High Foxp3 Research Articles

The prognostic and predictive role of tumor-infiltrating lymphocytes (FoxP3 + and CD8 +) and tumor-associated macrophages in early HER2 + breast cancer

PurposeIn HER2-positive (HER2 +) breast cancer, tumor-infiltrating lymphocytes (TILs) and tumor-associated macrophages (TAMs) may influence the efficacy of the HER2-antibody trastuzumab and the patient’s outcome. In this HER2 + patient cohort, our aim was to study the numbers of FoxP3 + regulatory TILs and CD8 + cytotoxic TILs, their correlations with CD68 + and CD163 + TAMs, and the prognostic and predictive value of the studied factors.MethodsWe evaluated 139 non-metastatic HER2 + breast cancer patients operated between 2001 and 2008. The FoxP3+TIL count (FoxP3+TILs) was assessed using the hotspot method, and the CD8 + TIL count (CD8+mTILs) utilizing a digital image analysis from invasive margin areas. The ratios between CD8+mTILs and FoxP3+TILs as well as CD8+mTILs and TAMs were calculated.ResultsFoxP3 + TILs and CD8 + mTILs correlated positively with each other (p<0.001). FoxP3+TILs had a positive correlation with CD68+and CD163+TAMs (p≤0.038), while CD8 + mTILs correlated only with CD68+TAMs (p<0.001). In the HER2 + and hormone receptor-positive Luminal B subgroup, high numbers of FoxP3+TILs were associated with shorter disease-free survival (DFS) (54% vs. 79%, p = 0.040). The benefit from adjuvant trastuzumab was extremely significant among patients with a high CD8 + mTILs/CD68 + TAMs ratio, with overall survival (OS) 84% vs. 33% (p = 0.003) and breast cancer-specific survival (BCSS) 88% vs. 48% (p = 0.009) among patients treated with or without trastuzumab, respectively.ConclusionIn the HER2 + Luminal B subgroup, high FoxP3 + TILs were associated with shorter DFS. A high CD8 + mTILs/CD68 + TAMs ratio seems to associate with impressive efficacy of trastuzumab.

CCL19/CCR7 drives regulatory T cell migration and indicates poor prognosis in gastric cancer

BackgroundGastric cancer is associated with significant morbidity and mortality in the world. Blocking programmed cell death protein 1 pathway have been approved for the treatment of a variety of tumors and have achieved remarkable clinical therapeutic effects. However, immune checkpoint inhibitors failed to achieve satisfactory results in gastric cancer. There is a need to identify novel immunotherapy targets in gastric cancer.MethodsWe analysed the correlation between Treg cells and CD8 + T cells in gastric cancer samples. We studied the relationship between chemokines and Treg cells or CD8 + T cells in gastric cancer. We compared CCL19/CCR7 expression in gastric cancer patients in TCGA database. We performed transwell experiments to determine the influence of CCL19 on Treg cells and CD8 + T cells migratory capacity. We conducted survival analysis of CCL19 and CCR7 in gastric cancer database.ResultsTreg cells show positive correlation with CD8 + T cells in gastric cancer. Treg cell expression was significantly upregulated in tumor tissues. Patients with high FOXP3 expression had worse overall survival than those with low FOXP3 expression. CCL19 had strong correlation with FOXP3 and weak correlation with CD8A. CCL19 had strong impact on the migratory capacity of Treg cells but weak impact on the migratory capacity of CD8 + T cells. Both CCL19 and CCR7 expression were significantly upregulated in gastric cancer tissues. Survival analysis demonstrated that both CCL19 and CCR7 indicate poor prognosis in gastric cancer.ConclusionsCCL19/CCR7 may be a potential novel therapeutic target in gastric cancer.

Regulatory T cells limit opportunistic colonization of Staphylococcus aureusto promote lesion resolution in cutaneous leishmaniasis

Abstract The severity and responsiveness to treatment in L. braziliensis patients is influenced by a variety of factors, including microbiota and cytolytic T cells. Recently, we studied the skin microbiome in patients and demonstrated that high levels of S. aureus are associated with delayed healing in patients. To understand how S. aureus might influence cutaneous leishmaniasis, we studied the immune responses in mice following colonization with a S. aureus isolate from a L. braziliensis patient. Colonization of the skin with S. aureus alone induced a dominant type-17 response as well as an accumulation of Foxp3 T regulatory cells, and subsequent infection of mice with L. braziliensis led to increased IL-17 dependent pathology. Unexpectedly, transient depletion of total Tregs or RORγtin Foxp3 Tregs in S. aureus colonized mice increased IFN-γ and decreased the IL-17A response in skin. These mice exhibited increased pathology, which was accompanied by epidermal cell death and loss of barrier integrity. Additionally, the mice had substantially more S. aureus in the skin, and in the lymph nodes. In mice colonized with S. aureus and infected with L. braziliensis partial reduction led to a substantial increase in disease and a higher burden of S. aureus. Consistent with these results, in a transcriptional analysis of lesions from L. braziliensis patients, we found that low Foxp3 expression in patients had increased expression of cytolytic genes and IFN-γ, increased S. aureus in the lesions and had a delay in lesion resolution compared to patients with high Foxp3. Taken, together, these results suggest that in L. braziliensis patients Tregs are critical to regulate type-1 responses that in the context of S. aureus can promote more severe disease.

Abstract 4638: Integrative spatial analysis of paired IHC and H&amp;E images identifies Foxp3 enriched tumor-infiltrating lymphocytes associated with disease-free survival in human papillomavirus (HPV)-related oropharyngeal squamous cell carcinoma

Abstract Background: Recent studies support that high levels of tumor-infiltrating lymphocytes (TILs) were associated with better prognosis in HPV(+) oropharyngeal squamous cell carcinoma (OPSCC). While these studies provide a potential role for TILs as a prognostic biomarker, the analyses were relied on manual quantification performed by pathologists, resulting in inter-observer variability. Deep Learning (DL)-based whole slide hematoxylin and eosin (H&amp;E) image analyses may overcome these challenges. However, this analysis only provides the presence of cell types without consideration for the functional roles of each cell within the tumor immune microenvironment (TME). In this work, we develop a computational pipeline to integrate paired H&amp;E and immunohistochemistry (IHC) images to functionally characterize TILs and investigate their prognostic utility. Methods: We analyzed 88 patients: 42 stage I, 39 stage II, and 7 stage III. Our data contain both H&amp;E and IHCs examining FoxP3, CD3, PD-L1, CD20, etc. on serial sections of the tissue. In-house DL-based H&amp;E analysis used to identify TILs, tumors, and stroma in each tumor, then performed registration between adjacent H&amp;E and IHC images from the same tissue. The patients were then classified into three basic immune phenotypes: immune inflamed (IN; high TILs in the tumor region), immune excluded (EX; TILs are mostly localized in stroma), and immune desert (ID; few/no TILs) based on TIL enrichment in the TME. To functionally characterize TILs, we quantified protein expression from the adjacent IHCs. For example, we further classified each patient into different subtypes based on enriched protein expression (e.g., FoxP3 high IN, FoxP3 low IN). We used the Kaplan-Meier method and multivariate Cox analysis to evaluate the prognostic value of different subtypes enriched with different proteins to predict disease-free survival (DFS). Results: The IN group with 43 patients was significantly associated with good prognosis. Interestingly, further stratification of the IN subgroup based on Foxp3 quantification on TIL regions (i.e., high FoxP3 IN and low FoxP3 IN) showed that high protein expression of FoxP3 in TILs in the IN subgroup is significantly associated with a better prognosis compared to other immune subgroups (HR, 0.16; p-value, 0.003). Multivariate analysis, including other clinical covariates showed that the immune subtypes associated with high FoxP3 are independently associated with DFS. These results demonstrate that DL-based integrative IHC and H&amp;E image analysis could be used to identify subgroups with distinct clinical outcomes. Furthermore, our results reveal unknown roles for Foxp3 expression in the TILs in HPV(+) OPSCC as a prognostic biomarker, a finding which should be evaluated in a larger cohort. Citation Format: Sumanth Reddy Nakkireddy, Inyeop Jang, Minji Kim, Linda X. Yin, Michael Rivera, Joaquin J. Garcia MD, Kathleen R. Bartemes, David M. Routman, Eric J. Moore, Daniel J. Ma, Kathryn M. Van Abel, Tae Hyun Hwang. Integrative spatial analysis of paired IHC and H&amp;E images identifies Foxp3 enriched tumor-infiltrating lymphocytes associated with disease-free survival in human papillomavirus (HPV)-related oropharyngeal squamous cell carcinoma. [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 4638.

Tumor Microenvironment and Immune Response in Lip Cancer.

Tumor-infiltrating lymphocytes (TILs) play a significant role in cancer progression and prognosis of patients. The tumor microenvironment (TME) may affect the anti-tumor immune response. We examined the TIL and tertiary lymphoid structure (TLS) density in the invading front and inner tumor stroma, and the lymphocyte subpopulation (CD8, CD4, FOXP3) density in 60 squamous cell carcinomas of the lip. Analysis was performed in parallel with markers of hypoxia (hypoxia-inducible factor (HIF1α), lactate dehydrogenase (LDHA)) and angiogenesis. Low TIL density in the invading tumor front was related with larger tumor size (p = 0.05), deep invasion (p = 0.01), high smooth-muscle actin (SMA) expression (p = 0.01), and high HIF1α and LDH5 expression (p = 0.04). FOXP3+ TILs infiltration and FOXP3+/CD8+ ratios were higher in inner tumor areas, linked with LDH5 expression, and higher MIB1 proliferation index (p = 0.03) and SMA expression (p = 0.001). Dense CD4+ lymphocytic infiltration in the invading front is related to high tumor-budding (TB) (p = 0.04) and angiogenesis (p = 0.04 and p = 0.006, respectively). Low CD8+ TIL density, high CD20+ B-cell density, high FOXP3+/CD8+ ratio and high CD68+ macrophage presence characterized tumors with local invasion (p = 0.02, 0.01, 0.02 and 0.006, respectively). High angiogenic activity was linked with high CD4+, FOXP3+, and low CD8+ TIL density (p = 0.05, 0.01 and 0.01, respectively), as well as high CD68+ macrophage presence (p = 0.003). LDH5 expression was linked with high CD4+ and FOXP3+ TIL density (p = 0.05 and 0.01, respectively). Further research is needed to explore the prognostic and therapeutic value of TME/TIL interactions.

Effect of spatial transcriptomic-derived intrinsic epithelial signatures on immune cell recruitment and behaviour in the microenvironment of colorectal cancer.

251 Background: Colorectal Cancer (CRC) remains a significant healthcare problem. Interest in the role of the Tumour Microenvironment (TME) in progression and metastasis is established, yet much remains to be learned about the mechanism of immune cell recruitment to the TME of primary and metastatic tumours. The Klintrup Makinen (KM) score measures the density of immune infiltrate to the invasive edge of tumours and is associated with good prognosis in primary CRC. Neutrophil infiltration to metastases is a poor prognostic feature suggesting phenotypic differences in the TME at primary and metastatic sites. We set out to examine epithelial determination of the TME in primary CRC. Methods: 180 patient’s primary CRC from a curated TMA underwent deep phenotyping using a multiplex-immunofluorescent panel (DAPI, Panck, CD3 (T cells), FOXP3 (T regulatory cells), CD68 (Macrophages), CD163 (Tumour associated macrophage), CD66b (Neutrophils), KI67). The Nanostring GeoMx Spatial Transcriptomic platform was used to obtain segmented (Tumour: Panck+; TME: Panck-) whole transcriptomic (18000 genes) readout from 54 matched cores from the same TMA. Results: No significant association was demonstrated between immune cell density of any cell types and clinicopathological variables including tumour side, MMR status, N-stage or survival. High KM grade was associated with high CD3 (P = 0.01) and FOXP3 (P = 0.002). Density of all immune cell types were highly corelated. Spatial transcriptomic analysis of the segmented epithelium of patients with high KM grade demonstrated over-expression of known neutrophil chemo-attractant CXCL5 (log2FC 3.76 Adj.P &lt; 0.005). The TME compartments of cores with high CD68 and low CD163 counts were associated with adaptive immune co-ordination with high expression of CXCL13 (log2FC -2.97, Adj.P &lt; 0.005) and CD74 (log2FC -1.42 Adj.P 0.007). IGF2 (log2FC 6.08 Adj.P &lt; 0.005) and LCN15 (log2FC 4.96 Adj.P &lt; 0.005) were globally over-expressed in both TME and epithelium of cores with generalised low immune infiltration. The epithelial compartment neutrophil dense cores also over-expressed IGF2 (log2FC -3.53 Adj.P 0.02). Conclusions: TMAs provide a unique opportunity for broad transcriptomic phenotyping of large numbers of patients simultaneously. The Nanostring GeoMx platform is a flexible tool which can be used in novel ways with other proteomic technologies to drive deeper analysis of the TME. CXCL5 expression in the epithelial compartment drives global immune infiltration to the TME and may be manipulated in different contexts therapeutically through CXCR2 signalling in future.

The Relationship Between FoxP3 and SOCs3 Gene Expressions and Disease Activity in Rheumatoid Arthritis.

Immune dysregulation plays an important role in the pathogenesis of rheumatoid arthritis (RA). The CD4+CD25 high FoxP3+ subset of regulatory T cells plays an essential role in preventing autoimmunity and maintaining immune homeostasis. Negative regulation of JAK/STAT signaling is controlled by Suppressor of Cytokine Signaling (SOCs3) proteins. SOCs is produced at lower levels in RA. Our aim was to evaluate the expressional dysregulation of SOCs3 and FoxP3 genes in RA patients in relation to disease activity. We have recruited 90 patients with RA and 60 healthy controls in case control study. Whole blood samples were collected from RA patients and healthy subjects. The measurement of FoxP3 and SOCs3 gene expression was performed by real-time PCR (qPCR). Patients with RA had significantly decreased expression levels of FoxP3 and SOCs3 genes in comparison with controls (P<0.001), in addition to the insignificance correlation of both genes with disease activity in RA patients. FoxP3 and SOCs3 genes showed significant defects in rheumatoid arthritis patients with no significant difference in disease activity.

Comprehensive immunoprofile analysis of prognostic markers in pancreaticobiliary tract cancers.

Pancreaticobiliary tract cancer has a poor prognosis with unmet needs in a new target treatment. Some studies have reported that an enhancement of T-cell immunity is associated with a good prognosis. The aim of this study is to investigate the immunoprofile as a prognostic marker of pancreaticobiliary tract cancers. Unresectable pancreatic ductal adenocarcinoma (PDAC, n=80) and biliary tract cancer (BTC, n=74) diagnosed between January 2012 and December 2018 in Samsung Medical Center were analyzed. Expression levels of CD8, FOXP3, PD-1, PD-L1, and CXCL13 in PDAC and BTC tissue samples were examined with immunohistochemical staining, which was evaluated with various clinical factors. In PDAC, higher degree of PD-L1 expression was significantly associated with shorter overall survival (OS) (p=0.0095). On the other hand, higher infiltrations of PD-1+ immune cells (p=0.0002) and CD8+ T cells (p=0.0067) were associated with longer OS. In BTC, higher FOXP3+ (p=0.0343) and CD8+ (p=0.0028) cell infiltrations were associated with better survival. Low infiltration of CD8+ (p=0.0148), FOXP3+ (p=0.0208), PD-1+ (p=0.0318) cells in PDAC, and FOXP3+ cells (p=0.005) in BTC were considerably related to metastasis. In a combined evaluation of clinical factors and immunoprofiles, univariate analysis revealed that operation after chemotherapy (p < 0.0001), mass size (p=0.0004), metastasis (p=0.006), PD-L1 (p < 0.0001), PD-1 (p=0.003) and CD8 (p=0.0063) was significantly associated with OS in PDAC, and CD8 (p=0.007) was statistically related to OS in BTC. In multivariate analysis, prognostic factors were operation after chemotherapy (p=0.021) in PDAC and CD8 (p=0.037) in BTC. Therefore, immunoprofile analysis of cells expressing CD8, FOXP3, PD-1, and PD-L1 might have prognostic values in patients with pancreaticobiliary tract cancers.

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.

Underlying IPEX syndrome in a patient with idiopathic juvenile arthritis and vitiligo

BackgroundIPEX syndrome is an X-linked inborn error of immunity clinically characterized by the triad of: enteropathy, polyendocrinopathy and eczema. However many other clinical presentations lacking the triad above described have been reported what underpin the need of careful clinical suspicion, immunological evaluation and genetic sequencing.Case presentationHere we report a case of a Brazilian boy with severe eczema as the first and only presentation requiring cyclosporin therapy. Progressive and cumulative symptoms of arthritis and enteropathy lead to the suspicion of an inborn error of immunity. Peripheral FOXP3 expression was normal (CD127−/CD4+/CD25+/FOXP3+—396 cells—63%) and a pathogenic mutation in FOXP3 gene (c.1150G>A; p.Ala384Thr), confirmed the diagnosis of IPEX syndrome.ConclusionsIPEX syndrome should be suspected in patients presenting with severe eczema associated or not with other autoimmune/hyper inflammatory diseases in life. Our study also reinforces that FOXP3 expression by flowcytometry seems not to be a good screening method, and genetic sequencing is mandatory even in those with high suspicion and normal peripheral FOXP3 expression.

CD3 high and FoxP3 - tumor-infiltrating lymphocytes in the invasive margin as a favorable prognostic marker in patients with invasive urothelial carcinoma of the bladder.

Tumor-infiltrating lymphocytes (TILs) have been extensively explored as prognostic biomarkers and cellular immunotherapy methods in cancer patients. However, the prognostic significance of TILs in bladder cancer remains unresolved. We evaluated the prognostic effect of TILs in bladder cancer patients. Sixty-four bladder cancer patients who underwent surgical resection between 2018 and 2020 in Zhejiang Provincial People's Hospital were analyzed in this study. Immunohistochemistry was used to evaluate CD3, CD4, CD8, and FoxP3 expression on TILs in the invasive margin of tumor tissue, and the presence of TIL subsets was correlated with the disease-free survival (DFS) of bladder cancer patients. The relationship between clinical-pathological features and DFS were analyzed. A high level of CD3 + TILs (CD3 high TILs) ( P = 0.027) or negative expression of FoxP3 TILs (FoxP3 - TILs) ( P = 0.016) was significantly related to better DFS in bladder cancer patients. Those with CD3 high FoxP3 - TILs had the best prognosis compared to those with CD3 high FoxP3 + TILs or CD3 low FoxP3 - TILs ( P = 0.0035). Advanced age [HR 4.57, (1.86-11.25); P = 0.001], CD3 low TILs [HR 0.21, (0.06-0.71); P = 0.012], CD8 low TILs [HR 0.34, (0.12-0.94); P = 0.039], and FoxP3 + TILs [HR 10.11 (1.96-52.27); P = 0.006] in the invasive margin were associated with a worse prognosis (DFS) by multivariate analysis. In conclusion, we demonstrated that CD3 high , FoxP3 - , and CD3 high FoxP3 - TILs in the invasive margin were significantly associated with better DFS. CD8 high and CD4 high TILs in the invasive margin tended to predict better DFS in bladder cancer. Patients with CD4 high CD8 high TILs in the invasive margin were likely to have a better prognosis.

The clinicopathological significance and relapse predictive role of tumor microenvironment of intrahepatic cholangiocarcinoma after radical surgery.

This study attempts to detect the expression of FoxP3, CD68, CD8α, and PD-L1 in the tumor microenvironment (TME) of intrahepatic cholangiocarcinoma (ICC), and analyze the relationship between the corresponding cells and clinicopathological characteristics as well as prognosis of ICC. RNA sequencing (RNA-seq) provided the general landscape of the TME in ICC. A total of 99 ICC patients and the corresponding specimens were used for multiplex immunofluorescence and relapse-free survival (RFS) was analyzed. Flow cytometry further validated the effect of regulatory T (Treg) cells on ICC relapse. RNA-seq data showed that the infiltration of Treg cells, CD8+ T cells, and macrophages were likely associated with ICC relapse. The survival analysis based on multiplex immunofluorescence showed that the high FoxP3(+) Treg cells ratio and low CD68(+) macrophages ratio in mesenchyme were associated with higher RFS rate, respectively. Low FoxP3(+) Τreg cells ratio was associated with more perineural invasion, and high CD68(+) macrophages ratio was correlated with more lymph node metastasis. Cox regression analysis revealed that FoxP3(+) Treg cells ratio was an independent predictive factor for ICC relapse. Flow cytometry showed that TregIII was the predominant Treg cell subtype in both tumor tissue and peripheral blood of ICC patients, and high TregIII abundance in peripheral blood was significantly associated with longer RFS of ICC patients. High FoxP3(+) Treg cells ratio in the mesenchyme of ICC tumor tissue predicted longer RFS and was an independent favorable prognostic factor for ICC patients. Among all Treg cell subtypes, TregIII in peripheral blood was correlated with the RFS of ICC patients.

Programmed death-ligand 1 expression in the immune compartment of colon carcinoma

AbstractProgrammed cell death ligand 1 (PD-L1) on tumor cells is a significant prognostic biomarker for a number of malignancies, although less is known about the significance of PD-L1 positive immune cells in colon carcinoma. The purpose of this study is to evaluate the role of PD-L1 in a large cohort of colon carcinomas to identify patterns of PD-L1 expression in the tumor microenvironment and its correlation with other key immune subsets to better understand the impact of these immune cells. We assessed 1218 colon carcinomas on representative tissue microarray sections, gathered relevant clinicopathologic information, and performed immunohistochemical staining for mismatch repair proteins, CD8, CD163, LAG3, PD-L1, FoxP3, and BRAF V600E. We then performed automated quantification; manual quantification was used for PD-L1 tumor cells and immune cells. Dual PD-L1/PU.1 immunostain was also performed. The majority of PD-L1 positive cells expressed PU.1 thus representing tumor-associated macrophages. Based on the median number of PD-L1 positive immune cells (7.6/mm2), we classified tumors into two classes: (1) PD-L1 immune cell low and (2) PD-L1 immune cell high. PD-L1 immune cell high colon carcinomas showed favorable prognostic pathologic features including less frequent extramural venous invasion (p = 0.0001) and lower AJCC stage (p = 0.0001); they were also more commonly associated with deficient mismatch repair (dMMR) (p = 0.0001) and BRAF V600E reactivity. PD-LI immune cell high tumors were associated with high CD8, CD163, and FoxP3 positive cells (p = 0.0001, respectively). PD-L1 immune cell high and LAG3 high colon carcinomas were associated with improved disease-specific survival (p = 0.0001 and 0.001, respectively). PD-L1 expression on tumor cells was not associated with disease-specific survival. On multivariate analysis of chemotherapy naïve stage 2 colon carcinomas, only extramural venous invasion (p = 0.002), perineural invasion (p = 0.001) and PD-L1 immune cell expression (p = 0.032) correlated with disease-specific survival. Resected colonic carcinomas with high expression of PD-L1 and LAG3 proteins on immune cells were associated with improved prognosis in colon carcinoma. The mechanism underlying the improved prognosis of colon carcinomas bearing high numbers of immunoregulatory cells needs further investigation.

Effect of FOXP2 transcription factor on immune infiltration of thyroid cancer and its potential clinical value.

BackgroundThe clinical outcomes are not always favorable in certain thyroid cancer patients. The effect of Forkhead-box family on immune cells infiltration and tumor microenvironment in thyroid cancer was explored. The role of FOXP2 in tumor invasion and recurrence was investigated consequently.MethodsTIMER and GEPIA were firstly employed to compare FOXPs expression in normal and cancer tissues from multiple human cancers. The results from database were confirmed by quantitative Real Time-PCR and Western blot in matched thyroid cancer and adjacent normal tissues, in addition to a panel of thyroid cancer cell lines and normal thyroid cell. GEPIA platform was employed to discover the possibility of FOXPs as prognostic indicator. TISIBD and UACLCAN were then employed to estimate the influence of FOXPs on lymph node metastasis and tumor staging. GEPIA analysis was initially employed to analyze correlation of FOXPs and tumor immune infiltrating cells, and TIMER dataset was then included for standardization according to tumor purity.ResultDifferent member of FOXPs showed divergence in expression in various cancer tissues. Lower FOXP1, FOXP2 and higher FOXP3, FOXP4 levels could be identified in thyroid cancer tissues when compared with matched normal tissue. There was an inverse correlation between FOXP2, FOXP4 and immune invasion, whereas FOXP1 and FOXP3 were positively correlated. FOXPs showed remarkable correlations with multiply immune cells. More importantly, only FOXP2 showed the significant effect on recurrence and tumor staging.ConclusionAs immune regulatory factor, the reduction of FOXP2 may affect tumor microenvironments and immune cells infiltration, enhance tumor immune escape, and promote recurrence of thyroid cancer. FOXP2 could be a new potential diagnostic and prognostic marker.

Consequences of adjusting cell density and feed frequency on serum-free expansion of thymic regulatory T cells

BackgroundGiven the promising results from phase 1/2 clinical trials of therapy involving regulatory T cells (Tregs), it is critical to develop Treg manufacturing methods that use well-defined reagents. MethodsSeeking to maximize expansion of human thymic Tregs activated with anti-CD3/CD28 antibody-coated beads and cultured in serum-free medium, the authors investigated the effect of adjusting process parameters including cell density and cell concentration, and feeding strategy on Treg yield and quality. ResultsThe authors found that levels of expansion and viability varied with cell density on the day of restimulation. Tregs restimulated at low cell densities (1 × 105 cells/cm2) initially had high growth rates, viability and FOXP3 expression, but these parameters decreased with time and were less stable than those observed in cultures of Tregs restimulated at high cell densities (5 × 105 cells/cm2), which had slower growth rates. High-density expansion was associated with expression of inhibitory molecules and lower intracellular oxygen and extracellular nutrient concentrations as well as extracellular lactate accumulation. Experiments to test the effect of low oxygen revealed that transient exposure to low oxygen levels had little impact on expansion, viability or phenotype. Similarly, blockade of inhibitory molecules had little effect. By contrast, replenishing nutrients by increasing the feeding frequency between 2 days and 4 days after restimulation increased FOXP3, viability and expansion in high-density cultures. ConclusionThese data show the previously undescribed consequences of adjusting cell density on Treg expansion and establish a Good Manufacturing Practice-relevant protocol using non-cell-based activation reagents and serum-free media that supports sustained expansion without loss of viability or phenotype.

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.

The expression landscape of FOXP3 and its prognostic value in breast cancer.

BackgroundForkhead Box Protein 3 (FOXP3), as an essential marker of regulatory T cell (Treg) development, is reportedly overexpressed in invasive breast carcinoma (BRCA) and could be a potential prognostic factor for BRCA. However, the biological function of FOXP3 in BRCA is still unclear. In this study, we comprehensively explored the expression landscape of FOXP3 and its prognostic value in BRCA.MethodsFOXP3 transcriptomic expression data were mainly obtained from The Cancer Genome Atlas (TCGA). The Kaplan-Meier plotter and receiver operating characteristic (ROC) curve were used to assess the prognostic and diagnostic value of FOXP3 in BRCA. UALCAN, cBio-Portal, and MethSurv were used to evaluate the genomic variation of FOXP3. Gene set enrichment analysis (GSEA) was performed to explore the FOXP3 pathways involved in BRCA. Morover, we detected the expression of FOXP3 in 123 BRCA specimens and 5 BRCA cell lines to verify the biological value of FOXP3 in BRCA. The Kaplan-Meier method was adopted for the overall survival (OS) analysis, and a Cox proportional hazards model was used to estimate the hazard ratio (HR) for OS.ResultsFOXP3 was more highly expressed in BRCA than in normal tissues (2.808±1.020 vs. 1.409±0.656, P<0.001), and overexpressed FOXP3 was associated with a better prognosis. The ROC curve demonstrated a significant diagnostic value of FOXP3 in BRCA (area under the ROC curve, AUC: 0.877). Genomic analysis revealed that promoter hypomethylation of FOXP3 may be the underlying mechanism of FOXP3’s upregulation in BRCA. GSEA found that FOXP3 coexpressed genes were mainly involved in the Toll-like receptor pathway, JAK/STAT pathway, cell cycle, and apoptosis. Moreover, high FOXP3 expression was an independent protective factor for OS in our 123 BRCA tissues (HR: 0.367; P=0.036). In vitro, we found that FOXP3 knockdown with siRNA promoted migration and invasion in MCF-7 cells.ConclusionsThis study demonstrated that FOXP3 shows prognostic and diagnostic value for BRCA. We provided evidence that promoter hypomethylation and a high expression of FOXP3 were both related to a favorable prognosis in BRCA, which maybe associated with the Toll-like receptor pathway, JAK/STAT pathway, cell cycle, and apoptosis.

Abstract 6116: Tumor immune environment in liver metastasis of colorectal cancer and its correlation with recurrence free survival

Abstract Introduction: The liver is a common site of metastases of colorectal cancer. Tumor infiltrating lymphocytes (TILs) play an important role in the process of colorectal cancer liver metastasis (CRCLM). A comprehensive investigation into TILs will provide more prognostic information for patients with colorectal liver metastases after liver resection. Methods: 15 CRC patients with liver metastases from our hospital were enrolled in the study. The density and percentage of the TILs [CD8+, CD3+, CD4+, CD20+, tumor associated macrophages (TAMs), dim CD56+NK, bright CD56+NK, PD-1+etc.] in the liver metastasis samples from operation were analyzed by multiplex immunofluorescence technique (3DMedcines Inc.). The level of the density and percentage of immune cells were determined by dichotomy method. The correlation between recurrence free survival (RFS) and immune cells were analyzed using the Kaplan-Meier curve and Log-rank test. Results: The existence of dim CD56+ NK cells in the stroma was associated with significant improvement in RFS (p=0.0461). The median RFS was 26.1 months (95% CI, 14.9 to 33.5) with the existence of dim CD56+ NK cells in the stroma versus 1.7 months (95% CI, -18.0 to 32.4) without dim CD56+ NK cells in the stroma. Nevertheless, dim CD56+ NK cells in the tumor-enriched segments was not significantly associated with improved outcome. The existence of bright CD56+ NK cells in the stroma was associated with significant improvement in RFS (p=0.0307) but not in the tumor-enriched segments. The median RFS was 34.6 months (95% CI, 15.0 to 35.2) with the existence of bright CD56+ NK cells in the stroma versus 7.9 months (95% CI, -5.3 to 23.1) without bright CD56+ NK cells in the stroma. However, low FoxP3+ cells in the tumor-enriched segments (p=0.0037) was significantly associated with improved outcome but not in the stroma. The median RFS was 36.5 months (95% CI, 23.1 to 40.1) with low FoxP3+ cells versus 8.2 months (95% CI, 0.5 to 21.8) with high FoxP3+ cells in the tumor-enriched segments. Low CD3+CD4+FoxP3+ cells (p=0.074) and the existence of PD-L1+CD68+ cells (p=0.0524) in the tumor-enriched segments showed a trend towards better RFS. No significant correlation was achieved for CD8+, CD3+, CD3+CD4+, CD20+, CD68+CD163+, CD68+CD163-, PD-1, PD-1+CD8+ cells either in the tumor-enriched segments nor in the stroma. Conclusion: We identified that CD56+ NK cells in stroma are associated with improved outcome in CRCLM. Nevertheless, FoxP3+ cells are associated with poor prognosis in CRCLM. Citation Format: Jiamin Zhou, Weiqi Xu, Miao Wang, Siqi Zhang, Ding Zhang. Tumor immune environment in liver metastasis of colorectal cancer and its correlation with recurrence free survival [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 6116.

Study of Oligonucleotides Access and Distribution in Human Peripheral Blood Mononuclear Cells.

Therapeutic oligonucleotides have achieved great clinical interest since their approval as drug agents by regulatory agencies but their access and distribution in blood cells are not completely known. We evaluated by flow cytometry the ability of short fluorescent scramble oligonucleotides (ON*) to access human peripheral blood mononuclear cells (PBMC) after incubating with ON* during 1 h and 7 days of culture follow-up ‘in vitro’. Blood samples were treated with chemically modified oligonucleotides (phosphorothioate backbone and 2′ O-Me ends) to resist nuclease digestion under culture conditions. The ON* internalization was determined after discarding the membrane-associated fluorescence by trypan blue quenching. Whereas the oligonucleotide accessed neutrophils and monocytes rapidly, achieving their maximum in 1 h and 24 h, respectively, lymphocytes required 7 days to achieve the maximum (80% of cells) transfection. The ON*ability to access lymphocyte types (T, B, and NK) and T cell subtypes (CD4+, CD8+, and CD4-CD8-) were similar, with T cells being more accessible. Regulatory CD4+ and CD8+ T cells were classified in low and high Foxp3 expressers, whose expression proved not to alter the ON* internalization during the first hour, achieving 53% of CD4+Foxp3+ and 40% of CD8+Foxp3+ cells. Our results contribute to understanding and improving the management of therapeutic ONs.

Regulatory T cells induce a suppressive immune milieu and promote lymph node metastasis in intrahepatic cholangiocarcinoma

BackgroundEmerging evidence indicates that immunogenicity plays an important role in intrahepatic cholangiocarcinoma (ICC). Herein, we systematically evaluated the clinical relevance of immunogenicity in ICC.MethodsHighly immunogenic ICCs identified in the public dataset and the Cancer Immunome Atlas (TCIA) were assessed to determine the prognostic impact of immunogenicity in ICC and key components after curative resection. We also investigated the clinical relevance of the immune milieu in ICC.ResultsUsing the Gene Expression Omnibus dataset 89749 and TCIA, we identified CD8+/forkhead box P3 (FoxP3)+ tumour-infiltrating lymphocytes (TILs), T-cell immunoglobulin and mucin domain 3 (TIM-3) and human leukocyte antigen-A (HLA-A) in highly immunogenic ICCs. Immunohistochemical analysis of the in-house cohort showed that intratumoral FoxP3+ TILs correlated with CD8+ TILs (P = 0.045, Fisher’s exact test) and that high FoxP3+/CD8+ ratio (FCR) was an important marker for poor survival (P < 0.001, log-rank test). Furthermore, the FCR was higher in tumour-free lymph nodes in ICCs with lymph node metastases than in those without lymph node metastases (P = 0.003, Mann–Whitney U test).ConclusionsFCR should be considered an important biomarker that represents the immune environment of ICC based on its potentially important role in tumour progression, especially lymph node metastasis.