mucosal barrier.

Association of Immune Dynamics and Treatment Outcome in Patients (pts) with Relapsed/Refractory Non-Hodgkin Lymphoma (R/R NHL) Treated with the Novel Cereblon (CRBN) E3 Ligase Modulator (CELMoD) Agent CC-99282

Introduction Compounds inducing CRBN-mediated degradation of Ikaros/Aiolos have demonstrated antitumor activity in pts with NHL. The pharmacodynamic (PD) effects of these agents include T-cell activation, increased trafficking of macrophages, and increased number of regulatory T cells (Tregs) in peripheral blood (PB). Furthermore, high baseline immune infiltration in tumors has been correlated with improved outcomes. CC-99282 (BMS-986369) is a novel CELMoD® agent that has enhanced antiproliferative, apoptotic, and immune-stimulatory effects compared with other immunomodulatory agents in various preclinical NHL models. Part A of the CC-99282-NHL-001 study (NCT03930953) showed CC-99282 monotherapy has a predictable and manageable safety profile, dominated by the class effect of neutropenia, and promising efficacy (objective response rate 42%) in heavily pretreated pts with R/R NHL. Methods CC-99282-mediated immune effects and the dependency of response on immune changes were evaluated using biomarker data from part A of CC-99282-NHL-001, including longitudinal substrate degradation, circulating tumor DNA (ctDNA), ex vivo cytokine secretion, immunophenotyping, and baseline tumor RNA-seq data. Results At steady state, CC-99282 induced deep (> 90%) and sustained degradation of Ikaros/Aiolos in monocytes, B cells, and T cells that was independent of response. PD effects were also confirmed in tumor cells obtained at screening and on treatment. PB T cells from pts dosed with CC-99282 showed increased secretion of IL-2, TNFα, INFγ, and GM-CSF on ex vivo stimulation with anti-CD3. Tumor and PB biomarkers were analyzed for correlation to response. ctDNA data available for 39/50 pts treated in part A showed high tumor burden (> 2.5 log10 of tumor-specific mutant sequence [Kurtz, et al. J Clin Oncol 2018]) in 28/39 pts (72%) at study entry, and yet 13/28 pts (43%) still achieved a partial or complete response. B-cell content estimation in screening biopsies, calculated from bulk RNA-seq data using cell-type-specific markers (Bindea, et al. Immunity 2013), confirmed similar B-cell content in responders and non-responders prior to CC-99282 treatment. In contrast to treatment with other CELMoD agents, this analysis showed that durable responses to CC-99282 were observed in pts with R/R NHL despite their high tumor burden and low immune infiltration. Baseline PB immunophenotype was not significantly associated with response. However, unsupervised clustering of changes in immune cell subsets in pts between baseline and cycle (C) 1 day (D) 15, revealed 5 clusters (Figure 1). Cluster 5, enriched in diffuse large B-cell lymphoma (DLBCL) non-responders, showed upregulation of peripheral Tregs without changes in T-cell activation. Clusters 1 and 4 also showed upregulation of Tregs, with cluster 1 showing additional immune activation. Cluster 2, enriched for follicular lymphoma (FL), only showed T-cell activation. Cluster 3 was enriched in responders without changes in immune subsets upon CC-99282 treatment, suggesting a stronger direct effect on tumor cells in the early stages of treatment. Baseline tumor biopsies from pts in all clusters had similar Treg levels, independent of their immune changes upon treatment. Further, longitudinal analysis of ctDNA during treatment showed that all clusters of responders had a similar decrease in ctDNA that was maintained until the end of treatment despite increases in Tregs. Immunophenotyping at C2D1 and C3D1 showed that immune activation was maintained in responders and increases in Tregs progressively extended to all pts independently of response. The implications of these differences remain unclear. The only previous systematic analyses of the prognostic role of Tregs in lymphoma concluded that increase in Tregs correlated with longer overall survival for pts with DLBCL, but found no significant association in pts with FL (Glowala-Kosinska, et al. Eur J Haematol 2013; Peng, et al. J Cancer Res Clin Oncol 2020). Conclusion Taken together, these results indicate a link between patterns of immune activation and response to CC-99282, but whether this link is causative, or correlative, remains to be elucidated. Responder subsets were identified based on effects on Tregs and immune activation; however, all cases led to durable responses. Further study and validation of these data could provide rationale for pt selection and combination strategies. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Dynamics of immune system parameters in development of SARS-CoV-2-specific immunity in a patient with common variable immune deficiency

With the accumulation of data on the evolving SARS-CoV-2 infection pandemic, it became clear that the risk factors for severe course of COVID-19 among the patients with primary immunodeficiency include disorders associated with dysregulation of the immune response. In this regard, it is of interest to identify possible predictors of the pronounced inflammatory reaction upon infection with coronavirus in the patients with general variable immune insufficiency. For this purpose, the dynamics of immune system parameters was studied in a patient with CVID who underwent a severe clinical variant of COVID-19, as an example of clinical case.
 We present patient K., 49 years old, with CVID diagnosis verified at the age of 35 years who received regular replacement therapy with IVIG. He suffered from COVID-19 in severe form, received anti-cytokine therapy and an additional course of IVIG during the treatment. He was discharged in satisfactory condition. The quantitative and functional parameters of the T and B lineages of immune system were evaluated by flow cytofluorimetry during routine examinations before the infection and three months after the discharge from the hospital after COVID-19. It has been shown that, before the disease, there were changes in the parameters of B cells characteristic of CVID manifesting as a decrease in switched-memory B cells and plasmoblasts. Alterations in the T cell subsets were also revealed, as redistribution of the subpopulation composition towards T effectors with an increased cytolytic potential of these cells and a weakening of T cell suppression, due to decreased Treg in peripheral blood. After undergoing COVID-19, the patient developed specific IgM and IgG antibodies. The development of immune response was accompanied by an increase in the number of un-switched and switched memory B cells. At the same time, we have registered an increase in memory T cells ready for the proliferative response of T helper cells and Treg cells. The initial pro-inflammatory pattern of the T cell lineage system in our patient with CVID is explained by the implementation of the compensatory capabilities of the immune system, thus leading to activation of the cytolytic effects of cellular compartment in adaptive immune response, along with attenuation of the humoral component. Moreover, it is likely that these changes contributed to the clinical course of COVID-19 in this clinical case. Development of a specific humoral response to SARS-CoV-2 in a patient with CVID after a COVID-19 infection is accompanied by an increased proportion of memory B cells, coordinated dynamics of T cell suppression and activation parameters.

Astragaloside IV Inhibits the Proliferation of Human Uterine Leiomyomas by Targeting IDO1.

Simple SummaryImmunotherapy is increasingly becoming a success strategy for oncology treatment. Indoleamine-2,3-dioxygenase1 (IDO1) is a tryptophan-degrading enzyme involved in immunological escape mechanisms, which is considered as a potential target for tumor therapy. However, the clinical efficacy of IDO1 inhibitors is not promising. Therefore, there is an urgent to investigate the mechanism between chemical drugs with antitumor effects and IDO1-mediated immunosuppression. The Chinese medicine AS-IV exerts antitumor effects with many advantages, including fewer toxic side effects and immunomodulatory effects. We noted the lack of studies of AS-IV on benign tumors. Therefore, our study demonstrates the Inhibitory effect of AS-IV on ULMs and elucidates the underlying mechanism.Astragaloside IV (AS-IV) is a chemical found in traditional Chinese medicine called Astragalus membranaceus (Fisch.) Bunge that has antitumor properties. However, the roles and mechanisms of AS-IV in uterine leiomyomas (ULMs) are unclear. The immunosuppressive enzyme indoleamine-2,3-dioxygenase-1 (IDO1) is involved in tumor formation. IDO1 is a new and reliable prognostic indicator for several cancers. In this work, AS-IV was applied to ULM cells in various concentrations. CCK-8, immunofluorescence, and flow cytometry were used to examine the proliferation and apoptosis of ULM cells caused by AS-IV. After lentiviral vector transduction with IDO1 short hairpin RNA (shRNA), the knockdown and overexpression of IDO1 were stable in ULM cells. To verify the antitumor effect of AS-IV in vivo, we established a rat model of uterine leiomyoma. HE staining, Masson staining, and transmission electron microscopy were used to observe pathological changes in the uterus, and the levels of serum sex hormones were measured by radio immune assay (RIA). The levels of CD3+T, CD4+T, and CD25+ Foxp3+Treg in rat peripheral blood were detected by flow cytometry. Western blotting and immunohistochemistry were used to examine protein expression. We found that AS-IV dramatically increased the apoptotic rate of ULM cells and reduced viability in a time- and dosage-dependent manner. After sh-IDO1 lentiviral transfection, we discovered that knocking down IDO1 reversed the effects of AS-IV on ULM cell proliferation and autophagy. We also found that AS-IV can effectively inhibit the growth of ULMs in vivo. AS-IV may promote apoptosis and autophagy in ULMs by activating PTEN/PI3K/AKT signaling through inhibition of IDO1. These findings imply that AS-IV exerts antifibroid effects, and the underlying mechanism may be IDO1, which is involved in proliferation, apoptosis, and autophagy.

Selected Articles from This Issue

Highlights| September 01 2022 Selected Articles from This Issue Author & Article Information Online ISSN: 1557-3265 Print ISSN: 1078-0432 ©2022 American Association for Cancer Research2022American Association for Cancer Research Clin Cancer Res (2022) 28 (17): 3631. https://doi.org/10.1158/1078-0432.CCR-28-17-HI Related Content A commentary has been published: A Cathepsin-Targeted Quenched Activity–Based Probe Facilitates Enhanced Detection of Human Tumors during Resection A commentary has been published: Circulating Tumor DNA Kinetics Predict Progression-Free and Overall Survival in EGFR TKI–Treated Patients with EGFR-Mutant NSCLC (SWOG S1403) A commentary has been published: Lenalidomide plus R-GDP (R2-GDP) in Relapsed/Refractory Diffuse Large B-Cell Lymphoma: Final Results of the R2-GDP-GOTEL Trial and Immune Biomarker Subanalysis View more A commentary has been published: First-in-Human Phase I/II ICONIC Trial of the ICOS Agonist Vopratelimab Alone and with Nivolumab: ICOS-High CD4 T-Cell Populations and Predictors of Response View less Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn MailTo Tools Icon Tools Get Permissions Cite Icon Cite Search Site Article Versions Icon Versions Version of Record September 1 2022 Citation Selected Articles from This Issue. Clin Cancer Res 1 September 2022; 28 (17): 3631. https://doi.org/10.1158/1078-0432.CCR-28-17-HI Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest Search Advanced Search Diffuse large B-cell lymphoma (DLBCL) is recognized as a heterogeneous disease that can be cured in up to 60–70% of the cases with upfront therapy. However, relapsed/refractory DLBCL (R/R DLBCL) still represents a great clinical challenge. Palazón-Carrión and colleagues conducted an open label multicenter phase 2 trial (R2-GDP-GOTEL) testing the synergism of combination lenalidomide (LEN) and rituximab (R2) plus gemcitabine, cisplatin, dexamethasone (R2-GDP schedule) in R/R DLBCL patients, not suitable for autologous stem cell transplantation. Results suggest that LEN in combination with R-GDP (R2-GDP schedule) is an immunomodulatory treatment option with manageable toxicity and promising clinical activity, with high proportion of complete responses and favorable survival outcomes in patients with R/R DLBCL. Additionally, evolution of MDSC and Treg in peripheral blood seem an alternative for monitoring immune profiling that is related to clinical outcomes of overall survival and complete responses. In the first-in-human phase I/II ICONIC trial, Yap and colleagues... You do not currently have access to this content.

1061P Static and dynamic tracking of radiomic and immunophenotypic features predicts the benefit of immune checkpoint inhibitors in advanced NSCLC

Parallel monitoring of radiomic and blood/tissue immunophenotypic cues may intercept the critical events implicated in Immune Checkpoint Inhibitors (ICIs) efficacy. Thus, we explored radio-immune features and their evolution to provide suitable predictors of ICI response in advanced NSCLC patients. On 58 ICI treated NSCLC cases, we prospectively evaluated: Tumor Immune Microenvironment (TIME) by PD-L1 expression and incidence and spatial distribution of infiltrating lymphocytes (TILs); peripheral blood (PB) at T0 and first disease assessment (T1) for the quantification of effector (NK, CD8, PD1, Granzyme B [GnzB], Perforin [Perf], Ki67) and suppressor (CD4+CD25+FOXP3+ Tregs) phenotypes (flowcytometry), soluble PD-L1 (sPD-L1) and Lung Immune Prognostic Index (LIPI); CT derived Radiomic Features (RFs, n: 851) at T0 and T1. Changes in PB parameters were expressed as Δ% = ([T1 - T0]/T0)*100, while ΔRFs as (T1-T0)/T0. Primary endpoint was tumor response (RECIST v.1.1): CR/PR or SD ≥ 6 months defined clinical benefit (CB), while SD < 6 months or PD non-responders (NR). Baseline immune profile of CB patients comprised: -TIME enriched of CD3+, CD8+ and PD1+ and poor of immune excluded (IE) TILs (Mann-Whitney, p<0.01 vs NR); -PB bearing prominent effector cells, low sPD-L1 and good LIPI, enclosing a highly prognostic model (Fisher, p<0.005). Among PB-TIME associations, PB Tregs correlated directly with IE and inversely with intratumor-CD8+ TILs (p<0.05). Mean Δ% NK, CD8+Ki67+ and CD8+GnzB/Perf+ were, respectively, -20%, -0.4% and -41% in NR and +22%, +170% and +65% in CB, which also displayed a greater boost of counterregulatory Tregs (p<0.05). Out of 657 ΔRFs resulting from pre-processing and Z-score standardization, 11 were differentially regulated in CB vs NR (Mann Whitney, p<0.05). Distinct ΔRFs principal components, encompassing wavelet-HLL_firstorder_Maximum and -HLL_firstorder_Skewness, showed, respectively, direct and inverse correlation with Δ% CD8+Ki67+ lymphocytes. Static and dynamic radio-immune signatures may discern ICI outcome in advanced NSCLC, ultimately enabling tailored therapeutic approaches.

The changes in peripheral blood Th17 and Treg ratios in Hashimoto's thyroiditis are accompanied by differential PD-1/PD-L1 expression.

ObjectiveThe aim of this study was to analyze the percentages of T helper 17 cells (Th17s) and T regulatory cells (Tregs) in autoimmune Hashimoto’s thyroiditis (HT), and the expression of the checkpoint molecules programmed death receptor 1/programmed death ligand 1 (PD-1/PD-L1) on these cells.MethodsThis is a case-control study involving 53 initially diagnosed HT patients (HT group) and 21 normal controls (NC group). The peripheral blood mononuclear cells from the individuals of the two groups were isolated and restimulated ex vivo; the percentage of Th17s, Tregs, PD-1+ Th17s, PD-L1+ Th17s, PD-1+ Tregs, and PD-L1+ Tregs was assessed by flow cytometric analysis.Results(1) The percentage of Th17s in the peripheral blood of the HT group was significantly higher than that of the NC group [(6.38 ± 1.32)% versus (3.12 ± 0.66)%; t = 14.110, P < 0.001], while the percentage of peripheral blood Tregs was significantly lower [(3.82 ± 1.48)% versus (5.61 ± 1.60)%; t = −4.599, P < 0.001]. (2) HT patients’ Th17s expressed PD-1 at a significantly lower frequency than their counterparts in the NC [(6.46 ± 2.77)% versus (18.51 ± 3.96)%; t = −14.842, P < 0.001], while no difference was observed for PD-L1 between the two groups. (3) In contrast, both PD-1 and PD-L1 were expressed at significantly higher frequency on HT patients’ Tregs than on NC [respectively: (17.01 ± 3.04)% versus (10.23 ± 2.77)%; t = 8.850, P < 0.001 for PD-1; (16.60 ± 9.58)% versus (11.36 ± 10.14)%; t = 2.089, P < 0.005, for PD-L1].Conclusion(1) The increased percentage of Th17s and decreased percentage of PD-1+ Th17s in the HT group suggest that a loss of control on Th17 activity through the checkpoint inhibitory axis PD-1/PD-L1 may participate in disease pathogenesis. (2) While the decreased percentage of Tregs in HT patients may explain a lack of regulatory functions able to prevent the autoimmune destruction of the thyroid, the significance of the increased frequency of Tregs expressing PD-1 and PD-L1, previously reported to boost Tregs differentiation, remains to be established. Elucidating this apparent contradiction may reveal important mechanisms underlying HT pathogenesis.

Herpesvirus entry mediator regulates the transduction of Tregs via STAT5/Foxp3 signaling pathway in ovarian cancer cells.

The ratio of regulatory T cells (Treg) in peripheral blood of cancer patients has a closely correlation to the occurrence and development of ovarian cancer. In this study, our aim to explore the expression of herpesvirus entry mediator (HVEM) in ovarian cancer and its correlation with Tregs. The expression of HVEM in peripheral blood of ovarian cancer patients was detected by ELISA, and the ratio of CD4+ CD25 + Foxp3 positive Tregs cells was detected by flow cytometry. Ovarian cancer cell lines with high- and low-HVEM expression were constructed. CD4+ cells were co-cultured with ovarian cancer (OC) cells, and the expressions of IL-2 and TGF-β1 in the supernatant of cells were detected by ELISA, and western blot was used to detect the expressions of STAT5, p-STAT5, and Foxp3. The results indicated that the number of Treg cells in the peripheral blood of OC patients increased, and the expression of HVEM increased, the two have a certain correlation. At the same time, the overexpression of HVEM promoted the expression of cytokines IL-2 and TGF- β1, promoted the activation of STAT5 and the expression of Foxp3, leading to an increase in the positive rate of Treg, while the HVEM gene silence group was just the opposite. Our results showed that the expression of HVEM in OC cells has a positive regulation effect on Tregs through the STAT5/Foxp3 signaling pathway. To provide experimental basis and related mechanism for the clinical treatment of ovarian cancer.

Clinical Significance of Negative Costimulatory Molecule PD-1/PD-L1 on Peripheral Blood Regulatory T Cell Levels among Patients with Pulmonary Tuberculosis.

Objective This study aimed to investigate the expression and clinical significance of negative costimulatory molecules programmed death-1 (PD-1) and programmed death ligand 1 (PD-L1) on CD4+CD25+CD127low regulatory T cells (Tregs) in peripheral blood of patients with active pulmonary tuberculosis (TB). Methods A total of 30 patients with active pulmonary TB and 20 healthy controls were enrolled. The proportions of peripheral blood CD4+CD25+CD127low Tregs and the expression of PD-1 and PD-L1 on CD4+CD25+CD127low Tregs were detected among active pulmonary TB patients using flow cytometry. The associations of proportions of CD4+CD25+CD127low Tregs with the demographic and clinical characteristics of active pulmonary TB patients were evaluated, and the correlation between PD-1/PD-L1 expression and proportions of peripheral blood CD4+CD25+CD127low Tregs was examined among patients with active pulmonary TB using Pearson correlation analysis. Results Flow cytometry detected a significantly higher proportion of peripheral blood CD4+CD25+CD127low Tregs in the TB group than in the control group (9.14% ± 2.66% vs. 6.39% ± 1.73%; t = 4.067, P < 0.001), and a higher proportion of peripheral blood CD4+CD25+CD127low Tregs among active pulmonary TB patients with a positive anti-M. tuberculosis antibody than in those with a negative antibody (Figure 2(a)); however, there were no gender, M. tuberculosis culture, tuberculin test, CT examination, or sputum smear test-specific proportions of CD4+CD25+CD127low Tregs among patients with active pulmonary TB. The PD-1 (6.13% ± 3.53% vs. 24.78% ± 7.73%, P < 0.05) and PD-L1 levels (2.97% ± 2.00% vs. 9.23% ± 5.76%, P < 0.05) were lower on peripheral blood CD4+CD25+CD127low Tregs among the TB group than in the control group. In addition, Pearson correlation analysis revealed a positive correlation between PD-1 and PD-L1 expression on peripheral blood CD4+CD25+CD127low Tregs among patients with active pulmonary TB (r = 0.435, P=0.016) and a negative correlation between the proportion of peripheral blood CD4+CD25+CD127low Tregs and PD-1 (r = ‒0.344, P=0.024) and PD-L1 expression among patients with active pulmonary TB (r = ‒0.310, P=0.043). Conclusion The proportion of CD4+CD25+CD127low Tregs is higher in patients with active pulmonary TB than in healthy controls, and the negative costimulatory signal PD-1/PD-L1 expression is downregulated among active pulmonary TB patients. Our findings provide insights into the illustration of pathogenic mechanisms and immunotherapy of active pulmonary TB.

Level of interleukin-35 in patients with idiopathic membranous nephropathy and its predictive value for remission time.

ObjectiveAs a member of interleukin-12 family, interleukin-35 (IL-35) plays an important regulatory role in immune response. The relationship between IL-35 and idiopathic membranous nephropathy (IMN) is still unclear, and the purpose of this study is to clarify the relationship between IL-35 and disease activity and remission of IMN.MethodsThis study was a single-center, retrospective study in which all patients were diagnosed with IMN by renal biopsy or aPLA2R titer and treated with Mahuang Fuzi and Shenzhuo Decoction (MFSD). A follow-up was conducted with the endpoint of clinical complete or partial remission (CR+PR). Levels of serum IL-35 were measured and its relationship with IMN remission were analyzed. The regulatory T cell (Treg) and inducible IL-35 producing Tregs (iTR35) in peripheral blood of IMN patients were detected by flow cytometry.ResultsA total of 76 IMN patients (age 51.95 ± 13.29) were followed-up for 18 (12, 24) months. The level of serum IL-35 in all patients increased after treatment, but the degree of increase in remission group was significantly higher than that in no remission (NR) group (117.6% vs 83.7%, P<0.01). The baseline IL-35 level in remission group was higher than that in NR group (174.87 vs.151.87 pg/ml, P=0.016). Cox regression analysis showed that baseline IL-35 level was a independent risk factor for IMN remission (HR 1.081, 95%CI 1.048-1.116, P<0.001). Patients with baseline IL-35 lower than the lower quartile (≤145.49 pg/ml) had an average remission time twice as long as those with baseline IL-35 higher than the upper quartile (> 203.05 pg/ml) (12mon vs. 24mon, P<0.01). The baseline IL-35 can predict the remission time of IMN patients with either aPLA2R positive (AUC=0.673) or negative (AUC=0.745). Analysis of 18 patients with IMN showed that IL-35 level had a higher correlation with iTR35, but not Treg (r=0.613, P<0.05).ConclusionsThe level of IL-35 in patients with IMN showed an increasing trend with the progress of treatment, and the baseline IL-35 could predict the remission time of IMN patients, including those patients with negative aPLA2R. The level of IL-35 is related to the number of iTR35 cells.

Presence of periodontitis may synergistically contribute to cancer progression via Treg and IL-6

A close causal relationship has been suggested to exist between cancer and periodontitis. We hypothesized that the immune surveillance system is impaired in patients with periodontitis, which contributes to cancer development and growth. Therefore, the present study investigated the relationship between immune surveillance mechanisms and periodontitis in cancer patients. The presence or absence of periodontitis was assessed and the peripheral blood (PB) concentrations of IL-6, immunosuppressive cytokines (VEGF, TGF-β1, and CCL22) and proportion of T regulatory cells (Treg, CD3 + CD4 + CD25 + Foxp3 +) were measured. Subjects were classified into the following four groups: non-cancer patients without periodontitis (C − P −), non-cancer patients with periodontitis (C − P +), cancer patients without periodontitis (C + P −), and cancer patients with periodontitis (C + P +). The results of a multivariate analysis showed that the PB concentration of IL-6 was significantly higher in C + than in C- and higher in C + P + than in C + P −. The PB proportion of Treg was significantly higher in C + P + than in C + P −, C − P + , and C − P −. The results of this study suggested that the presence of periodontitis and cancer synergistically increased Treg in PB, which may be one of the underlying causes of immunosuppression and immune evasion in cancer. It was also suggested that the presence of periodontal disease and/or cancer also increases IL-6 in PB, which would be associated with cancer progression. These results suggest the possibility that the presence of periodontitis might synergistically contribute to cancer progression.

Regulatory T Cell Proportion and Phenotype Are Altered in Women Using Oral Contraception.

Regulatory T (Treg) cells are a specialized CD4+ T cell subpopulation that are essential for immune homeostasis, immune tolerance, and protection against autoimmunity. There is evidence that sex-steroid hormones estrogen and progesterone modulate Treg cell abundance and phenotype in women. Since natural oscillations in these hormones are modified by hormonal contraceptives, we examined whether oral contraception (OC) use impacts Treg cells and related T cell populations. T cells were analyzed by multiparameter flow cytometry in peripheral blood collected across the menstrual cycle from healthy women either using OC or without hormonal contraception and from age-matched men. Compared to naturally cycling women, women using OC had fewer Treg cells and an altered Treg cell phenotype. Notably, Treg cells exhibiting a strongly suppressive phenotype, defined by high FOXP3, CD25, Helios, HLADR, CTLA4, and Ki67, comprised a lower proportion of total Treg cells, particularly in the early- and mid-cycle phases. The changes were moderate compared to more substantial differences in Treg cells between women and men, wherein women had fewer Treg cells—especially of the effector memory Treg cell subset—associated with more T helper type 1 (Th1) cells and CD8+ T cells and lower Treg:Th1 cell and Treg:CD8+ T cell ratios than men. These findings imply that OC can modulate the number and phenotype of peripheral blood Treg cells and raise the possibility that Treg cells contribute to the physiological changes and altered disease susceptibility linked with OC use.

O-030 Disturbed homeostasis of regulatory T-cells and imbalanced signal transducer and activator of transcription 5 (STAT5) in women with ovarian endometriosis

Abstract Study question Do women with endometriosis have altered cytokine signalling and regulatory T-cell (Treg) homeostasis compared to controls? Summary answer Altered homeostasis of circulating Treg cells seems to be associated with STAT5 phosphorylation, which is significantly increased in women with ovarian endometriosis compared to controls. What is known already Cytokine signaling influences homeostasis of regulatory T-cells (Treg), which play a crucial role in cell-mediated immunity and are responsible for certain immune disorders. Endometriosis is a condition associated with altered immune response to endometrial cells. While some animal studies have shown that Treg deficiency facilitates ectopic implantation of endometriosis, a recent study performed in a small number of women with endometriosis has shown the decreased proportion of a specific-activated subset of Treg cells in the endometrioma compared to controls. There is no data characterising cytokine signalling in circulating Treg subsets, which could underlie their altered homeostasis in women with endometriosis. Study design, size, duration A prospective cohort study including 14 women with endometriosis and 10 controls at a university-based tertiary care centre for endometriosis. The data were collected from August 2020 to September 2021. Serum blood samples were collected in the early follicular phase of the menstrual cycle, in women not using any hormonal treatment. Early follicular steroid hormone levels were measured. A detailed expert ultrasound examination was performed to diagnose and accurately map endometriotic lesions. Participants/materials, setting, methods We investigated signal transducer and activator of transcription (STAT) signalling in peripheral blood Treg subsets, including CD45RA+FOXP3low+ naive Tregs (nTregs). Expression of proliferation marker Ki-67 and STAT activation also in FOXP3- conventional (Tcon) CD4+T-cells was monitored, hypothesising that defects in this pathway could be associated with altered Treg/Tcon homeostasis in endometriosis. Immunophenotyping and flow cytometry methods with phospho-specific antibodies that allow analysis of basal STAT activation/phosphorylation also in rare subsets of Treg cells were used. Main results and the role of chance Among women with endometriosis, 5 had evidence of ovarian and deep infiltrating endometriosis (DIE), 5 had only ovarian endometriosis and 4 had only evidence of DIE. Groups were comparable in their clinical characteristics, there was no difference in the median age of women with endometriosis and controls; 31 (range 23-45) vs. 34 (range 27-39). We demonstrated significantly higher levels of phosphorylated STAT5 (pSTAT5) in nTreg from women with ovarian endometriosis compared to controls (p = 0.026). To assess the homeostatic balance between the nTreg and Tcon, we compared pSTAT5 levels between these cell subsets in each patient. STAT5 phosphorylation was significantly higher in Tcon than in nTreg subset (p &amp;lt; 0.001). Considering STAT5 activation in nTreg subset, this was positively correlated with the proportion of nTreg among CD4+T-cells (rs=0.54, p = 0.027) in all women with endometriosis. We also found a significant positive correlation between pSTAT5 levels in Tcon from women with endometriosis and the percentage of Ki-67+ cells in the Tcon subset (rs=0.66, p = 0.005) as well a significant correlation between luteinizing hormone (LH) levels and pSTAT5 in Tcon subset of CD4 cells (rs=0.71, p = 0.02). This implies LH-dependent imbalanced STAT5 signalling that could be responsible for increased Tcon homeostatic proliferation. Limitations, reasons for caution Our sample size is relatively small and the findings should be validated in future prospective studies. Due to the sample size, there is a risk for Type I error, although the results are coherent in indicating disturbance of Treg homeostasis. Wider implications of the findings Our study suggests that altered STAT5 signalisation may be associated with disturbed nTreg/Tcon homeostasis, which seems to be mostly prominent in women with ovarian endometriosis. If confirmed by further studies in the future, targeted treatment options could be developed to supress progress of endometriosis in this group of women. Trial registration number not applicable

P-392 Peripheral blood naïve Treg (nTreg) cell subsets and implantation success in patients undergoing in vitro fertilization (IVF) treatment

Abstract Study question Which populations of circulating Treg cells, based on immunophenotyping with thirteen color flow cytometry and analysis with clustering algorithms play a role in implantation? Summary answer The phenotype of subpopulation within Treg cells that was most significantly increased in women who became pregnant was nTreg-like phenotype, which lacked CXCR5 expression. What is known already There are indications that Treg lymphocytes mediated immuno-regulatory mechanisms contribute to the occurrence of idiopathic infertility. Findings of studies indicate that higher nTreg cell subsets were associated with successful IVF treatment. These findings suggest that changes in the composition of the nTreg subset may allow increased tolerance induction during implantation. More detailed characterization of the Tregs in blood samples from women undergoing IVF/ICSI treatment has not been performed and the degree of heterogeneity of among these cells has not been studied to date. Using 13-color flow cytometry Tregs cells can be divided into multiple complex subsets based on different markers. Study design, size, duration This prospective observational study included 36 patients undergoing IVF/ICSI treatment. Blood samples for 13-color flow cytometry, UMAP and FlowSOM cell clustering analyses were obtained from 19 patients undergoing controlled ovarian hyperstimulation and 17 patients undergoing vitrified-warmed blastocysts transfer (FET) in the natural cycle on the day of optimal blastocysts transfer, for data analysis GraphPad Prism was used. The study was performed between March 2020 and October 2021. Participants/materials, setting, methods In this study peripheral blood Treg cells composition was determined. We analyzed the phenotype of populations over or under-represented in samples from women who became pregnant compared to the women who did not within Treg cells. Treg cells were analyzed by both standard gating flow cytometry analysis and clustered by algorithms according to their level of expression of cell surface markers: CD25 (IL-2Rα), CD127 (IL-7Rα), CCR7, CXCR5, CD28, CD38, CD161, CD31, CD45RA, HLA-DR and CD15s. Main results and the role of chance Thirteen color flow cytometry and dimensionality reduction analyses were carried out, using the UMAP and FlowSOM cell clustering algorithms. Combining these tools, we were able to analyze the phenotype of populations in samples from women who became pregnant ( n = 23) compared to the women who did not (n = 13) within Treg cells. FlowSOM identified 15 phenotypically distinct populations within Treg cells. The most prevalent population that was increased in women who became pregnant was nTreg-like phenotype, which lacked CXCR5 expression. This related to our results by standard gating, where we observed that the percentage of CXCR5-nTreg cells and CD31+RTEnTreg cells within CD25+CD127low Treg cells was significantly higher in pregnant women (p = 0.003; p = 0.0162, respectively). Pregnant females had significantly higher percentage of CD45RA+nTreg cells within CD25hiCD127low Treg cells (p = 0.0162) which was in good correlation with the percentage of CD45RA+FOXP3low nTreg cells within CD25+FOXP3+ Treg cells (p &amp;lt; 0.0001). Furthermore, the percentage of CD45RA+FOXP3+low nTreg cells within CD25+FOXP3+ Treg cells was significantly higher in pregnant women (p = 0.045). Meanwhile, the percentage of CD45RA-FOXP3hi effector (eTreg) Treg cells within CD25+FOXP3 Treg cells was lower in pregnant women (p = 0.0238). Finally, women who achieve a pregnancy had significantly lower percentage of CD45RA-FOXP3hiCD15s+eTreg cells within CD25+FOXP3+ Treg cells (p = 0.0233). Limitations, reasons for caution This study has some limitations. The sample size is too small. Standard gating in flow cytometry has the limitation of analysing only few markers at a time in a flow plot, the levels of expression of the markers are not considered, as an alternative, dimensionality reduction analyses were carried out. Wider implications of the findings Our findings imply that the composition of distinct populations within nTreg cells can influence the women’s chance of becoming pregnant, especially nTreg cells, lacking CXCR5 expression, which distingushes T-follicular regulatory cells. Findings help us to understand the role of nTreg subpopulations in process of implantation in women undergoing IVF/ICSI treatment. Trial registration number not applicable

Abstract 2096: Natural killer cells inhibit CD4+ T cell mediated IL-9 production in head and neck Cancer

Abstract Head and neck squamous cell carcinoma (HNSCC) is the 6th most common cancer in the world, yet 5 year survival remains poor for those with recurrence despite advances in treatment such as immunotherapy. Checkpoint blockade substantially improved survival in ~20% of recurrent and/or metastatic HNSCC patients. However, given the lack of efficacy in most patients, and the known immunosuppressive nature of HNSCC, exploring other potential immune cell therapeutic targets is of interest. Decreased natural killer (NK) as well as increased immunosuppressive CD4+ regulatory T cells (Tregs) in both peripheral blood and the tumor microenvironment are associated with a poor prognosis in HNSCC. We discovered that the anti-diabetes drug, metformin, which is also known to improve immune cell function, not only increases peripheral NK cells in HNSCC patients to the level of healthy controls, but also suppresses IL-9 secretion from NK cells. IL-9 has been implicated as a driving factor in allergic disease, a poor prognosis factor in hematologic malignancies, and an enhancer of T-reg immunosuppressive abilities. However, the function of IL-9 in solid tumors is poorly understood. Interestingly, upon depletion of CD56+ NK and NKT cells from peripheral blood mononuclear cells (PBMCs), there was a significant increase of IL-9 secretion. This effect was negated upon depletion of CD4+ T cells suggesting that NK cells may directly or indirectly inhibit CD4+ T cell IL-9 production. In order to understand potential consequences of IL-9 overproduction, we subjected HNSCC cell lines, which we demonstrated express IL9 receptor, to exogenous IL-9 which resulted in enhanced proliferation. We also found that IL-9 plasma levels are high in HNSCC patients compared to healthy controls corroborating a potential tumorigenic role for IL-9. In order to elucidate potential mechanism(s) by which NK/NKT cells regulate CD4+ T cell production, we performed single-cell RNAseq on PBMCs upon NK and NKT cell depletion. We also performed flow cytometry to identify specific CD4+ cell types responsible for increased IL-9 production and the consequential changes in other immune cell phenotypes upon the absence of NK/NKT cells. Understanding the role of IL-9 in HNSCC will be important for utilizing IL-9 as a potential marker for HNSCC prognosis as well as a possible target for future immunotherapy approaches. Citation Format: McKenzie Crist, Maria Lehn, Vinita Takiar, Trisha Wise-Draper. Natural killer cells inhibit CD4+ T cell mediated IL-9 production in head and neck Cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2096.

First-in-human (FIH) phase I study of the highly selective phosphoinositide 3-kinase inhibitor delta (PI3Kδ) inhibitor IOA-244 in patients with advanced cancer: Safety, activity, pharmacokinetic (PK), and pharmacodynamic (PD) results.

3107 Background: T regulatory (Tregs) cells contribute to immune suppression in cancer. The highly selective inhibitor of PI3Kδ, IOA-244, blocks the activity of Tregs among other things, thus reprograms the anti-tumor immune response. Methods: IOA-244 was investigated in a two-part FIH study. Part A explored the continuous daily dosing of IOA-244 at 10, 20, 40 and 80 mg. Part B consists of expansion cohorts of specific tumor indications, including pre-treated uveal melanoma patients (pts). Primary objective: safety of the anticipated biologically effective dose (BED), or the recommended phase 2 dose (RP2D). Secondary objectives: PK; PD (e.g., inhibition of CD63 expression on basophils, changes in immune cell subsets in peripheral blood); RECIST 1.1.-based responses; PFS and OS. Exploratory studies: changes in circulating immune cells by Cytometry by Time of Flight (CyTOF); response assessments by radiomics Results: Part A Solid Tumor (completed): Sixteen pts were treated in 4 cohorts each with 4 pts. Pts characteristics: uveal melanoma (9/16; 56%), cutaneous melanoma (5/16; 31%) and pleural mesothelioma (2/16; 13%). Four pts had at least one serious TEAE, none considered related to IOA-244. There was no treatment-emergent adverse events (TEAE) leading to study drug discontinuation, immune related toxicity or Dose Limiting Toxicity. CTCAE v5 Grade 1 and 2 were observed, including 2 cases of transient diarrhoea and 2 of AST/ALT elevation. Part A (Completed) – Subgroup Uveal Melanoma Pts (progressed ≥1 line prior therapy): 9 pts treated (3/9 pts ongoing). Mean time on treatment: 7.7 mo (range: 1.8-16.0 mo with 3 pts ongoing). ORR (RECIST 1.1): CR+PR: 0/9 (0%); SD: 6/9 (67%). Median OS: 5.4 mo - not determined (% alive at 1 year: 44% with 3 pts ongoing). CT images from 7/9 patients were assessed for changes in their metastatic lesions by radiomics (baseline and Week 8). Based on 147 matched lesions, 19% had complete responses and 16% had new lesions. In the liver, non-progressive disease was observed in 61% of all lesions, including 42% with either complete response or volume reduction of more than 30%. Using CyTOF, circulating Tregs were reduced while CD8 and NK cells were increased. Part B Uveal Melanoma Expansion Cohort (ongoing): 7 patients (7/7 pts ongoing); mean time on treatment 3.7 mo. ORR (RECIST 1.1): SD in 4/7 pts (57%). Part A Follicular Lymphoma Cohort (ongoing): At 20 mg: 4/4 pts. No DLT. At 80 mg: recruiting. Conclusions: In addition to being well tolerated, IOA-244 at the 80 mg dose shows reduction in peripheral blood Tregs and anti-tumor responses based on radiomics. Therefore, RECIST 1.1.-derived SD may underestimate anti-tumor activity of IOA-244 in treatment-resistant uveal melanoma. Additional patients will be treated to further refine this radiomics-based observation. Clinical trial information: NCT04328844.

Short‐chain fatty acids regulate B cells differentiation via the FFA2 receptor to alleviate rheumatoid arthritis

Short-chain fatty acids (SCFAs) are metabolites from gut microbes involved in the host's inflammatory response and immunity. The aim of this study was to investigate the role of SCFAs in rheumatoid arthritis (RA) and possible mechanisms. Gut microbiota diversity in mice was analysed by 16S rDNA sequencing. SCFAs levels were analysed by gas chromatography mass spectrometry. T and B cells were analysed by flow cytometry. Bone damage was analysed by micro-CT and X-ray. Histopathological status was analysed by HE staining. Proteins in tissues were analysed by immunohistochemistry and PCR. Mice with CD19+ B cells deficient in FFA2 receptors were used to explore the molecular mechanisms involved. Levels of acetate, propionate, butyrate, and valerate were decreased in RA patients, and the first three correlated positively with the frequency of Bregs but not Tregs in peripheral blood. Administration of the three SCFAs prior to the onset of collagen-induced arthritis in mice improved arthritic symptoms, increased the Bregs frequency, and decreased transitional B and follicular B cell frequency. However, the preceding phenomena could not be observed in mice with CD19+ B cells deficient in FFA2 receptors. The effects of the three SCFAs in RA were dependent on FFA2 receptors but were independent of the other five B cell receptors (FFA3 receptor, HCA2 receptor, PPARγ, Olfr-78, and AhR). SCFAs regulate B cells differentiation via FFA2 receptors to alleviate RA. This provides new insights into the treatment of RA from an immunological and microbiological perspective.

Upregulated TIGIT+ and Helios+ regulatory T cell levels in bronchoalveolar lavage fluid of NSCLC patients

BackgroundThe tumour microenvironment reshapes the specific gene expression of regulatory T cells (Tregs). A better definition of Treg subpopulations in the non-small-cell lung cancer (NSCLC) milieu is expected to clarify the identity and functional mode of Tregs and lead to the identification of better therapeutic targets. MethodsA total of 53 peripheral blood (PB) samples from 36 NSCLC patients and 17 control subjects and 42 matched bronchoalveolar lavage fluid (BALF) samples from 31 NSCLC patients and 11 control subjects were obtained to examine the frequencies of Treg subgroups through flow cytometry. Fifteen PB samples from healthy individuals were collected to explore the differential functions of Treg subsets. The PB samples of 5 patients after chemotherapy were obtained to evaluate the effect of chemotherapy on Treg subsets. Serum CYFRA 21-1 levels in NSCLC patients were determined using an electrochemiluminescence immunoassay. ResultsThe proportions of CD4+CD25+FoxP3+ Tregs in both PB and BALF were increased in NSCLC patients compared to controls. In BALF, the TIGIT+, Helios+, and TIGIT+Helios+ Treg subset levels were significantly elevated; the levels of the last two subsets were associated with NSCLC development, while the level of TIGIT-Helios- Tregs was decreased. The proportions of overall Tregs and TIGIT+, Helios+, and Helios+TIGIT+ Tregs were positively correlated with the serum CYFRA 21-1 levels in all patients. Functional differences were observed between Helios+TIGIT+ and Helios-TIGIT- Tregs. After chemotherapy, regardless of the reduction in serum CYFRA 21-1 levels, the proportions of Tregs and Treg subsets did not change. ConclusionsElevated TIGIT+Helios+ and Helios+ Treg levels may play a role in NSCLC tumour progression, and targeting TIGIT and Helios on Tregs may be an effective treatment for NSCLC.

Effects of SOCS1-overexpressing dendritic cells on Th17- and Treg-related cytokines in COPD mice

BackgroundIn this study, we established a chronic obstructive pulmonary disease (COPD) model by stimulating mice with cigarette smoke, and observed the effects of dendritic cells (DCs) overexpressing SOCS1 on Th17, Treg and other related cytokines in peripheral blood, bronchoalveolar lavage fluid and lung tissues of COPD mice.MethodsAfter successfully transfecting DCs with overexpressing SOCS1 (DC-SOCS1), the mice were injected with DC-SOCS1 (1 × 106), DC-SOCS1 (2 × 106) and immature DCs (1 × 106) via tail vein on days 1 and 7 of COPD fumigation modeling. After day 28 of modeling, the peripheral blood, BALF and lung tissue samples were extracted from the mice, and the changes of DCs, Th17 and Treg cells and related cytokines were detected by immunohistochemistry, immunofluorescence, HE staining, flow cytometry and ELISA.ResultsThe results showed that DC-SOCS1 was able to reduce the secretion of pro-inflammatory factors and increase the anti-inflammatory factors in the COPD mice, and the effect of high concentration (2 × 106 DC-SOCS1) was better than low concentration (1 × 106 DC-SOCS1). Moreover, the intervention effect was significant on day 1 compared with day 7. In the mice injected with DC-SOCS1, the expression of CD83, IL-4, Foxp3, and CCR6 was increased on day 1 than those on day 7, while IL-17 and IFN-γ was decreased.ConclusionsIntervention of COPD mice with high concentrations of DCs-SOCS1 reduced pro-inflammatory factor secretion and attenuated the inflammatory response in COPD.Trial registration Not applicable.

DNA Methyltransferase 3b Accelerates the Process of Atherosclerosis.

Background DNA methylation plays a key role in establishing cell type–specific gene expression profiles and patterns in atherosclerosis. The underlying mechanism remains unclear. Previous studies have shown that DNA methyltransferase 3b (DNMT3b) may play an important role in atherosclerosis. This study aimed to establish the regulatory role of DNMT3b in the development of atherosclerosis. Methods We constructed a viral vector carrying Dnmt3b shRNA to transduce ApoE−/− mice. Meanwhile, healthy human peripheral blood Treg cells were treated with inhibitor of DNMT3b (AZA and EGCG) or transduced with DNMT3b shRNA. Results It showed that Dnmt3b silencing attenuated atherosclerosis, including decreased lesion size and macrophage content and increased collagen and smooth muscle cells content in ApoE−/− mice. To further investigate the possible mechanisms, combined with previous studies by our group, we showed that Foxp3-TSDR methylation level was significantly reduced Foxp3 expression and peripheral blood Treg levels were significantly increased by Dnmt3b shRNA vector transduction in animals committed to western diet for 12 and 18 weeks. Consistently, inhibition of DNMT3b (AZA and EGCG) decreased the expression levels of DNMT3b, which can increase the expression levels of FOXP3, and increase the levels of TGF-β and IL-10 and decrease the levels of IL-β and IFN-γ. After transduction with DNMT3b shRNA, the effect was more obvious. Conclusions DNMT3b accelerated atherosclerosis, and may be associated with FOXP3 hypermethylation status in regulatory T cells.