Number Of Foxp3 Research Articles

Characterization of pathological features and immune microenvironment in hepatic tuberculosis and pulmonary tuberculosis.

Hepatic tuberculosis (HTB) is rare extrapulmonary tuberculosis that is clinically similar to liver malignancy, making it difficult for correct diagnoses. Pathology is the gold standard for tuberculosis diagnosis. However, there are few reports on the pathological features of HTB. A total of 32 HTB cases were considered and the differences in pathological features and drug resistance were analyzed and compared with those for pulmonary tuberculosis (PTB). Enhanced CT scans showed ring-shaped delayed enhancement during the arterial, venous, and delayed phases. Most HTB cases were single lesions, with the highest incidence in the right lobe, and the average lesion volume was smaller than that of PTB. The frequency of granuloma in pathological changes, the overall share of the lesion area in the HTB group, and the number of foxp3+ cells were significantly higher than in the PTB group. However, no statistically significant differences were observed between the two groups' other pathological features and immune cell numbers. The immune microenvironment of the normal tissues surrounding the lesion was further analyzed. The findings showed that the number of macrophages and foxp3+ cells in the HTB group was significantly higher than in the PTB group. No significant difference in drug resistance was detected between the HTB and PTB groups. In conclusion, there are substantial differences in the characterization of pathological feature and immune microenvironment between HTB and PTB. The frequency of granuloma and subsequent overall share of the lesion area was significantly higher in HTB compared to PTB.

LINC00942 inhibits ferroptosis and induces the immunosuppression of regulatory T cells by recruiting IGF2BP3/SLC7A11 in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is a common malignant tumor with a high recurrence rate and a poor prognosis. Long intergenic nonprotein coding RNA 942 (LINC00942) is reported to be related to ferroptosis and the immune response in HCC and serves as an oncogene in various cancers. This research aimed to explore the contribution of LINC00942 in HCC progression. Functional assays were used to evaluate the functional role of LINC00942 in vitro and in vivo. Mechanistic assays were conducted to assess the association of LINC00942 with insulin-like growth factor 2 mRNA binding protein 3 (IGF2BP3) and solute carrier family 7 member 11 (SLC7A11) and the regulatory pattern of LINC00942 in HCC cells. LINC00942 was found to exhibit upregulation in HCC tissue and cells. LINC00942 facilitated HCC cell proliferation, suppressed ferroptosis, and converted naive CD4+ T cells to inducible Treg (iTreg) cells by regulating SLC7A11. Furthermore, SLC7A11 expression was positively modulated by LINC00942 in HCC cells. IGF2BP3 was a shared RNA-binding protein (RBP) for LINC00942 and SLC7A11. The binding between the SLC7A11 3′ untranslated region and IGF2BP3 was verified, and LINC00942 was found to recruit IGF2BP3 to promote SLC7A11 mRNA stability in an m6A-dependent manner. Moreover, mouse tumor growth and proliferation were inhibited, and the number of FOXP3+CD25+ T cells was increased, while ferroptosis was enhanced after LINC00942 knockdown in vivo. LINC00942 suppresses ferroptosis and induces Treg immunosuppression in HCC by recruiting IGF2BP3 to enhance SLC7A11 mRNA stability, which may provide novel therapeutic targets for HCC.

The Relationship of Prognostic Factors with Regulatory T cells in Langerhans Cell Histiocytosis

Aim: Langerhans cell histiocytosis(LCH), is a clonal disorder characterized by abnormal proliferation of dendritic cells, which has an intense inflammatory microenvironment. There is limited information about contribution of microenvironment to this rare disease. We aimed to compare regulatory T cells in microenvironment and BRAFV600E mutation with prognostic data.
 Material and Methods: Overall, 26 cases were included to the study. The number FOXP3+ regulatory T cell(Treg)and presence of BRAFV600E mutation were assessed according to age, gender, localization, unifocal or multifocal, involvement of organ at risk, and recurrence status in a histochemical manner. 
 Results: The number of adult cases was higher than pediatric cases. Bone was the most common localization, and 81% of cases were unifocal. Risk organ involvement was observed in 3 cases, 2 of which showed recurrence. It was found that the number of FOXP3+ Tregs was higher in adults, those with unifocal localization, and those with bone involvement. In addition, the number of FOXP3+ Tregs was higher in the group with recurrence than those without recurrence. BRAFV600E mutation was higher in children when compared to adults (p=0.003), but, no significant correlation was found when compared with remaining prognostic parameters (p>0.05). 
 Conclusions: Although BRAFV600E mutation is more common in pediatric patients, it can also be seen in adult patients. The number of FOXP3+ Tregs is proportional to CD3+ and CD4+ cells. T cells, which present at varying rates in microenvironment, play an essential role in the pathogenesis of LCH.

Ileocecal involvement in intestinal Behçet's disease and Crohn's disease: comparison of clinicopathological and immunophenotypic features.

Intestinal Behçet's disease (BD) predominantly affects the ileocecal region and is currently diagnosed based on endoscopic features and clinical manifestations. It is difficult to distinguish between intestinal BD and Crohn's disease (CD) due to similar patient populations, gastrointestinal involvement, extraintestinal manifestations, and long-term recurrent course. In this study we aimed to compare the clinicopathological and immunophenotypic features of intestinal BD to CD. The medical and pathological records of 29 cases of intestinal BD and 120 cases of CD diagnosed at Sir Run Run Shaw Hospital were retrospectively analyzed. Immunohistochemistry for CD3, CD20, FOXP3, myeloperoxidase, and quantitative analysis of the infiltrating inflammatory cells was conducted. Intestinal BD with ileocecal ulcer had a higher incidence of abdominal pain and a higher erythrocyte sedimentation rate than CD, while chronic diarrhea was more common in CD. Excessive neutrophils in the mucosal lamina propria, neutrophilic exudate on the ulcer surface, and prominent lymphocytic infiltration in ulcer tissues were statistically more frequent in intestinal BD than in CD. The numbers of FOXP3+ T cells, CD3+ T cells, and CD20+ B cells in biopsy tissue from intestinal BD were significantly higher than CD, but the ratio of FOXP3+ T cells to CD3+ T cells was not statistically different. Besides the typical clinical and endoscopic findings, diagnostic biopsies from the ileocecal region in intestinal BD show some histological and immunophenotypic features that are different from CD, which may be useful in distinguishing these two entities.

Fibroblast growth factor inhibition by molecular-targeted agents mitigates immunosuppressive tissue microenvironment in hepatocellular carcinoma

Background & aimsCombination immunotherapy refers to the use of immune checkpoint inhibitors (ICI) and molecular-targeted agents (MTA), which have recently been approved for the treatment of advanced hepatocellular carcinoma (HCC). Owing to its relatively low antitumor effect (up to 30%), sequential therapy following ICIs treatment is required in patients with HCC. This study aimed to determine the impact of MTAs on the tumor immune microenvironment (TIME).MethodsWe established immune syngeneic orthotopic HCC mouse models using Hep-55.1C and Hep-53.4, and treated them with MTAs (lenvatinib, sorafenib, regorafenib, cabozantinib, and DC101 as anti-vascular endothelial growth factor receptor-2 antibodies, and AZD4547 as a fibroblast growth factor receptor (FGFR)-1/2/3/4 inhibitor) for 2 weeks. Subsequently, alterations in the TIME caused by MTAs were evaluated using immunohistochemistry (antibodies for CD3, CD8, Foxp3, Granzyme B, Arginase-1, NK1.1, F4/80, CD11c, PD-1, and PD-L1). We conducted RNA-seq analysis using lenvatinib- and AZD4547-treated tumors. To confirm the clinical relevance of these findings, we analyzed the transcriptome data of human HCC cells (MHCC-97H) treated with various concentrations of lenvatinib for 24 h using RNA-seq data from the Gene Expression Omnibus database.ResultsThe number of Foxp3- and F4/80-positive cells in the TIME was decreased in many MTAs. Cabozantinib increased the numbers in NK1.1-, Granzyme B, and CD11c-positive cells. Lenvatinib and AZD4547 increased the number of CD8, Granzyme B, and PD-L1-positive cells. Gene ontology enrichment analysis revealed that lipid metabolism-related genes were downregulated by lenvatinib and AZD4547. In total, 161 genes downregulated by FGFR inhibition in rodent models overlapped with those downregulated by lenvatinib in human HCC cells.ConclusionsIn this study, we showed that cabozantinib activated the innate immune system, and lenvatinib and AZD4547, which commonly inhibit FGFR signaling, altered TIME to a hot immune state by downregulating lipid metabolism-related genes. These findings support the therapeutic use of combination immunotherapies.

Crosstalk within peripheral blood mononuclear cells mediates anti-inflammatory effects of n-3 PUFA-rich lipid emulsions in parenteral nutrition

Parenteral nutrition (PN) rich in n-6 and n-3 long-chain fatty acids is used in clinical practice for nourishing patients who are unable to receive adequate nutrition through their digestive systems. In this study, we compare the effect on inflammation of the commonly used lipid emulsions Omegaven (n-3-rich) and Intralipid (n-6-rich) in human peripheral blood mononuclear cells (PBMCs). PBMCs were treated with different doses of n-3-rich Omegaven and n-6-rich Intralipid and the immune cells were characterized by flow cytometry. We show that incubation of PBMCs with n-3-rich Omegaven leads to an increase in expression of CD1d and CD86 in CD14+monocytes. At the same time, an increased number of NKT cells expressing cytotoxic T cell antigen 4 is observed, suggesting immunological synapse formation. Both CD14+monocytes and NKT cells showed an increase in IL-10 production and a reduction in the pro-inflammatory cytokines IFN-γ, TNF-α, and IL-4, which led to an increase in the number of FOXP3+T regulatory cells. In addition, we show that n-3-rich Omegaven reduces the expression of TNFα, IFNγ and IL-4 in CD4+T and CD8+T cells independent of the presented interaction between CD14+monocytes and NKT cells. The described mechanism of n-3 rich lipid emulsions was confirmed in PBMCs from patients with inflammatory bowel disease but not in colorectal cancer patients which seem to lack the interaction between CD14+monocytes and NKT cells. These results show a mechanism for the beneficial effect of the n-3-rich Omegaven in patients with inflammatory conditions but questions its use in patients with cancer. Hence, our results may assist in choosing the best lipid emulsion for patients who require PN.

The Balance of CD8-Positive T Cells and PD-L1 Expression in the Myocardium Predicts Prognosis in Lymphocytic Fulminant Myocarditis

Plain Language SummaryMyocarditis is a heart condition caused by inflammation of the heart muscle. Fulminant myocarditis (FM) is a severe form that can lead to sudden heart failure, shock, or dangerous heart rhythms. Understanding how the immune system is involved in FM is crucial for finding better treatments. In this study, we examined heart tissue from 16 patients with lymphocytic FM to understand the role of specific immune cells (T cells) and molecules (PD-1 and PD-L1) in the disease. We followed the patients for about 5 months to track their outcomes. We found that patients with more CD8+ T cells (a type of immune cell) in their heart tissue had a higher risk of cardiac events such as heart failure. Furthermore, when a specific molecule called PD-L1 was more abundant in heart cells, it indicated a poorer prognosis. This suggests that high levels of CD8+ T cells and PD-L1 in the heart tissue can predict a worse outcome for patients with lymphocytic FM. Understanding these markers may help doctors identify patients at higher risk and tailor treatments to improve their prognosis. However, more research with larger groups of patients is needed to confirm these findings and guide treatment decisions.

Time restricted feeding in diet induced obesity restores diurnal T cell rhythms in the small intestine

Abstract Diet induced obesity (DIO) causes circadian disruption and dysregulation of T cells in the gut. Previous studies have also shown that immune cells, including T cells, have rhythms of migration through tissues. We previously found that time-restricted feeding (TRF) in DIO leads to reduced cardiovascular target organ damage. However, it is unknown how TRF in DIO alters intestinal T cells, specifically anti-inflammatory regulatory T cells (FoxP3) and inflammatory Th17 cells (RORgt). We used a 20-week model of high fat (DIO) with TRF intervention with food restricted to the 12-hour active (dark) phase in weeks 18–20. We assessed T cells in the small intestine via flow cytometry in normal diet (ND), DIO, and DIO+TRF groups at Zeitgeber time (ZT) 3 (light period) and ZT13 (dark period). Interestingly, there is a significant diurnal difference of total immune cells (CD45 +), total T cells (TCRb +), and CD4 +T cells in the small intestine of ND mice, with increases at ZT13 compared to ZT3. No diurnal rhythm in T cells is seen in DIO mice. DIO+TRF restores a significant time-of-day difference in T cellsin the small intestine. We investigated CD4 +T cell transcription factors, RORgt and Foxp3. RORgt expression was suppressed at ZT13 compared to ZT3 in DIO. DIO+TRF led to increased RORgt +CD4 +T cells at ZT13 compared to ZT3. The number of Foxp3 +CD4 +T cell was also increased at ZT13 compared to ZT3 in DIO+TRF. CXCR3, a T cell migration marker, was found to be significantly downregulated on CD4 +T cells at ZT13 compared to ZT3 in DIO+TRF mice but not DIO. These findings indicate that intestinal T cell rhythms are lost in DIO mice and TRF restores the T cell rhythm and suggest that the TRF-induced restoration of T cell rhythms in the gut may lead to reduced target organ damage.

Number of FoxP3+ regulatory T-cells are associated with recurrence in vulvar squamous cell carcinoma.

Surgical management is essential in early-stage vulvar squamous cell carcinoma (SCC), but these surgical procedures often cause significant morbidity. Immunotherapy may be a new treatment option in these patients. FoxP3+ Tregs suppress anti-tumor immune responses. High intratumoral FoxP3+ Treg infiltration has been reported to be associated with poor prognosis in most solid tumors. However, there are also conflicting results. We evaluated FoxP3+ lymphocyte infiltration in vulvar SCC and aimed to determine its relationship with prognosis and clinicopathological parameters. Cases diagnosed with vulvar SCC in our department were retrospectively reviewed. The paraffin block that best reflects the morphology was selected, and immunohistochemical studies were performed in accordance with the manufacturer's instructions. FoxP3+ lymphocyte counts were made in tumoral stroma and within tumoral cell islands separately in hot-spot areas. We found a positive correlation between high FoxP3+ lymphocyte count and good prognostic parameters. There was less recurrence in the group with high FoxP3+ lymphocyte counts in tumoral cell islands. Overall survival was not statistically different between these groups. Less lymphovascular invasion was observed in the group with high lymphocyte count in the tumoral stroma. In vulvar SCC, FoxP3+ Treg infiltration into the tumor stroma and into tumoral cell islands is associated with good prognostic features. In these tumors, stage appeared as the only independent prognostic parameter. Studies to be conducted in larger series may reveal whether Tregs can be targeted in cancer treatment.

Histopathologic and transcriptomic phenotypes of a conditional RANKL transgenic mouse thymus.

Although conventional knockout and transgenic mouse models have significantly advanced our understanding of Receptor Activator of NF-κB Ligand (RANKL) signaling in intra-thymic crosstalk that establishes self-tolerance and later stages of lymphopoiesis, the unique advantages of conditional mouse transgenesis have yet to be explored. A main advantage of conditional transgenesis is the ability to express a transgene in a spatiotemporal restricted manner, enabling the induction (or de-induction) of transgene expression during predetermined stages of embryogenesis or during defined postnatal developmental or physiological states, such as puberty, adulthood, and pregnancy. Here, we describe the K5: RANKL bigenic mouse, in which transgene derived RANKL expression is induced by doxycycline and targeted to cytokeratin 5 positive medullary thymic epithelial cells (mTECs). Short-term doxycycline induction reveals that RANKL transgene expression is significantly induced in the thymic medulla and only in response to doxycycline. Prolonged doxycycline induction in the K5: RANKL bigenic results in a significantly enlarged thymus in which mTECs are hyperproliferative. Flow cytometry showed that there is a marked enrichment of CD4+ and CD8+ single positive thymocytes with a concomitant depletion of CD4+ CD8+ double positives. Furthermore, there is an increase in the number of FOXP3+ T regulatory (Treg) cells and Ulex Europaeus Agglutinin 1+ (UEA1+) mTECs. Transcriptomics revealed that a remarkable array of signals-cytokines, chemokines, growth factors, transcription factors, and morphogens-are governed by RANKL and drive in part the K5: RANKL thymic phenotype. Extended doxycycline administration to 6-weeks results in a K5: RANKL thymus that begins to display distinct histopathological features, such as medullary epithelial hyperplasia, extensive immune cell infiltration, and central tissue necrosis. As there are intense efforts to develop clinical approaches to restore thymic medullary function in the adult to treat immunopathological conditions in which immune cell function is compromised following cancer therapy or toxin exposure, an improved molecular understanding of RANKL's involvement in thymic medulla enlargement will be required. We believe the versatility of the conditional K5: RANKL mouse represents a tractable model system to assist in addressing this requirement as well as many other questions related to RANKL's role in thymic normal physiology and disease processes.

Modification of Regulatory T Cell Epitopes Promotes Effector T Cell Responses to Aspartyl/Asparaginyl β-Hydroxylase.

Cancer is a leading cause of death worldwide. The search for innovative therapeutic approaches is a principal focus of medical research. Vaccine strategies targeting a number of tumor-associated antigens are currently being evaluated. To date, none have garnered significant success. Purportedly, an immunosuppressive tumor microenvironment and the accumulation of regulatory T cells contribute to a lack of tumor vaccine efficacy. Aspartyl/asparaginyl β-hydroxylase (ASPH), a promising therapeutic target, is overexpressed in a variety of malignant tumors but is expressed negligibly in normal tissues. Computer analysis predicted that ASPH expresses four peptide sequences (epitopes) capable of stimulating regulatory T cell activity. The abolition of these putative regulatory T cell epitopes increased the CD4+ and CD8+ effector T cell responses to monocyte-derived dendritic cells pulsed with a modified, epitope-depleted version of ASPH in an ex vivo human lymphoid tissue-equivalent coculture system while simultaneously decreasing the overall number of FoxP3+ regulatory T cells. These findings suggest that the efficacy of all new vaccine candidates would profit from screening and eliminating potential tolerogenic regulatory T cell epitopes.

Local assessment of the immunohistochemical expression of Foxp3+ regulatory T lymphocytes in the different pathological forms associated with bovine paratuberculosis

BackgroundMycobacterium avium subsp. paratuberculosis infected animals show a variety of granulomatous lesions, from focal forms with well-demarcated granulomas restricted to the gut-associated lymphoid tissue (GALT), that are seen in the initial phases or latency stages, to a diffuse granulomatous enteritis, with abundant (multibacillary) or scant (paucibacillary) bacteria, seen in clinical stages. Factors that determine the response to the infection, responsible for the occurrence of the different types of lesion, are still not fully determined. It has been seen that regulatory T cells (Treg) play an important role in various diseases where they act on the limitation of the immunopathology associated with the immune response. In the case of paratuberculosis (PTB) the role of Treg lymphocytes in the immunity against Map is far away to be completely understood; therefore, several studies addressing this subject have appeared recently. The aim of this work was to assess, by immunohistochemical methods, the presence of Foxp3+ T lymphocytes in intestinal samples with different types of lesions seen in cows with PTB.MethodsIntestinal samples of twenty cows showing the different pathological forms of PTB were evaluated: uninfected controls (n = 5), focal lesions (n = 5), diffuse paucibacillary (n = 5) and diffuse multibacillary (n = 5) forms. Foxp3+ lymphocyte distribution was assessed by differential cell count in intestinal lamina propria (LP), gut-associated lymphoid tissue (GALT) and mesenteric lymph node (MLN).ResultsA significant increase in the number of Foxp3+ T cells was observed in infected animals with respect to control group, regardless of the type of lesion. However, when the different categories of lesion were analyzed independently, all individuals with PTB lesions showed an increase in the amount of Foxp3+ T lymphocytes compared to the control group but this increase was only significant in cows with focal lesions and, to a lesser extent, in animals with diffuse paucibacillary forms. The former showed the highest numbers, significantly different from those found in cows with diffuse lesions, where no differences were noted between the two forms. No specific distribution pattern was observed within the granulomatous lesions in any of the groups.ConclusionsThe increase of Foxp3+ T cells in focal forms, that have been associated with latency or resistance to infection, suggest an anti-inflammatory action of these cells at these stages, helping to prevent exacerbation of the inflammatory response, as occurs in diffuse forms, responsible for the appearance of clinical signs.

NFAT Factors Are Dispensable for the Development but Are Critical for the Maintenance of Foxp3+ Regulatory T Cells.

The transcription factors of the nuclear factor of activated T cell (NFAT) family play a crucial role in multiple aspects of T cell function. It has recently been reported that NFATs play an important role in the suppressive function of CD4+CD25+Foxp3+ regulatory T (Treg) cells. In this study, we have investigated the role of NFATs in the thymic development of Treg cells in mice. We show that NFAT factors are dispensable for the development of Foxp3+ Treg cells in the thymus but are critical for the maintenance of both the phenotype and survival of Treg cells in the thymus as well as in peripheral lymphoid organs. Specifically, the homeostasis of CD4+CD25+Foxp3+ but not the CD4+CD25−Foxp3+ fraction is severely perturbed when NFAT signaling is blocked, leading to a strongly reduced Treg population. We underscored this intriguing effect of NFAT on CD4+CD25+Foxp3+ Treg cells to the disruption of survival signals provided by interleukin 2 (IL-2). Accordingly, blocking Treg cell death by abolishing the activity of pro-apoptotic Bcl-2 family member Bim, compensated for the survival defects induced due to a lack of NFAT-IL-2-IL-2R signaling. Inhibition of NFAT activity led to a strong reduction in the number of Foxp3+ Treg cells; however, it did not influence the level of Foxp3 expression on an individual cell basis. In addition, we show a differential effect of IL-2 and IL-7 signaling on Foxp3+ Treg versus CD4+CD25− T cell development, again underlining the dispensability of NFAT signaling in the development, but not in the maintenance of Foxp3+ Treg cells.

Prognostic impact of count of extratumoral lymphatic permeation in lung adenocarcinoma and its relation to theimmune microenvironment.

Extratumoral lymphatic permeation (ly‐ext) has been reported as an independent poor prognostic factor for lung adenocarcinoma, but whether or not the number of ly‐ext foci is associated with prognosis and its relationship to the immune microenvironment is unclear. We counted the number of ly‐ext foci on pathological slides from patients with completely resected lung adenocarcinoma with ly‐ext, and divided them into two groups: a group with a high number of ly‐ext foci (ly‐ext high) and one with a low number of ly‐ext foci (ly‐ext low). Among the patients with ly‐ext, only a high number of ly‐ext foci was an independent poor prognostic factor. The 3‐year recurrence‐free survival (RFS) rate of the ly‐ext high group was significantly lower than that of the ly‐ext low group (14.7% vs. 50.0%, P < 0.01). Then, we analyzed the immune microenvironment of pT1 lung adenocarcinoma with ly‐ext (13 cases of ly‐ext high and 11 cases of ly‐ext low tumor) by immunohistochemistry using antibodies for stem cell markers (aldehyde dehydrogenase 1 A1 and CD44), tumor‐promoting mucin (MUC1), tumor‐infiltrating lymphocytes (CD4, CD8, FOXP3, and CD79a), and tumor‐associated macrophages (CD204). The number of CD8+ TILs within the primary lesion was significantly lower and the number of FOXP3+ TILs within the primary lesion was significantly higher in the ly‐ext high group (P < 0.05 and P < 0.01, respectively). Our results indicated that a high number of ly‐ext foci was an independent poor prognostic factor. Moreover, tumors with high numbers of ly‐ext foci had a more immunosuppressive microenvironment.

The correlation between infiltration of FoxP3+ Tregs, CD66b+ TANs and CD163+ TAMs in colorectal cancer.

IntroductionThe infiltration of immune cells in tumor tissue is affected by the tumor microenvironment. However, the relationship between the infiltration of regulatory T cells (Tregs), tumor-associated neutrophils (TANs) and tumor-associated macrophages (TAMs) in colorectal cancer (CRC) remains unclear.Material and methodsTissue microarray and immunohistochemistry were used to detect the infiltration of FoxP3+ Tregs, CD66b+ TANs and CD163+ TAMs in 249 CRC samples (training cohort) and 243 CRC samples (validation cohort). The relationship between two cells was evaluated by Spearman’s rank correlation coefficient and comparison between two groups was analyzed by Mann-Whitney U test.ResultsThe continuous variable positive cell numbers were non-normally distributed. Spearman correlation analysis showed that CD66b+ TAN level in cancer tissues was negatively related to FoxP3+ Treg level (correlation coefficient: –0.495, p < 0.05) and CD163+ TAM level (correlation coefficient: –0.266, p < 0.05), and FoxP3+ Treg level was positively related to CD163+ TAM level (correlation coefficient: 0.467, p < 0.05) in the training cohort. The numbers of FoxP3+ Tregs were significantly different between low and high CD66b+ TAN level groups (p < 0.001), as well as that of CD66b+ TANs in low and high CD163+ TAM level groups and CD163+ TAMs in different FoxP3+ Treg level groups. The results of the validation cohort were similar to those of the training cohort.ConclusionsThere is a negative correlation between infiltration of CD66b+ TANs and that of FoxP3+ Tregs or CD163+ TAMs, and a positive correlation between infiltration of FoxP3+ Tregs and CD163+ TAMs in CRC tissues.

Correlation between aryl hydrocarbon receptor and IL-17+ and Foxp3+ T-cell infiltration in bladder cancer.

Bladder cancer (BC) is one of the most prevalent cancers around the world and, if not treated well, has high morbidity and mortality. Many studies have indicated that there may be various roles for the aryl hydrocarbon receptor (AHR) in the immune system. The aim of this study was to determine the frequency of Foxp3+ regulatory T (Treg) and T helper 17 cells (Th17) in BC tissue in comparison with controls and determine the relationship between AHR, Foxp3+ Treg and Th17 cells in BC. A total of 40 patients with BC were enrolled in this study. The control group was selected from non-tumoural parts of bladder tissues from the patients who have undergone cystoscopy. The percentage of regulatory T cells (Foxp3+ /CD4+ ) and Th17 (IL-17+ /CD4+ ), as well as AHR+ cells in BC tissues and controls, were determined by immunohistochemistry. The results of this study showed that the number of Foxp3+ Treg and Th17 is significantly higher in bladder tumour tissues in comparison with non-tumoural tissues. Also, the percentage of AHR+ lymphocytes and AHR+ cells was increased significantly in bladder tumour tissues rather than non-tumoural tissues. This study also found a relation between AHR and Foxp3+ /CD4+ T lymphocytes ratio cells in BC. The percentage of Foxp3+ Tregs and AHR+ cells were significantly correlated with the grade and stage of BC. An increase in the percentage of Foxp3+ Treg and Th17 cells may play an important role in tumour immunity; and determining the relationship between AHR and differentiation of Th17/Foxp3+ Treg in BC can lead to a potential cancer therapeutic possibility.

Gamma-ray irradiation modulates PGRMC1 expression and the number of CD56+ and FoxP3+ cells in the tumor microenvironment of endometrial endometrioid adenocarcinoma.

PurposeAlthough the conventional gamma ray brachytherapy has been successful in treating endometrioid endometrial adenocarcinoma (EC), the molecular and cellular mechanisms of this anti-tumorigenic response remain unclear. Therefore, we investigated whether gamma ray irradiation induces changes in the number of FoxP3+ T-regulatory lymphocytes (Tregs), CD56+ natural killer cells (NK), and the expression of progesterone receptor membrane component 1 (PGRMC1) in the tumor microenvironment (TME).Materials and MethodsAccording to the inclusion criteria, 127 cases were selected and grouped into irradiation-treated (Rad+) and control (underwent surgery) groups and analyzed using immunohistochemistry. Predictive prognostic values were analyzed using Mann-Whitney U test, ROC analysis, relative risk, log-rank, Spearman rank tests and multivariate Cox’s regression.ResultsWe observed significant differences (p < 0.001) between the radiation-treated patients and the control groups in FoxP3+ Tregs numbers, CD56+ NK cells and PGRMC1 expression. Gamma ray induced a 3.71- and 3.39-fold increase in the infiltration of FoxP3+ cells, CD56+ NK cells, respectively and 0.0034-fold change in PGRMC1 expression. Univariate and multivariate analyses revealed predictive role of the parameters. In the irradiated patients’ group, inverted correlations between clinical unfavorable outcome, FoxP3+ Tregs and CD56+ NK cells were observed.ConclusionOur results suggest an immune-modulating role, specifically by increasing immune cell infiltration, of gamma radiation in the TME which may potentially be utilized as biomarkers in prognostic values.

P–418 Different immunoregulatory components at the decidua basalis of oocyte donation pregnancies

Abstract Study question Is the number of regulatory T-cells (Tregs) and immunoregulatory cytokines in the decidua basalis of oocyte donation (OD) pregnancies different compared to naturally conceived pregnancies? Summary answer This study suggests that the immunoregulation at the fetal-maternal interface in OD pregnancies with a higher amount of fetal-maternal HLA mismatches appears to be altered. What is known already Tregs and related immunoregulatory cytokines, such as interleukins, transforming growth factor-β, and galectin–1, play a key role in maintaining tolerance at the decidua basalis in human pregnancy. Previous studies observed decreased numbers of decidual Tregs in miscarriage and preeclamptic pregnancies. These complications occur in higher frequencies in OD pregnancies, which are characterized by more fetal-maternal human leukocyte antigen (HLA) mismatches compared with naturally conceived (NC) and non-donor in vitro fertilization (IVF) pregnancies, since the fetus obtains paternal and donor-derived HLA genes. Consequently, the maternal immune system has to cope with greater immunogenetic dissimilarity. Involved immunoregulatory mechanisms however remain poorly understood. Study design, size, duration: This case-control study included 27 OD, 11 IVF, and 16 NC placentas of uncomplicated pregnancies, which were collected after delivery at 37–42 weeks of gestation between 2005 and 2013. Clinical data, maternal peripheral blood and umbilical cord blood were collected. Participants/materials, setting, methods Decidua basalis was dissected from the placentas, and processed to formalin-fixed, paraffin-embedded slices (4 µm). Immunohistochemical staining for FOXP3, interleukin 10, interleukin 6, galectin–1, transforming growth factor-β, and Flt–1 was performed. Semi-quantitative (FOXP3+ Tregs) and computerized analysis (cytokines), using Image-J software, were executed. Maternal peripheral blood and fetal umbilical cord blood were typed for HLA class I and II, using the Sequence Specific Oligonucleotides PCR technique, to calculate the number of fetal-maternal HLA mismatches. Main results and the role of chance All the deciduae basalis of OD, IVF and NC pregnancies showed FOXP3+ Tregs. No significant differences were found when comparing the three groups for the mean number of FOXP3+ Tregs. However, when the amount of fetal-maternal HLA mismatches was related to the percentage of FOXP3+ Tregs, the Tregs were significantly higher in pregnancies with 4–6 HLA class I mismatches (n = 16), than in those with 0–3 HLA class I mismatches (n = 38; p = 0.029). Furthermore, OD pregnancies express less interleukin 10, interleukin 6, galectin–1 and Flt–1 in the decidua basalis compared to NC pregnancies. Moreover, the amount of interleukin 10 was significantly lower with 3–4 fetal-maternal HLA class II mismatches (p = 0.032). Limitations, reasons for caution This study is limited by a small sample size. Moreover, only term placentas were collected. It would be worthwhile investigating immunological alterations in the decidua throughout the whole gestation, since maternal adaptation of the fetal allograft could be more prominent early in pregnancy. Wider implications of the findings: Unravelling the mechanisms of immunomodulation during OD pregnancy, reflected by a high level of fetal-maternal dissimilarity, could help to reach the ultimate goal in transplantation; the induction of donor-specific tolerance. In addition, it might help to understand the development of complications in OD pregnancy. Trial registration number Not applicable

Immunohistochemical evaluation of immune cell infiltration in canine gliomas.

Evasion of the immune response is an integral part of the pathogenesis of glioma. In humans, important mechanisms of immune evasion include recruitment of regulatory T cells (Tregs) and polarization of macrophages toward an M2 phenotype. Canine glioma has a robust immune cell infiltrate that has not been extensively characterized. The purpose of this study was to determine the distribution of immune cells infiltrating spontaneous intracranial canine gliomas. Seventy-three formalin-fixed, paraffin-embedded tumor samples were evaluated using immunohistochemistry for CD3, forkhead box 3 (FOXP3), CD20, Iba1, calprotectin (Mac387), CD163, and indoleamine 2,3-dioxygenase (IDO). Immune cell infiltration was present in all tumors. Low-grade and high-grade gliomas significantly differed in the numbers of FoxP3+ cells, Mac387+ cells, and CD163+ cells (P = .006, .01, and .01, respectively). Considering all tumors, there was a significant increase in tumor area fraction of CD163 compared to Mac387 (P < .0001), and this ratio was greater in high-grade tumors than in low-grade tumors (P = .005). These data warrant further exploration into the roles of macrophage repolarization or Treg interference therapy in canine glioma.

Differential Regulation of T Cells By PI3K Delta Inhibitors in a CLL Murine Model

In the microenvironment of chronic lymphocytic leukemia (CLL), T cells are typically dysfunctional. Idelalisib, a PI3Kδ inhibitor, is approved for R/R CLL but trials in treatment-naïve patients were discontinued due to immune-mediated severe adverse events (SAE). Duvelisib is a PI3Kγ/δ inhibitor also in clinical development with a similarly high incidence of immune-mediated SAEs. By contrast, umbralisib (TGR-1202) is a next-generation PI3Kδ inhibitor associated with lower rates of SAEs in clinical trials, even with long-term followup, which has also demonstrated activity against CK1e. Previously, we demonstrated in normal human T cells that ex vivo treatment with umbralisib relatively preserved Treg number (CD4+ CD25hi CD127lo FoxP3+) and function (Treg co-culture supression assay) compared to treatment with idelalisib or duvelisib. FoxP3 mRNA levels and IL-10 secretion from T cells also remained closer to normal after umbralisib treatment compared to idelalisib or duvelisib.The purpose of the current study was to compare the effects of umbralisib, duvelisib, and idelalisib on T cells in a CLL mouse model and analyze immune-mediated adverse events following oral administration. We hypothesized that umbralisib may preserve the number and function of the regulatory T cell (Treg) population, translating to decreased immune-mediated side effects after treatment. Leukemic euTCL1 splenocytes were adoptively transferred into wildtype mice to induce CLL disease and treated via oral administration with vehicle, umbralisib, idelalisib or duvelisib. Tcon:Treg and Teff:Treg ratios in periphery and spleen measured by flow cytometry were found to be spared in the umbralisib-treated group and decreased on duvelisib and idealisib groups. Expression of functional Treg markers TGFB-1, CD39, CD103, PD-1 and CTLA-4 was also spared in the umbralisib group but affected in the other two. To assess immune-mediated toxicity, GI tract and liver tissues were collected and H&E stained. Pathology analysis was performed and immune-mediated toxicity was scored according to the following parameters: inflammatory cell infiltrate, kuppfer cell hyperplasia, microvesicular steatosis, cell degeneration (liver) and denuded mucosa, chronic inflammation, acute inflammation (GI tract). Overall toxicity grade was significantly lower in umbralisib-treated group compared to duvelisib-treated group in both liver (p=0.0009) and GI tract (p=0.0025). Toxicity grade in these tissues negatively correlated with total Treg count in spleen (R-squared~0.7). Immunohistochemistry staining of FoxP3+ Tregs in liver and GI tract was concordant with the assessment of Treg count in spleen by flow cytometry, as the number of FoxP3+ cells was closer to normal in umbralisib-treated mice compared to duvelisib-treated mice. Next, we investigated whether co-inhibition of CK1e by umbralisib may be involved in the differential regulation of T cells. Combination of a selective CK1e inhibitor, SR-4471, with duvelisib, prevented the reduction of total Treg number and functional markers in ex vivo culture of murine euTCL1 T cells, mimicking the effect of umbralisib. We have found that canonical Wnt signaling is inhibited dose-dependantly in euTCL1 T cells treated with umbralisib, demonstrated by lower levels of B-catenin and downstream TCF-1/7. These data determine Tregs to be a major player involved in immune-mediated toxicity characteristic of the PI3K inhibitor class of drugs. Umbralisib may differentially regulate CLL T cells through complimentary inhibition of both PI3Kδ and CK1e, potentially preserving Treg number and function to provide protection from immune-mediated SAEs. DisclosuresMiskin:TG Therapeutics, Inc.: Employment, Equity Ownership. Maryanski:TG Therapeutics, Inc.: Employment, Equity Ownership. Pinilla-Ibarz:ARIAD: Consultancy, Honoraria; BMS: Honoraria, Speakers Bureau; Pfizer: Honoraria, Speakers Bureau.