Proportion Of Tregs Research Articles

CD73 mediated host purinergic metabolism in intestine contributes to the therapeutic efficacy of a novel mesenchymal-like endometrial regenerative cells against experimental colitis.

The disruption of intestinal barrier functions and the dysregulation of mucosal immune responses, mediated by aberrant purinergic metabolism, are involved in the pathogenesis of inflammatory bowel diseases (IBD). A novel mesenchymal-like endometrial regenerative cells (ERCs) has demonstrated a significant therapeutic effect on colitis. As a phenotypic marker of ERCs, CD73 has been largely neglected for its immunosuppressive function in regulating purinergic metabolism. Here, we have investigated whether CD73 expression on ERCs is a potential molecular exerting its therapeutic effect against colitis. ERCs either unmodified or with CD73 knockout (CD73-/-ERCs), were intraperitoneally administered to dextran sulfate sodium (DSS)-induced colitis mice. Histopathological analysis, colon barrier function, the proportion of T cells, and maturation of dendritic cells (DCs) were investigated. The immunomodulatory effect of CD73-expressing ERCs was evaluated by co-culture with bone marrow-derived DCs under LPS stimulation. FACS determined DCs maturation. The function of DCs was detected by ELISA and CD4+ cell proliferation assays. Furthermore, the role of the STAT3 pathway in CD73-expressing ERCs-induced DC inhibition was also elucidated. Compared with untreated and CD73-/-ERCs-treated groups, CD73-expressing ERCs effectively attenuated body weight loss, bloody stool, shortening of colon length, and pathological damage characterized by epithelial hyperplasia, goblet cell depletion, the focal loss of crypts and ulceration, and the infiltration of inflammatory cells. Knockout of CD73 impaired ERCs-mediated colon protection. Surprisingly, CD73-expressing ERCs significantly decreased the populations of Th1 and Th17 cells but increased the proportions of Tregs in mouse mesenteric lymph nodes. Furthermore, CD73-expressing ERCs markedly reduced the levels of pro-inflammatory cytokines (IL-6, IL-1β, TNF-α) and increased anti-inflammatory factors (IL-10) levels in the colon. CD73-expressing ERCs inhibited the antigen presentation and stimulatory function of DCs associated with the STAT-3 pathway, which exerted a potent therapeutic effect against colitis. The knockout of CD73 dramatically abrogates the therapeutic ability of ERCs for intestinal barrier dysfunctions and the dysregulation of mucosal immune responses. This study highlights the significance of CD73 mediates purinergic metabolism contributing to the therapeutic effects of human ERCs against colitis in mice.

Dynamic comparison of early immune reactions and immune cell reconstitution after umbilical cord blood transplantation and peripheral blood stem cell transplantation.

Umbilical cord blood transplantation (UCBT) and peripheral blood stem cell transplantation (PBSCT) are effective allogeneic treatments for patients with malignant and non-malignant refractory hematological diseases. However, the differences in the immune cell reconstitution and the immune reactions during initial stages post-transplantation are not well established between UCBT and PBSCT. Therefore, in this study, we analyzed the differences in the immune reactions during the early stages (days 7-100 post-transplantation) such as pre-engraftment syndrome (PES), engraftment syndrome (ES), and acute graft-versus-host disease (aGVHD) and the immune cell reconstitution between the UCBT and the PBSCT group of patients. We enrolled a cohort of patients that underwent UCBT or PBSCT and healthy controls (n=25 each) and evaluated their peripheral blood mononuclear cell (PBMC) samples and plasma cytokine (IL-10 and GM-CSF) levels using flow cytometry and ELISA, respectively. Our results showed that the incidences of early immune reactions such as PES, ES, and aGVHD were significantly higher in the UCBT group compared to the PBSCT group. Furthermore, in comparison with the PBSCT group, the UCBT group showed higher proportion and numbers of naïve CD4+ T cells, lower proportion and numbers of Tregs, higher proportion of CD8+ T cells with increased activity, and higher proportion of mature CD56dim CD16+ NK cells during the early stages post-transplantation. Moreover, the plasma levels of GM-CSF were significantly higher in the UCBT group compared to the PBSCT group in the third week after transplantation. Overall, our findings demonstrated significant differences in the post-transplantation immune cell reconstitution between the UCBT and the PBSCT group of patients. These characteristics were associated with significant differences between the UCBT and the PBSCT groups regarding the incidences of immune reactions during the early stages post transplantation.

Tumor immunosuppression relief via acidity modulation combined PD-L1 siRNA for enhanced immunotherapy

The efficacy of immune checkpoint therapy is limited by the immunosuppressive tumor microenvironment (TME), and lactate, the most universal component of TME, has been rediscovered that plays important roles in the regulation of metabolic pathways, angiogenesis, and immunosuppression. Here, a therapeutic strategy of acidity modulation combined with programmed death ligand-1 (PD-L1) siRNA (siPD-L1) is proposed to synergistically enhance tumor immunotherapy. The lactate oxidase (LOx) is encapsulated into the hollow Prussian blue (HPB) nanoparticles (NPs) prepared by hydrochloric acid etching followed by the modification with polyethyleneimine (PEI) and polyethylene glycol (PEG) via sulfur bonds (HPB-S-PP@LOx), siPD-L1 is loaded via electrostatic adsorption to obtain HPB-S-PP@LOx/siPD-L1. The obtained co-delivery NPs can accumulate in tumor tissue with stable systemic circulation, and simultaneous release of LOx and siPD-L1 in intracellular high glutathione (GSH) environment after uptake by tumor cells without being destroyed by lysosome. Moreover, LOx can catalyze the decomposition of lactate in the hypoxic tumor tissue with the aid of oxygen release by the HPB-S-PP nano-vector. The results show that the acidic TME regulation via lactate consumption can improve the immunosuppressive TME, including revitalizing the exhausted CD8+ T cells and decreasing the proportion of immunosuppressive Tregs, and synergistically elevating the therapeutic effect of PD1/PD-L1 blockade therapy via siPD-L1. This work provides a novel insight for tumor immunotherapy and explores a promising therapy for triple-negative breast cancer.

Abstract 4463: Prognostic significance of systemic inflammatory markers in recurrent or metastatic head and neck cancer patients treated with nivolumab

Abstract Treatment strategies for head and neck cancer has been improved with the contribution of immunotherapy. In spite of the fact, the mechanisms underlying the response to immune checkpoint inhibitors (ICIs) remain unclear, and discovery of the biomarker predicting therapeutic efficacy of ICIs is urgently required. Our previous study indicated that in patients with recurrent or metastatic head and neck squamous cell carcinoma treated with nivolumab, circulating T cells showed dynamic alterations depending on treatment efficacy using flow cytometry and mass cytometry. However, these analyses are somewhat complicated to apply each patient in clinical setting. In this study, we focused on systemic inflammatory markers, including inflammatory makers such as blood cell fractions and C-reactive protein(CRP) in peripheral blood. Firstly, we assessed the pretreatment systemic inflammatory markers in 61 recurrent or metastatic head and neck cancer patients treated with nivolumab, and determined whether these markers were associated with treatment responses by Kaplan-Meier method, multivariate analysis, regression analysis and classification and regression trees(CART). Next, we investigated circulating T cell subsets in 36 recurrent or metastatic head and neck cancer patients using flow cytometry. High neutrophil to lymphocyte ratio(NLR), platelet to lymphocyte ratio(PLR), and systemic immune-inflammation index(SII) significantly associated with poor prognosis in overall survival (NLR: p=0.0047, PLR: p=0.0357, SII: p=0.0011). Furthermore, high albumin, lymphocyte to monocyte ratio(LMR), and prognostic nutritional index(PNI) were significant good prognostic markers of overall survival (albumin: p=0.0037, LMR: p=0.0088, PNI: p=0.037). Overall survival time were estimated the values that were defined systemic inflammatory markers by multivariate analysis. These systemic inflammatory markers were assigned importance by each coefficient. Notably, the number of monocyte and lymphocyte have a strong influence on overall survival time. In addition, the index that were depending on the number of white blood cells and monocyte, the percentage of lymphocyte, platelet count, albumin, and PNI were useful prognostic tools in analyzing regression analysis. Furthermore, the evaluation of PNI were contributed to the predict treatment efficacy in analyzing CART. Moreover, NLR, PLR, and SII were positively correlated with the proportion of Treg (NLR: R2=0.3622/p=0.0001, PLR: R2=0.4198/p<0.0001, SII: R2=0.3654/p<0.0001). We concluded that in recurrent or metastatic head and neck cancer patients treated with nivolumab, systemic inflammatory markers could provide new insight into rational therapeutic strategies in cancer immunotherapy for head and neck cancer. Citation Format: Hiroe Tada, Kazuaki Chikamatsu. Prognostic significance of systemic inflammatory markers in recurrent or metastatic head and neck cancer patients treated with nivolumab. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4463.

Mesenchymal Stem Cells Attenuate Sepsis-associated Acute Kidney Injury by Changing the Balance of Th17 cells/Tregs via Gal-9/Tim-3.

The aim of the present study was to investigate the protective effect of MSCs on CLP-induced SA-AKI and determine the mechanisms of this effect. The expression of Gal-9 and Tim-3 was assayed by qPCR and western blot. IL-10, IL-17, RORγt, and FOXP3 were assayed by qPCR and TNFα, INFγ, IL-4, and IL-6 were assayed by ELISA in renal samples after CLP with or without MSCs treatment. The expression of Gal-9 in MSCs was knocked down in vivo using RNA interference, and si-Gal-9-MSCs were injected in SA-AKI mice. The effect of MSCs on the differentiation of lymphocytes into Th17 cells and Tregs was evaluated in vitro by FAC in coculture of MSCs and CD4+ T cells and after blockade of the Gal-9/Tim-3 pathway. MSCs decreased serum creatinine and urea nitrogen levels and relieved tubular injury. Additionally, MSCs significantly improved the survival rate and markedly attenuated the infiltration of neutrophils and the levels of TNF-α, IFN-γ, IL-4, and IL-6 in the kidneys of septic mice (P < 0.05). Treatment with MSCs also reduced the proportion of Th17 cells and the levels of IL-17 and RORγt (P < 0.05). In contrast, MSCs increased the proportion of Tregs and the levels of IL-10 and FOXP3 related to these cells (P < 0.05). Furthermore, we determined whether Gal-9/Tim-3 and Th17 cells/Tregs are involved in the protective effects of MSCs in an SA-AKI model. The results of Western blot and real-time PCR indicated that MSCs inhibited the expression of Tim-3 and increased the expression of gal-9 (P < 0.05). Knockdown of gal-9 in MSCs using small interfering RNA blunted the therapeutic effect of MSCs, and blockade of the Gal-9/Tim-3 pathway using α-lactose or anti-Tim-3 inhibited the induction of Tregs and suppressed the inhibition of the differentiation to Th17 cells by MSCs after coculture of MSCs with CD4+ T cells (P > 0.05). Treatment with MSCs can protect against SA-AKI. The results suggested that the relieving effect of MSCs against SA-AKI may be partially mediated by the induction of Tregs and inhibition of Th17 cells via the Gal-9/Tim-3 pathway.

Research Highlights.

Research Highlights.

The significance of Th1,Th2,Th17and treg cells in the prediction and evaluation of ulcerative colitis

Objective This study aimed to investigate clinical significance of Th1, Th2, Th17 and Tregs proportions in predicting and evaluating UC. Methods A total of 101 UC patients diagnosed by the Department of Gastroenterology of the Shanxi Provincial People’s Hospital were recruited. This is a retrospective study. The proportions of Th1, Th2, Th17 and Tregs in the peripheral blood were detected by flow cytometry. Results The proportions of Th1, Th2 and Th17 cell in UC patients were higher than healthy controls ( p &lt; 0.001); The area under the curve (AUC) values of Th1, Th1/Treg and Th17/Treg were all &gt;0.900 in predicting UC ( p &lt; 0.001), with the cut off values being 15.25%, 4.885 and 0.425, respectively. In addition, Th1, Th17, Treg, Th17/Treg, Th2/Treg, Th1/Treg and Th17/Treg were statistically significant among the mild to severe group ( p &lt; 0.05). The percentage of Treg cells was negatively correlated with Mayo Score, while the percentages of Th17 cell, Th17/Treg, Th1/Treg, Th2/Treg were positively correlated with Mayo score ( p &lt; 0.05). Notably, Th17/Treg was closely related to Mayo score (r = 0.513, p &lt; 0.001). Conclusions The dysregulation of Th1, Th2, Th17 and Tregs is a significant phenomena of immune disorder in UC, and these auxiliary indicators correlate with increased disease severity. The analysis of Th1, Th2, Th17 and Tregs possesses certain clinical significance in the prediction and evaluation of UC.

Expansion and characterization of regulatory T cell populations from Korean kidney transplant recipients.

The development of immunosuppressants has enabled remarkable progress in kidney transplantation (KT). However, current immunosuppressants cannot induce immune tolerance, and their nonspecific immunosuppressive effects result in many adverse effects. Regulatory T cells (Tregs) play crucial roles in controlling all specific immune responses. This study evaluated the distribution of Tregs and their effects on kidney allograft function in Korean KT recipients. We enrolled 113 KT recipients with stable graft function. The differentiation and expansion of Tregs were examined by flow cytometry to compare the Tregs subpopulations. Among the 113 patients, 73 (64.6%) were males, and the mean follow-up period from KT to Tregs collection was 147.5 + 111.3 months. Patients receiving lower doses of cyclosporine had higher proportions of Tregs than those with higher doses of cyclosporine (36.3 + 21.6 vs 17.0 + 12.7, P = .010, respectively). Patients taking cyclosporine tended to have higher Tregs numbers than those taking tacrolimus (94.7 + 158.1 vs 49.3 + 69.4, P = .095, respectively). However, no significant association was observed between Tregs and allograft dysfunction in the cox proportional hazard model. Tregs counts may be associated with the type and dose of immunosuppressants. However, no significant relationship was found between Tregs and kidney allograft function in stable KT recipients.

BATF sustains homeostasis and functionality of bone marrow Treg cells to preserve homeostatic regulation of hematopoiesis and development of B cells.

Bone marrow Treg cells (BM Tregs) orchestrate stem cell niches crucial for hematopoiesis. Yet little is known about the molecular mechanisms governing BM Treg homeostasis and function. Here we report that the transcription factor BATF maintains homeostasis and functionality of BM Tregs to facilitate homeostatic regulation of hematopoiesis and B cell development. Treg-specific ablation of BATF profoundly compromised proportions of BM Tregs associated with reduced expression of Treg effector molecules, including CD44, ICOS, KLRG1, and TIGIT. Moreover, BATF deficiency in Tregs led to increased numbers of hematopoietic stem cells (HSCs), multipotent progenitors (MPPs), and granulocyte-macrophage progenitors (GMPs), while reducing the functionality of myeloid progenitors and the generation of common lymphoid progenitors. Furthermore, Tregs lacking BATF failed to support the development of B cells in the BM. Mechanistically, BATF mediated IL-7 signaling to promote expression of effector molecules on BM Tregs and their homeostasis. Our studies reveal a previously unappreciated role for BATF in sustaining BM Treg homeostasis and function to ensure hematopoiesis.

Akkermansia muciniphila and Bifidobacterium bifidum Prevent NAFLD by Regulating FXR Expression and Gut Microbiota.

Non-alcoholic fatty liver disease (NAFLD) is closely associated with gut microbiota and has become the most common chronic liver disease worldwide, but the relationship between specific strains and NAFLD has not been fully elucidated. We aimed to investigate whether Akkermansia muciniphila and Bifidobacterium bifidum could prevent NAFLD, the effects of their action alone or in combination, possible mechanisms, and modulation of the gut microbiota. Mice were fed with high-fat diets (HFD) for 20 weeks, in which experimental groups were pretreated with quadruple antibiotics and then given the corresponding bacterial solution or PBS. The expression of the glycolipid metabolism indicators, liver, and intestinal farnesol X receptors (FXR), and intestinal mucosal tight junction proteins were detected. We also analyzed the alterations of inflammatory and immune status and the gut microbiota of mice. Both strains were able to attenuate mass gain (p<0.001), insulin resistance (p<0.001), and liver lipid deposition (p<0.001). They also reduced the levels of the pro-inflammatory factors (p<0.05) and the proportion of Th17 (p<0.001), while elevating the proportion of Treg (p<0.01). Both strains activated hepatic FXR while suppressing intestinal FXR (p<0.05), and elevating tight junction protein expression (p<0.05). We also perceived changes in the gut microbiota and found both strains were able to synergize beneficial microbiota to function. Administration of A. muciniphila or B. bifidum alone or in combination was protective against HFD-induced NAFLD formation and could be used as alternative treatment strategy for NAFLD after further exploration.

Regulatory T cell enhancement in adults with cystic fibrosis receiving Elexacaftor/Tezacaftor/Ivacaftor therapy.

Cystic fibrosis (CF), especially CF lung disease, is characterized by chronic infection, immune dysfunction including impairment of regulatory T cells (Tregs) and an exaggerated inflammatory response. CF transmembrane conductance regulator (CFTR) modulators have shown to improve clinical outcomes in people with CF (PwCF) with a wide range of CFTR mutations. However, it remains unclear whether CFTR modulator therapy also affects CF-associated inflammation. We aimed to examine the effect of elexacaftor/tezacaftor/ivacaftor therapy on lymphocyte subsets and systemic cytokines in PwCF. Peripheral blood mononuclear cells and plasma were collected before and at three and six months after the initiation of elexacaftor/tezacaftor/ivacaftor therapy; lymphocyte subsets and systemic cytokines were determined using flow cytometry. Elexacaftor/tezacaftor/ivacaftor treatment was initiated in 77 PwCF and improved percent predicted FEV1 by 12.5 points (p<0.001) at 3 months. During elexacaftor/tezacaftor/ivacaftor therapy, percentages of Tregs were enhanced (+18.7%, p<0.001), with an increased proportion of Tregs expressing CD39 as a marker of stability (+14.4%, p<0.001). Treg enhancement was more pronounced in PwCF clearing Pseudomonas aeruginosa infection. Only minor, non-significant shifts were observed among Th1-, Th2- and Th17-expressing effector T helper cells. These results were stable at 3- and 6-month follow-up. Cytokine measurements showed a significant decrease in interleukin-6 levels during treatment with elexacaftor/tezacaftor/ivacaftor (-50.2%, p<0.001). Treatment with elexacaftor/tezacaftor/ivacaftor was associated with an increased percentage of Tregs, especially in PwCF clearing Pseudomonas aeruginosa infection. Targeting Treg homeostasis is a therapeutic option for PwCF with persistent Treg impairment.

Inhibition of phosphoglycerate kinase 1 attenuates autoimmune myocarditis by reprogramming CD4+ T cell metabolism.

CD4+ T cells are the major drivers of cardiac-specific autoimmunity in myocarditis, specifically Th1, Treg, and most significant Th17 cells. But the molecular mechanisms of their activation remain unclear. We aimed to elucidate the regulatory role of phosphoglycerate kinase 1 (PGK1) in CD4+ T cells and experimental autoimmune myocarditis (EAM). EAM was induced in BALB/c mice by subcutaneous injections with alpha myosin heavy chain peptide emulsified in complete Freund's adjuvant. Single-cell sequencing analysis found that glycolysis and PGK1 expression were elevated in cardiac CD4+ T and Th17 cells from myocarditis mice. Mice treated with PGK1 inhibitor NG52 showed less cardiac inflammation and fibrosis and better contractile function, as well as reduced cardiac infiltrating Th17 and Th1 cells and increased proportion of Treg. NG52 suppressed CD4+ T cell activation and differentiation of mice and myocarditis patients in vitro. Mechanistically, inhibition of PGK1 suppressed glycolytic activity and decreased pyruvate dehydrogenase kinase 1 (PDHK1) phosphorylation, thereby increasing reactive oxygen species (ROS) production in mitochondria and thus preventing Th17 cell differentiation. PGK1 may act as a key metabolic regulator of CD4+ T cell differentiation and regulates Th17 cell differentiation by regulating glycolysis and the PDHK1-ROS axis. Targeting PGK1 might be a promising strategy for the treatment of myocarditis.

ADAR1 improved Treg cell function through the miR-21b/Foxp3 axis and inhibits the progression of acute graft-versus-host disease after allogeneic hematopoietic stem cell transplantation.

Th17/Treg equilibrium towards the pro-inflammatory Th17 side contributes greatly to the rejection during allogeneic hematopoietic stem cell transplantation (allo-HSCT). Forkhead box P3 (Foxp3) is important in the pathogenic conversion between Th17 and Treg cells. However, how Foxp3 expression was regulated is largely unknown. Here, we investigated the role of RNA-editing enzyme ADAR1 in Foxp3-mediated Th17/Treg imbalance and progression of acute graft-versus-host disease (aGVHD), a most serious complication in patients received allo-HSCT. Th1, Th17 and Treg cells were respectively isolated from peripheral blood CD4+T cells of allo-HSCT patients, and we found that proportions of Th1 and Th17 were markedly increased, while Treg proportion was significantly decreased in aGVHD patients post transplantation compared with non-aGVHD patients, accompanied by decreased ADAR1 and increased miR-21b levels. RNA-immunoprecipitation (RIP) combined with gain- and loss-of-function experiments demonstrated that ADAR1 improved Treg cell functions and negatively regulated the production of miR-21b, a Foxp3-targeting miRNA. Inhibition of miR-21b improved Treg functions, and Foxp3 knockdown could eliminate the effect of miR-21b inhibition or ADAR1 overexpression on Treg function. Finally, an aGVHD mouse model was established and Ad-O/E-ADAR1 was injected into aGVHD mice to verify the effect of ADAR1 on aGVHD progression in vivo. The results showed that ADAR1 overexpression decreased Th17 proportion and increased Treg proportion in aGVHD mice and obviously improved tissue necrosis and reticular structure of aGVHD liver and lung in vivo. Collectively, ADAR1 suppresses miR-21b production and improves Foxp3-mediated Treg cell function to inhibit the progression of aGVHD after allo-HSCT.

Use of high-dimensional and spatial immune profiling to explore sotigalimab (CD40 agonist) activation of antigen presenting cells and T cells in the tumor microenvironment in patients with esophageal/gastroesophageal junction cancer.

450 Background: Neoadjuvant chemoradiation (CRT) followed by surgical resection is standard of care for patients with locally advanced esophageal/gastroesophageal junction (E/GEJ) cancer. A pathologic complete response (pCR) at surgery is associated with improved survival outcomes. Sotigalimab (sotiga) is a potent CD40 agonist mAb capable of inducing and expanding anti-tumor immune responses. A recently reported non-randomized phase II clinical trial of sotiga combined with CRT in E/GEJ cancer patients showed promising pCR rates that compared favorably to historical data1. Here, deep immune profiling was performed on a subset of patients to gain insight into the mechanism of sotiga. Methods: In the initial safety cohort (n=6) of this phase II E/GEJ clinical trial, tumor and blood samples were obtained pre-treatment and again following a single lead-in dose of sotiga, prior to initiation of CRT. Immune modulation in the tumor microenvironment and blood of patients was examined using high dimensional techniques, including mass cytometry, multiplexed ion beam imaging, and single cell RNA sequencing. Results: Sotiga administration dramatically re-models the tumor microenvironment, inducing immune infiltration and shifting the immune composition predominantly toward an inflammatory phenotype. At baseline, E/GEJ tumors were infiltrated with myeloid cells and T cells, the majority of which were Tregs. Sotiga increased antigen-presenting cell (APC) infiltration in tumors and activated dendritic cells as demonstrated by expression of MHCII and CD86. Sotiga treatment also induced infiltration of activated cytotoxic T cells and decreased the proportion of Tregs. There was also evidence of T cell activation in peripheral blood with increased expression of 4-1BB, CTLA-4, ICOS and PD-L1. Therapeutic responses to sotiga were associated with distinct T cell signatures both at baseline and on-treatment. At baseline, pCR patients (representing 4 of the 6 pts (67%) in this cohort) had a higher ratio of memory CD4+ T cells to Tregs which further increased following sotiga treatment, whereas patients who did not have a pCR had a higher proportion of myeloid cells at baseline and did not show a decrease in Treg cells after sotiga treatment. Conclusions: Sotiga induced dramatic changes in the tumor microenvironment including increased frequency of activated T cells and APCs, and decreased frequency of Tregs. A distinct signature of T cell infiltration in baseline tumor biopsies was observed in patients who achieved a pCR versus those who did not, potentially identifying patients that may benefit from this novel treatment strategy.

Biological effects of prostaglandin E2-EP4 antagonist (AAT-008) in murine colon cancer in vivo: enhancement of immune response to radiotherapy and potential as a radiosensitizer.

Prostaglandin E2 (PGE2) promotes tumor growth and metastasis by acting on a family of four receptors (EP1-4). We investigated the radiosensitizing effects of a newly developed antagonist of PGE2-EP4 (AAT-008) in mouse colon cancer cells in vivo and explored the mechanism using flow cytometry (FCM). CT26WT cells grown in Balb/c mice were used. AAT-008 at doses of 0, 3, 10, and 30 mg/kg/day was orally administered once or twice daily for up to 19 days. On day 3, the tumors were irradiated at 9 Gy in the radiotherapy (RT) group. Tumor sizes were measured every other day. For the first FCM series, AAT-008 (10 mg/kg/day) was administered from day 0 to 18 and RT (9 Gy) was given on day 3. The population of effector T cells (Teff), defined as CD45+CD8+CD69+, in the tumors was investigated on day 19. For the second FCM series, AAT-008 (30 mg/kg/day) was administered from day 0 to 12. The populations of Teff and regulatory T cells (Treg), and the ratio of Teff/Treg were investigated on day 13. The growth delay effect of AAT-008 administered alone (3-30 mg/kg/day) appeared minimal. In the first growth delay experiment where AAT-008 was administered once daily, the combined effect of AAT-008 (30 mg/kg/day) and RT appeared additive. In the second growth delay experiment where AAT-008 was administered twice daily, the combined effect appeared additive at 3 and 10 mg/kg/day and supra-additive at 30 mg/kg/day. In the first FCM series, the mean Teff proportions in the tumors were 43% and 31% in the 10 mg + RT and 0 mg + RT groups, respectively. Notably, 67% Teff was observed in responsive mice in the 10 mg + RT group. In the second FCM series, the mean Treg proportion and Teff/Treg ratio in the 0 mg + RT and 30 mg + RT groups were 4.0% and 1.5%, respectively (P=0.04) and 10 and 22, respectively (P=0.04). AAT-008 potentially enhances the radiosensitivity of colon cancer cells, apparently by stimulating the immune system against the cancer cells.

Acupuncture regulates the Th17/Treg balance and improves cognitive deficits in a rat model of vascular dementia

ObjectiveThe present study was developed to explore the impact of acupuncture on the Th17/Treg balance in the brain and the periphery and associated changes in cognitive deficits in a rat model of vascular dementia (VD). MethodsMale Wistar rats (8 weeks old) were randomly assigned to sham-operated (Gs, n = 10), and operation (n = 30) groups. A VD model was established for all rats in the operation group via the permanent bilateral occlusion of the common carotid artery. Behavioral screening of these rats was conducted via a hidden platform trial at 2 months post-operation. These operation group rats were then further subdivided into impaired (Gi) and acupuncture (Ga) groups (n = 10/group). Acupuncture was performed over a 21-day period for rats in the Ga group. A Morris water maze (MWM) test was used to assess cognitive function for rats in all groups. Flow cytometry and fluorescent staining were used to detect Th17 and Treg cells in samples from these animals based on IL-17/FoxP3 or CD4+FoxP3+/CD4+RORγt+ staining profiles. ResultsRelative to the Gs group, escape latency values for rats in the Gi group were significantly increased. Following treatment, rats in the Ga group exhibited significant reductions in escape latency values as compared to rats in the Gi group (P < 0.05). The relative Treg proportion in the peripheral blood and spleen additionally trended upwards in these Ga rats as compared to those in the Gi group (P > 0.05), whereas the frequency of Th17 cells in the peripheral blood and spleen of Ga group rats trended downward relative to the Gi group (P > 0.05). Significantly fewer CD4+RORγt+ and RORγt+ cells were detected in the Ga group relative to the Gi group, whereas CD4+FoxP3+ and FoxP3+ cell counts were increased (P < 0.01). ConclusionIn summary, VD model rats exhibited dysregulated Th17/Treg homeostasis. Acupuncture treatment was sufficient to reduce the frequency and numbers of Th17 cells in these animals while increasing Treg cell levels, thereby alleviating cognitive deficits with respect to both spatial learning and memory impairment. Consequently, the therapeutic benefits of such acupuncture treatment may be attributable to the regulation of the Th17/Treg balance and associated improvements in cognitive function.

Urinary T Cells Identify Renal Antineutrophil Cytoplasmic Antibody-Associated Vasculitis and Predict Prognosis: A Proof of Concept Study

Necrotizing crescentic glomerulonephritis is a major contributor to morbidity and mortality in Antineutrophil cytoplasmic antibodies (ANCA)-associated vasculitis (AAV). Because therapy relies on immunosuppressive agents with potentially severe adverse effects, a reliable noninvasive biomarker of disease activity is needed to guide treatment. We used flow cytometry to quantify T cell subsets in blood and urine samples from 95 patients with AAV and 8 controls to evaluate their biomarker characteristics. These were compared to soluble markers, monocyte chemoattractant protein-1 (MCP-1), soluble CD163 (sCD163), soluble CD25 (sCD25), and complement C5a (C5a), measured using multiplex analysis. Available kidney biopsies (n= 21) were classified according to Berden. Patients with active renal AAV (rAAV) showed significantly higher urinary cell counts than those in remission, or those with extrarenal manifestation, or healthy controls. Urinary T cells showed robust discrimination of disease activity with superior performance compared to MCP-1 and sCD163. Patients whose kidney biopsies had been classified as "crescentic" according to Berden classification showed higher urinary T cell counts. Discordant regulatory T cells (Treg) proportions and CD4+/CD8+ ratio in blood and urine suggested that urinary cells reflect tissue migration rather than mere micro-bleeding. Furthermore, urinary Treg and T helper cells (TH17) patterns were associated with clinical response and risk of renal relapse. Urinary T cells reflect the renal inflammatory milieu in AAV and provide further insights into the pathogenesis of this chronic condition. Their promising potential as noninvasive diagnostic and prognostic biomarkers deserves further exploitation.

Bioinformatic analysis identifies the immunological profile of turner syndrome with different X chromosome origins.

Turner syndrome (TS) is a chromosomal disorder that affects phenotypic females who have one intact X chromosome and complete or partial absence of the second sex chromosome in association with one or more clinical manifestations. However, the immunological profile of TS with different X chromosome origins is incompletely understood. In this study, transcriptomic expression profiles of 26 TS (45,X) samples and 10 normal karyotype (46,XX) samples derived from GSE46687 cohort were employed. Differentially expressed immune-related genes (DEIRGs) between monosomy X TS patients with different X chromosome origins and normal females were investigated respectively. Subsequently, functional annotation, protein-protein interaction (PPI) network analysis, immunocyte infiltration evaluation, tissue-specific gene expression and Weighted gene co expression network analysis (WGCNA) were performed to explore the immunological characteristic in TS with different X chromosome origins. 34 and 52 DEIRGs were respectively identified in 45,Xm and 45,Xp patients compared with normal individuals. The identified DEIRGs in Xm group were significantly enriched in pathways associated with cancer. In Xp TS patients, the most enriched signals were immune response-related. A majority of genes involved in the above pathways were downregulated. PPI analysis identified 4 (FLT3, IL3RA, CSF2RA, PIK3R3) and 6 (PDGFRB, CSF2, IL5, PRL, CCL17 and IL2)hub genes for Xm and Xp groups, respectively. CIBERSORT results showed that the proportion of Tregs in the Xm group and the naive B cells and resting NK cells in the Xp group significantly increased, respectively. Tissue-specific expression results indicated that BDCA4+_dentritic cells and CD19+ B cells were the prominent specific expressed tissues in Xp patients. Results of WGCNA support the above analysis. This study aims at studying the immunological characteristics of TS with different X chromosome origins. Pathways in cancer in Xm group and immune response in Xp group were suppressed. 4 and 6 hub IRGs were identified as biomarkers for Xm and Xp patients, respectively. B cells played important roles in Xp patients. Further studies are needed to draw more attention to the functional validation of these hub genes and the roles of B cells.

Abstract B040: Leader cells mediate tumor engraftment and promote immunosuppression to drive ovarian cancer progression in vivo

Abstract Epithelial ovarian cancer (EOC) is most frequently diagnosed at an advanced stage, and is characterized by its high recurrence rate and almost-universal acquisition of chemoresistance. Metastasis of EOC is driven by heterogenous spheroids that passively disseminate throughout the peritoneal cavity, promoting tumor spread and recurrence. We previously identified a sub-population of highly motile and invasive “Leader cells” (LCs) that express the basal epithelial protein KRT14 as an absolute determinant of invasive potential. We demonstrated KRT14 knockout in EOC cells does not impact viability or proliferation, however completely ablates their ability to invade through Matrigel in 2D and 3D format in vitro. Although a role for LCs in mesothelial clearance and invasion has been established, these mechanisms remain poorly understood. Here we show a critical role for KRT14+ LCs in immunosuppression and tumor-immune privilege in vivo. In the ID8 syngeneic EOC mouse model, tumor-specific LC (KRT14) loss (LCKO) impaired ovarian intrabursal primary tumor engraftment and subsequent metastatic spread. Immune phenotyping by flow cytometry revealed that mice bearing LCKO tumors displayed decreased proportions of immunosuppressive tumor-adjacent Tregs and M2 macrophages, and improved CD8+ T-cell/Treg ratios. Conversely, forced LC-overexpression (LCOE) in EOC tumors accelerated metastasis, and promoted tumor-immune evasion via upregulated secretion of suppressive chemokines including CCL22 and CCL5. In addition to their critical role in migration and invasion, the current study provides in vivo evidence that leader cells are mediators of immune suppression in ovarian cancer. This work supports the basis that defining novel strategies to target LCs to promote anti-tumor immunity and limit metastatic progression may be a useful strategy for improved epithelial ovarian cancer therapy. Citation Format: Amy L. Wilson, Brittany R. Doran, Bashirah Basri, Laura R. Moffitt, Magdalena Plebanski, Andrew N. Stephens, Maree Bilandzic. Leader cells mediate tumor engraftment and promote immunosuppression to drive ovarian cancer progression in vivo [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr B040.

Effects of lactate on metabolism and differentiation of CD4+T cells

BackgroundLactate accumulation caused by abnormal tumor metabolism can induce the formation of an inhibitory immune microenvironment through a variety of pathways, which is characterized by regulatory T cells (Treg) infiltration and effector T cells (Teff) depletion. Studies have found that the key reason why Treg cells can survive in harsh environments lies in their flexible metabolic mode, which can use lactate in tumor microenvironment (TME) as an alternative energy substance to maintain their inhibitory activity. In addition, lactate could also promote the differentiation of CD4+T cells into Treg, but the mechanism was not completely clear. The purpose of this study was to investigate the possible mechanism by which lactate is utilized by CD4+T cells to influence Th17/Treg ratio. MethodsBasal cytokines (anti-CD3, anti-CD28, TGF-β) and 10 mM lactate was added into Naïve CD4+T cells basal medium for 3 days. After TCR stimulation, Naïve CD4+T converted to CD4+T. Flow cytometry was used to detect the proportion of Treg cells; ELISA was used to detect the activity of LDHA, LDHB and NADH and the amount of α -Ketoglutaric Acid (α-KG) and 2-Hydroxyglutaric Acid (2HG) after lactate entered the cells; Western Blot and RT-PCR were used to detect the protein and gene expression of Foxp3, RORγt, LDHA and LDHB. In the validation experiment, lactate uptake inhibitor AZD3965, LDHA inhibitor GSK2837808A and NADH conversion inhibitor Rotenone were added respectively to observe the differentiation ratio of Treg cells and confirm the key points of metabolism; the degradation of Treg cell transcription factor Foxp3 was interfered with ubiquitination inhibitors to observe whether it co-ubiquitinated with HIF-1α; the expression and activity of LDHA, LDHB and NADH in mitochondria and cytoplasm were detected to confirm cell localization. ResultsWhen basal cytokines (anti-CD3, anti-CD28, TGF-β) stimulated, lactate was added to the culture medium, and CD4+T cells absorbed a large amount of lactate not only through MCT1 (monocarboxylic acid transporter), but also increased the expression of lactate dehydrogenase and accelerated the intracellular metabolism of lactate. LDHB in cytoplasm mainly catalyzed the dehydrogenation of lactate to pyruvate, accompanied by the transformation reaction between NAD+ and NADH. The latter further entered the mitochondria and participates in the tricarboxylic acid cycle metabolism. In addition, lactate could significantly increase the level of LDHA in mitochondria and promote the transformation of α-KG to 2HG, accompanied by the transformation of NADH to NAD+. These metabolic changes eventually led to an increase in the intracellular 2HG/α-KG ratio. Abnormal 2HG increased the proportion of Treg by inhibiting ATP5B-mediated phosphorylation of mTOR and the synthesis of HIF-1α, causing it not be enough to ubiquitinate and degrade with Foxp3. ConclusionsLactate plays an important role in regulating the differentiation of Treg cells, inducing the expression and function of LDHA and promoting the transformation of α-KG to 2HG may be an important mechanism.