Presence Of Foxp3 Research Articles

A positive FOXP3/lncRNA SNHG1 feedback axis ameliorates cardiomyocytes hypertrophy by negatively regulating Parkin-mediated mitophagy

In this study, we identified FOXP3 as a transcription factor for lncRNA SNHG1, which exerts a significant protective role against cardiomyocyte hypertrophy. Through DNA-pull down experiments and ChIP analysis, we confirmed that FOXP3 could bind to the promoter of SNHG1. Luciferase reporter and RT-qPCR experiments validated that FOXP3 overexpression promoted SNHG1 expression in cardiomyocytes. Furthermore, in a model of cardiomyocyte hypertrophy, FOXP3 expression was upregulated, particularly in cardiomyocytes. Functional assays demonstrated that FOXP3 overexpression inhibited cardiomyocyte hypertrophy, while FOXP3 knockdown held the opposite effect. Additionally, we revealed that lncRNA SNHG1 acted as a sponge for miR-182, miR-326, and miR-3918, thereby stabilizing FOXP3 mRNA in cardiomyocytes. The protective role of SNHG1 against cardiomyocyte hypertrophy was found to depend on the presence of FOXP3, forming a positive FOXP3/SNHG1 feedback axis. Moreover, we unveiled this positive FOXP3/SNHG1 feedback axis suppressed cardiomyocyte hypertrophy by negatively regulating Parkin-mediated mitophagy. These findings provide novel insights into the molecular mechanisms underlying cardiomyocyte hypertrophy and offer potential therapeutic targets for related interventions.

Immunohistochemical analysis of the tumor microenvironment in acral lentiginous melanoma (ALM).

e21588 Background: Acral lentiginous melanoma (ALM) is a rare subtype of melanoma occurring on the palms, soles and nail beds. Molecularly, ALM is characterized by a higher frequency of chromosomal structural and copy number variants, but lower tumor mutational burden (TMB). Meanwhile, immune checkpoint inhibitors (ICI) are less efficacious in this subtype. In order to better understand these differences in outcomes to ICI, we conducted an immunohistochemical analysis of ALM to explore putative immune biomarkers. Methods: Clinically annotated samples of ALM were identified from patients treated at the USC Keck School of Medicine between 2010-2018. Immunohistochemistry stains included CD8, PD-L1, CD56, CD68, FOXP3, CTLA-4, HLA-ABC and HLA-G, and were characterized based on the intensity of staining. The percentage of tumor cells and infiltrating immune cells (IC) within the tumor microenvironment for the aforementioned antigens were recorded. Data are reported as percent of total cells ( > 1%, > 5%, > 25%, > 50%) expressing marker in the entire sample. Results: A total of 28 ALM specimens from 21 different patients were analyzed, including 19 primary and 9 metastatic tumors. The median age of all patients was 67 years (range 32-92); the majority were men (66%) and Hispanic (66%). Primary ALM tumors were located more often on the plantar foot (62%), compared to the plantar hand (29%). The median tumor thickness was 2.8 mm (range 0.25 – 6.5 mm). Among all patients, 57% had localized disease, while 19% had regional metastases and 24% had distant metastases. CD8+ TIL cells were observed in 95% (18/19) of samples, with 68.4% (13/19) showing > 50% CD8+ staining on IC. PD-L1 expression > 1% on tumor cells was noted in 37% (10/27) of samples, and 19% (5/27) on IC; PD-L1 > 5% was noted in only 18.5% (5/27) on tumor cells and on 11% (3/27) on IC. Both CD56 and CD68 were noted to have at least 1% expression on IC among nearly all (94%) of samples analyzed (7/8 and 9/9, respectively). FoxP3 expression > 1% was noted on IC in 92.3% (24/26) of samples, and > 5% in 50% (13/26). CTLA-4 expression > 1% was observed among 81.5 % (22/27) of samples, while 63% (17/27) had expression > 5%; CTLA-4 expression > 50% was noted among 100% on melanophages (7/7 samples). HLA-ABC expression was detected in 100% of samples analyzed (10/10). The percentage of samples with HLA-G expression > 1% was 60.7% (17/28), while 28.6% (8/28) demonstrated > 5% expression. Further correlations with clinical outcomes will be reported. Conclusions: Our study suggests a unique immune tumor microenvironment in ALM, characterized by low PD-L1 expression, despite infiltration by CD8+, as well as CD56 and CD68 immune cells. Furthermore, the presence of FOXP3+ Tregs and HLA-G expression, both which mediate immune response evasion, may explain the poor outcomes of ICI therapy among these patients and warrant further investigation.

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.

Final results of adjuvant nivolumab for hepatocellular carcinoma (HCC) after surgical resection (SR) or radiofrequency ablation (RFA) (NIVOLVE): A phase 2 prospective multicenter single-arm trial and exploratory biomarker analysis.

416 Background: The NIVOLVE trial was designed to assess the efficacy and safety of nivolumab as an adjuvant therapy for HCC, and to identify biomarkers predictive of recurrence in patients after SR or RFA (Registration # UMIN 000026648). Methods: The trial involved 11 sites and was conducted in patients with HCC who showed a complete response after SR (n = 33) or RFA (n = 22) (ITT). Overall, 53 of 55 patients with Child-Pugh A received nivolumab (240 mg/body every 2 weeks (8 cycles)), followed by nivolumab (480 mg/body every 4 weeks [8 cycles]) within 6 weeks after SR or RFA. The primary endpoint was the 1-year recurrence-free survival rate (RFSR). The key secondary endpoint was RFS. Exploratory biomarker analysis included mutations, copy number alterations, and positivity of immune cells. Techniques included next generation sequencing, immunohistochemical staining (IHC) of formalin-fixed paraffin-embedded tissues (n = 31 [13 with recurrence and 18 without]) for CD8, PD-1, PD-L1, Foxp3, and β-catenin, and ctDNA analysis using pre-nivolumab whole blood by deep sequencing (CAPP-seq; Avenio). Results: The 1-year RFSR and median RFS were 78.6% and 26.3 months (95% confidence interval (CI), 16.8-NR), respectively, with no difference between SR and RFA. Copy number gains (CNGs) in WNT/β-catenin related genes ( APC, CTNNB1, TCF7L1, TCF7L2) (n = 30) correlated with shorter RFS (positive: 10.6 months vs. negative: not reached [NR]; p < 0.0001). IHC revealed that negative staining for PD-1 (p < 0.0001), a low combined positive score for PD-L1 (p = 0.005), a low number of CD8+ tumor infiltrating lymphocytes (TILs) (p < 0.001), and positivity for Foxp3+ cells (p = 0.015) correlated with recurrence. HCC cases with low numbers of CD8+ TILs or cases positive for Foxp3+ cells (n = 16) showed a significantly shorter RFS (16.8 months [95% CI, 8.7-25.2]) than those with high numbers of CD8+ TILs and those positive for PD-1/PD-L1 expression (n = 15) (NR [95% CI,26.2months–NR]) (p < 0.0001). HCC cases with activation of the WNT/β-catenin pathway assessed by IHC (n = 9) showed shorter RFS (17.0 months [95% CI,1.1–26.2]) than those without activation (n = 22) (NR [95% CI,23.0 months–NR]) (p = 0.014). Patients positive for ctDNA (n = 15) before nivolumab tended to have shorter RFS than those without ctDNA (n = 9) (26.3 vs. NR). There was no correlation between TMB and RFS. Treatment-emergent adverse events (AEs) (n = 53) were as follows: all grades, 93%; grades 3–4 (18.9%); and immune related AEs, 25%. Conclusions: The 1-year RFSR and RFS in the NIVOLVE trial were 78.6% and 26.3 months, respectively. No new safety signal was observed. CNG in WNT/β-catenin-related genes, activation of the WNT/β-catenin pathway, the presence of Foxp3+ cells, and low numbers of CD8+ TILs may predict recurrence after SR or RFA with adjuvant nivolumab. Clinical trial information: UMIN 000026648.

Adjuvant nivolumab for hepatocellular carcinoma (HCC) after surgical resection (SR) or radiofrequency ablation (RFA) (NIVOLVE): A phase 2 prospective multicenter single-arm trial and exploratory biomarker analysis.

4070 Background: The NIVOLVE trial was designed to assess the efficacy and safety of nivolumab as an adjuvant therapy for HCC, and to identify biomarkers predictive of recurrence in patients after SR or RFA (Registration # UMIN 000026648). Methods: The trial involved 11 sites and was conducted in patients with HCC who showed a complete response after SR (n = 33) or RFA (n = 22) (ITT). Overall, 53 of 55 patients with Child-Pugh A received nivolumab (240 mg/body every 2 weeks (8 cycles)), followed by nivolumab (480 mg/body every 4 weeks (8 cycles)) within 6 weeks after SR or RFA. The primary endpoint was the 1 year recurrence-free survival rate (RFSR). The key secondary endpoint was RFS. Exploratory biomarker analysis included mutations, copy number alterations, and tumor mutation burden in tumor tissues. Techniques included next generation sequencing, immunohistochemical staining (IHC) of formalin-fixed paraffin-embedded tissues (n = 31, 13 with recurrence and 18 without) for CD8, PD-1, PD-L1, Foxp3, and β-catenin, and ctDNA analysis using pre-nivolumab whole blood by deep sequencing (CAPP-seq; Avenio). Results: The 1-year RFSR and median RFS were 76.7% and 26.0 months (95% confidence interval (CI), 23.9–28.1), respectively, with no difference between SR and RFA. Copy number gains (CNGs) in WNT/β-catenin related genes ( APC, CTNNB1, TCF7L1, TCF7L2) (n = 8) correlated with shorter RFS (positive: 11.8 months vs. negative: not reached [NR]; p = 0.0003). IHC revealed that negative staining for PD-1 (p < 0.0001), a low combined positive score for PD-L1 (p = 0.0113), a low number of CD8+ tumor infiltrating lymphocytes (TILs) (p = 0.0130), and positivity for Foxp3+ cells (p = 0.0076) correlated with recurrence. Treatment-related adverse events (AEs) (n = 53) were as follows: all grades, 68%; grades 3–4 (18.9%); and immune related AEs, 25%. HCC cases with low numbers of CD8+ TILs or cases positive for Foxp3+ cells (n = 16) showed a significantly shorter RFS (16.8 months [95% CI, 8.7–25.1]) than those with high numbers of CD8+ TILs and those positive for PD-1/PD-L1 expression (n = 15) (NR [95% CI,26.2months–NR]) (p < 0.0001). HCC cases with activation of the WNT/β-catenin pathway assessed by IHC (n = 9) showed shorter RFS (17.0 months [95% CI,1.1–26.2]) than those without activation (n = 22) (NR [95% CI,24.7 months–NR]) (p = 0.0191). Patients positive for ctDNA (n = 10) before nivolumab tended to have shorter RFS than those without ctDNA (n = 30) (26.2 vs. NR). There was no correlation between TMB and RFS. Conclusions: The 1 year RFSR and RFS in the NIVOLVE trial were 76.6% and 26.0 months, respectively. No new safety signal was observed. CNG in WNT/β-catenin-related genes, activation of the WNT/β-catenin pathway, the presence of Foxp3+ cells, and low numbers of CD8+ TILs may predict recurrence after SR or RFA with adjuvant nivolumab. Clinical trial information: 000026648.

Heart Allograft Quilty Lesions are Associates with a Tolerant Gene Profile and Normal Allograft Function

Purpose Quilty lesions (lymphoid aggregates) are frequently encountered in heart allograft biopsies without morphologic evidence of rejection. However, their biologic nature and precise function remains unclear. Previously, in 42 cardiac allograft biopsies, we demonstrated that the presence of Foxp3+ innate and TGF- β+ adaptive regulatory lymphocytes in Quilty lesions (QL) are associated with higher allograft acceptance. In the current study, we aimed to characterize immunomodulatory pathways associated with QL by measuring mRNA expression. Methods Endomyocardial biopsies of heart allograft from nine patients were included in this study, four with unremarkable endomyocardium (control group) and five with at least one QL (Quilty group). No acute T-cell or antibody mediated rejection was present histologically in any of these specimens. Total RNA was extracted from formalin fixed paraffin tissue. Multiplexed mRNA measurement was performed using the nCounter system (NanoString Technologies, Seattle, WA), and data were analyzed with nSolver software (NanoString Technologies, Seattle, WA). Results Of 771 gene mRNA levels measured in the NanoString Transplant Immunology Panel, 274 were upregulated in the QL group over the control group, with approximately one third related to adaptive immunity and 5% to innate immunity. Higher levels of mRNA expression in the QL group were also shown in pathways for hematopoiesis (11%), cytokine (9%), chemokine (7%), cell-extracellular matrix interaction (7%), and apoptosis & cell cycle regulation (5%). More specifically, the mRNA expression of TH2 tolerance-associated immunity markers, including Foxp3, TGF-β, and CTLA4, were higher in the Quilty group (2.82, 1.42, and 3.97 fold increase, respectively, with p Conclusion Heart allografts with QL have dominant adaptive immunity related mRNA expression with significantly higher expression of TH2 tolerant immunity markers. These data suggest that QL, far from passive bystanders, may serve an immunomodulatory role in cardiac allografts. The presence of intra-allograft regulatory T-lymphocyte related signaling in QL may help to reduce the risk of rejection and foster allograft acceptance.

A novel Foxp3-related immune prognostic signature for glioblastoma multiforme based on immunogenomic profiling.

Foxp3+ regulatory T cells (Treg) play an important part in the glioma immunosuppressive microenvironment. This study analyzed the effect of Foxsp3 on the immune microenvironment and constructed a Foxp3-related immune prognostic signature (IPS)for predicting prognosis in glioblastoma multiforme (GBM). Immunohistochemistry (IHC) staining for Foxp3 was performed in 72 high-grade glioma specimens. RNA-seq data from 152 GBM samples were obtained from The Cancer Genome Atlas database (TCGA) and divided into two groups, Foxp3 High (Foxp3_H) and Foxp3 Low (Foxp3_L), based on Foxp3 expression. We systematically analyzed the influence of Foxp3 on the immune microenvironment. Least Absolute Shrinkage and Selection Operator (LASSO) Cox analysis was conducted for immune-related genes that were differentially expressed between Foxp3_H and Foxp3_L GBM patients. We found a differential expression of Foxp3 in high-grade glioma tissues. The presence of Foxp3 was significantly associated with poor OS. From the four-gene IPS developed, GBM patients were stratified into low-risk and high-risk groups in both the training set and validation sets. Furthermore, we developed a novel nomogram to evaluate the overall survival in GBM patients. This study offers innovative insights into the GBM immune microenvironment and these findings contribute to individualized treatment and improvement in the prognosis for GBM patients.

Transcriptional regulation of VEGFA expression in T-regulatory cells from breast cancer patients.

The initiation of new bloodvessel formation (neo-angiogenesis) is one of the primary requirements for the establishment of tumor. As the tumor grows beyond a certain size, a hypoxic-condition arises in the inner core of tumor, triggering the release of chemokines, which attract T-regulatory (Treg) cells in the tumor-site. The presence of FOXP3, a lineage-specific transcription factor, expressing Treg cells in various types of tumor implements immunosuppressive and tumor-promoting strategies. One such strategy is the invitation of endothelial cells for neo-vascularization in the tumor site. Here we report that as the disease progresses, Treg cells from breast cancer patients are capable of secreting high-amount of VEGFA. The VEGFA promoter lacks Treg-specific transcription factor FOXP3 binding site. FOXP3 in associationwith locus-specific transcription factor STAT3 binds to VEGFA promoter to induce its transcription in Treg cells obtained from breast cancer patients. Treg cell-secreted VEGFA induces neo-angiogenesis from endothelial cells under in-vitro conditions. Targeting Tregs in mice with breast tumor reduces tumor growth as well as the level of neo-angiogenesis in the tumor tissue.

Abstract A26: Humanized mouse model: A model to understand mechanisms of immune non-responsiveness to immune checkpoint inhibitors in melanoma

Abstract Immune checkpoint inhibitor therapy (anti-CTLA4 or anti-PD1 antibodies) is rapidly emerging as a front-line treatment option for many solid tumors. However, only a third of melanoma patients respond to immune checkpoint blockade. Currently available mouse xenograft and transgenic models have many shortcomings and are unable to address the basis of therapy resistance and immune nonresponsiveness that are observed in patients. Thus, there is an urgent need to establish an in vivo model with a human immune microenvironment that can address issues of therapy resistance. Our laboratory has developed a novel humanized mouse melanoma model. Immunodeficient NSG mice were reconstituted with human CD34+ cells and after 8-12 weeks, mice are fully reconstituted with human innate (monocyte/myeloid lineage cells, dendritic cells and NK cells) and adaptive (T and B cells) immune cells. Humanized mice were then challenged with HLA-matched melanoma cells, and the functional ability of human immune cells to restrict tumor growth was monitored. Delayed tumor growth was observed in humanized mice, indicating in vivo sensitization of human immune cells to melanoma. This was confirmed by in vitro demonstration of human lymphocytes from tumor-bearing mice showing enhanced cytokine expression after stimulation with melanoma antigen peptides. In therapy studies, tumor-bearing humanized mice treated with anti-PD-1 showed restricted tumor growth. Anti-PD-1 therapy resulted in enhanced infiltration of T cells that correlated with tumor response. MassCyTOF studies were performed using a panel of immune markers to understand the mechanism of therapy nonresponsiveness in some tumors. Downmodulation of HLA-class I molecules and increased presence of FOXP3+ cells in the tumor region were seen. Our results suggest that humanized mouse melanoma model can be explored further to understand the causes of therapy resistance and immune nonresponsiveness. Citation Format: Rajasekharan Somasundaram, Anastasia Samarkina, Thomas Connelly, Robin Choi, Hedy Choi, Kar Muthumani, Xiaowei Xu, Klaus Kaestner, Meenhard Herlyn. Humanized mouse model: A model to understand mechanisms of immune non-responsiveness to immune checkpoint inhibitors in melanoma [abstract]. In: Proceedings of the AACR Special Conference on Melanoma: From Biology to Target; 2019 Jan 15-18; Houston, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(19 Suppl):Abstract nr A26.

Prognostic value of FoxP3 and CTLA-4 expression in patients with oral squamous cell carcinoma.

Tumor-infiltrating lymphocytes include tumor-reactive lymphocytes and regulatory T-cells. However, the prognostic value of tumor-infiltrating lymphocytes in oral squamous cell carcinoma (OSCC) remains unclear. We used immunohistochemistry to evaluate the presence of tumor-infiltrating FoxP3⁺ T-cells and CTLA-4⁺ cells in four distinct histological compartments (tumor parenchyma and stroma at the tumor center, and parenchyma and stroma at the invasive front) and assessed the association between the prevalence of these cells and the histopathological status of 137 patients with OSCC. Five-year overall survival, disease-specific survival, and recurrence-free survival were favorable in patients with high numbers of FoxP3⁺ T-cells in the parenchyma of the invasive front. Recurrence-free survival and metastasis-free survival were decreased in patients with high numbers of CTLA-4⁺ cells in the parenchyma of the invasive front. The presence of FoxP3⁺ T-cells in the parenchyma of the invasive front may be a useful prognostic factor. Our results indicate that FoxP3⁺ T-cells may exert site-specific anti-tumor effects but may not play an immunosuppressive role in OSCC. In addition, our results suggest that CTLA-4+ cells suppress the function of FoxP3+ T-cells and promote anti-tumor immunity in OSCC.

The possible regulatory effect of the PD-1/PD-L1 signaling pathway on Tregs in ovarian cancer.

Aim of this study was to investigate the possible regulatory effect of the programmed death-1 (PD-1)/programmed death ligand-1 (PD-L1) signaling pathway on Tregs in ovarian cancer. Immunohistochemistry was used to detect the expression of PD-L1 and PD-1 and the presence of FOXP3+ Tregs in ovarian cancer. Then, ovarian cancer HO8910 cells were subjected to transfection with PD-L1 siRNA in vitro. CCK-8, Transwell and wound healing assays were performed to detect the biological behaviors of ovarian cancer cells. Human T-cells isolated from human peripheral blood were cocultured with HO8910 cells, which were divided into the Control, TGF-β, and TGF-β+ anti-PD-L1 groups. The proportion of differentiated Tregs was detected by flow cytometry. Mouse models of ovarian cancer were established, and PD-L1 antibody therapy was administered. Tumor growth and Treg recruitment were observed. PD-L1, PD-1 and FOXP3+ Tregs were found in ovarian cancer tissue. Patients with tumors with an advanced stage and low differentiation and lymph node metastasis had significantly higher levels of PD-1, PD-L1 and FOXP3+ Tregs. After transfection with PD-L1 siRNA, HO8910 cells showed a significant reduction in PD-L1 expression, proliferation, migration and invasion. After T-cells were cocultured with ovarian cancer cells, the TGF-β+ anti-PD-L1 group showed a substantial decline in the differentiation of T-cells into Tregs compared with the TGF-β group. Moreover, mice in the anti-PD-L1 group had significantly reduced tumor growth rates, Treg proportions in the tumor microenvironment, and FOXP3 expression.

Abstract LB-063: Validation of humanized PD-1 knock-in mice as an emerging model to evaluate human specific PD-1 therapeutics

Abstract Over the past decade there has been an increasing demand in the use of syngeneic models for evaluating the efficacy of checkpoint inhibition-based cancer immunotherapies. During tumor development, immune cells can became unresponsive to the presence of tumor cells due to chronic activation and expression of the programmed cell death protein-1 (PD-1) or the T-lymphocyte-associated antigen 4 (CTLA4) in T-cells or the presence of FoxP3+ Treg cells resulting in tumor immune-tolerance. Our previous studies have demonstrated that murine anti-PD-1 and CTLA-4 therapy can effectively re-activate the antitumor response against multiple syngeneic tumor models. While these models proved instrumental for evaluating murine immune-checkpoint inhibitors (ICI), there is a clear need for additional mouse models to evaluate the efficacy of ICI specific for human targets. To address this need, we describe the development of a humanized PD-1 knock-in (KI) mouse model. This mouse model has the advantage of expressing the human PD-1 protein in the context of a fully functional immune system. We also show the response to clinically relevant immune checkpoint inhibitors in two preclinical tumor models. We evaluated the anti-tumor activity of pembrolizumab in the MC38 colorectal carcinoma and the GL261 glioblastoma models. We observed significant tumor growth inhibition and growth delay in the MC38 tumor model when treated with pembrolizumab monotherapy, but not when treated with the murine counterpart (anti-PD-1 clone RPM1-14). To extend our validation studies to other tumor models, we implanted GL261 glioblastoma orthotopically in the brain of PD-1 KI mice and achieved a significant increased life span in the group treated with pembrolizumab compared to both the control group and the group treated with murine anti-PD-1 antibody. The immune profile from control and treated these animals were also characterized in these studies by flow cytometry. In summary, the results shown here underscore the value of the humanized PD-1 knock-in (KI) mouse model as a tool to evaluate human specific immune-checkpoint based therapeutics alone and in combination with other agents. Citation Format: Anya Avrutskaya, Fabiane Sonego, Jacob Hauser, Emily O’Koren, Gaëlle Martin, Julie Chaix, Robin Ball, Thi Bui, Ian Belle, Chassidy Hall, Kader Thiam, Paula L. Miliani De Marval. Validation of humanized PD-1 knock-in mice as an emerging model to evaluate human specific PD-1 therapeutics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-063.

A novel direct co-culture assay analyzed by multicolor flow cytometry reveals context- and cell type-specific immunomodulatory effects of equine mesenchymal stromal cells.

The immunomodulatory potential of multipotent mesenchymal stromal cells (MSC) provides a basis for current and future regenerative therapies. In this study, we established an approach that allows to address the effects of pro-inflammatory stimulation and co-culture with MSC on different specific leukocyte subpopulations. Equine peripheral blood leukocyte recovery was optimized to preserve all leukocyte subpopulations and leukocyte activation regimes were evaluated. Allogeneic labeled equine adipose-derived MSC were then subjected to direct co-culture with either non-stimulated, concanavalin A (ConA)-activated or phosphate 12-myristate 13-acetate and ionomycin (PMA/I)-activated leukocytes. Subsequently, production of the cytokines interferon-γ (IFN- γ), interleukin-1 (IL-1) and tumor necrosis factor-α (TNF-α) and presence of FoxP3 were determined in specific cell populations using multicolor flow cytometry. Prostaglandin E2 (PGE2) was measured in the supernatants. ConA-stimulation induced mild activation of leukocytes, whereas PMA/I-stimulation led to strong activation. In T cells, PMA/I promoted production of all cytokines, with no distinct suppressive effects of MSC. However, increased numbers of CD25/FoxP3-positive cells indicated that MSC supported regulatory T cell differentiation in PMA/I-activated leukocyte cultures. MSC also reduced numbers of cytokine-producing B cells and granulocytes, mostly irrespective of preceding leukocyte activation, and reversed the stimulatory effect of ConA on IFN-γ production in monocytes. Illustrating the possible suppressive mechanisms, higher numbers of MSC produced IL-10 when co-cultured with non-stimulated or ConA-activated leukocytes. This was not observed in co-culture with PMA/I-activated leukocytes. However, PGE2 concentration in the supernatant was highest in the co-culture with PMA/I-activated leukocytes, suggesting that PGE2 could still mediate modulatory effects in strongly inflammatory environment. These context- and cell type-specific modulatory effects observed give insight into the interactions between MSC and different types of immune cells and highlight the roles of IL-10 and PGE2 in MSC-mediated immunomodulation. The approach presented could provide a basis for further functional MSC characterization and the development of potency assays.

High CD8+ and absence of Foxp3+ T lymphocytes infiltration in gallbladder tumors correlate with prolonged patients survival

BackgroundGallbladder cancer (GBC), although infrequent in industrialized countries, has high incidence rates in certain world regions, being a leading cause of death among elderly Chilean women. Surgery is the only effective treatment, and a five-year survival rate of advanced-stage patients is less than 10%. Hence, exploring immunotherapy is relevant, although GBC immunogenicity is poorly understood. This study examined the relationship between the host immune response and GBC patient survival based on the presence of tumor-infiltrating lymphocytes at different disease stages.MethodsTumor tissues from 80 GBC patients were analyzed by immunohistochemistry for the presence of CD3+, CD4+, CD8+, and Foxp3+ T cell populations, and the results were associated with clinical stage and patient survival.ResultsThe majority of tumor samples showed CD3+ T cell infiltration, which correlated with better prognosis, particularly in advanced disease stages. CD8+, but not CD4+, T cell infiltration correlated with improved survival, particularly in advanced disease stages. Interestingly, a < 1 CD4+/CD8+ T cell ratio was related with increased survival. Additionally, the presence of Foxp3+ T cells correlated with decreased patient survival, whereas a ≤ 1 Foxp3+/CD8+ T cell ratio was associated with improved patient survival.ConclusionsDepending on the disease stage, the presence of CD8+ and absence of Foxp3+ T cell populations in tumor tissues correlated with improved GBC patient survival, and thus represent potential markers for prognosis and management of advanced disease, and supports testing of immunotherapy.

Lambda phage nanoparticles displaying HER2-derived E75 peptide induce effective E75-CD8+ T response.

We have investigated the in vitro immunogenicity and in vivo prophylactic and therapeutic potential of lambda (λ) phage particles displaying the E75 peptide (derived from HER2 protein) in an implantable TUBO breast tumor model of BALB/c mice. The mice were immunized with the E75-displaying phage (λF7-gpD::E75) every 2-week intervals over a 6-week period, and the generated immune responses were studied. Results showed in vitro induction of immune responses by the λF7 (gpD::E75) construct compared to the control λF7 and buffer groups. In the in vivo prophylactic study, all the control and vaccinated mice groups developed tumors. However, in the therapeutic experiments, we observed a significant difference in tumor size at days 14-36 for mice immunized with λF7 (gpD::E75) compared to control groups (P < 0.05). Moreover, the survival time prolonged in mice immunized with λF7 (gpD::E75). The discrepancy between the results obtained from the in vitro and in vivo studies may have been a result of the induction of Foxp3 CD4+CD25+ which has been previously reported to hamper effective T cell functionality. In conclusion, we observed a significant immune stimulatory response in the in vitro study, while in vivo, the vaccine was not able to exert significant tumor inhibitory effects. We suggest that the presence of Foxp3+ CD4+CD25+ cells may have impaired the anti-tumor response in mice challenged in vivo with the TUBO xenograft tumor.

Presence of immune cells, low tumor proliferation and wild type BRAF mutation status is associated with a favourable clinical outcome in stage III cutaneous melanoma

BackgroundThe variable prognosis in stage III cutaneous melanoma is partially due to unknown prognostic factors. Improved prognostic tools are required to define patients with an increased risk of developing metastatic disease who might benefit from adjuvant therapies. The aim was to examine if cellular immune markers in association with tumor proliferation rate and BRAF mutation status have an impact on prognosis in stage III melanoma.MethodsWe have used two sets of case series with stage III disease: 23 patients with short survival (≤ 13 months) and 19 patients with long survival (≥ 60 months). Lymph node metastases were analyzed for Ki67, CD8 and FOXP3 protein expression using immunohistochemistry. BRAF mutation status was analyzed in a previous study on the same samples.ResultsLow tumor proliferation rate was significantly associated with a better prognosis (p = 0.013). Presence of FOXP3+ T cells was not correlated to adverse clinical outcome. A highly significant trend for a longer survival was found in the presence of an increasing number of markers; CD8+ and FOXP3+ T cells, low tumor proliferation and BRAF wildtype status (p = 0.003). Presence of at least three of these four markers was found to be an independent favorable prognostic factor (OR 19.4, 95% CI 1.9-197, p = 0.012), when adjusting for ulceration and number of lymph node metastases. Proliferation alone remained significant in multivariate analyses (OR 26.1, 95% CI 2.0-344, p = 0.013) but with a wider confidence interval. This panel still remained independent when also adjusting for a previously identified prognostic glycolytic-pigment panel.ConclusionsWe have demonstrated that presence of immune cells in association with tumor proliferation and BRAF mutation status may further contribute to identify stage III melanoma patients with high risk of relapse.

In situ localization of tumor cells associated with the epithelial-mesenchymal transition marker Snail and the prognostic impact of lymphocytes in the tumor microenvironment in invasive ductal breast cancer

Purpose: Tumor surgery is aimed at complete resection of the lesion while ensuring a sufficient tumor-specific safety distance. Nevertheless, in many cases the most peripheral part - the invasion front - remains in situ. Tumor cells at the tumor margin have been reported to lose their epithelial properties and acquire features of mesenchymal cells. The process of epithelial-to-mesenchymal transition (EMT) is believed to be of prime importance for tissue and vessel invasion. Furthermore, the detection of tumor-infiltrating lymphocytes in the microenvironment of breast cancer might serve as a reliable prognostic marker.Methods: We investigated tissue microarrays of 352 breast cancer patients with regard to the presence and distribution of the EMT factor Snail, and the presence of FoxP3, CD3 and CD8 in the immune microenvironment.Results: The expression of the transcription factor Snail is strongly associated with longer disease-free and overall survival. The presence of CD3, CD8 or FoxP3 is associated with a better outcome, although statistically significant results were noted only for FoxP3. The prognostic significance of FoxP3 and Snail were also proven in multivariate analysis.Conclusions: Based on previous studies concerning the intratumoral heterogeneity of EMT, our results suggest that Snail and FoxP3 are possible prognostic markers for breast cancer. The diverse presence of lymphocytes in the tumor microenvironment (CD3 and CD8) was confirmed. Although the importance of these markers is known, their specific role in tumor invasion and metastasis as well as their hierarchical organization in these tumors remain unclear.

PD-L1 Expression Is Associated with FOXP3+ Regulatory T-Cell Infiltration of Soft Tissue Sarcoma and Poor Patient Prognosis.

Background: Programmed death ligand-1(PD-L1) functions as a negative mediator of immune response through different pathways in anti-tumor immunity. Recent studies have reported that PD-L1 plays a pivotal role in the function of regulatory T-cells (Tregs). Although increases in FOXP3+ Tregs infiltration and PD-L1 expression have been revealed in several cancers, their correlation with soft tissue sarcoma remains unknown.Methods: We included 163 cases of soft tissue sarcoma who were diagnosed and underwent extensive and radical resection at the Sun Yat-sen University Cancer Center, Guangzhou, China, from 2000-2010. PD-L1 and FOXP3 expression was evaluated by immunohistochemistry. Correlation between their expressions and associations with clinicopathological features were studied.Results: Among 163 STS samples, 19 (11.7%) exhibited PD-L1 positivity, and 41 (25.2%) cases expressed high FOXP3+ Treg infiltration. Significant correlation between PD-L1 expression and FOXP3+Treg infiltration in STS was identified (r=0.450, p<0.001). In univariate analysis, PD-L1 expression was significantly associated with high tumor grade and the age of patients, while the presence of FOXP3+ in tumor infiltrating Tregs was significantly associated with the age of patients, high tumor stage, higher tumor grade and tumor depth. Multivariate analysis revealed PD-L1 and FOXP3 as independent prognostic indicators significantly associated with OS and DFS.Conclusions: Our study revealed that PD-L1 and FOXP3+Tregs may work synergistically in promoting immune evasion of the tumors in soft tissue sarcoma. A combined strategy to block PD-L1/PD-1 with simultaneous depletion of Tregs may show promise in enhancing the therapeutic efficacy of these patients.

Analysis of the immune microenvironment in resected non-small cell lung cancer: the prognostic value of different T lymphocyte markers.

The prognosis of non-small cell lung cancer (NSCLC) remains poor and heterogeneous and new biomarkers are needed. As the immune system plays a pivotal role in cancer, the study of immune-related markers may provide valuable prognostic information of NSCLC. In 122 formalin-fixed, paraffin-embedded tumor tissue samples from early-stage NSCLC, tumor and tumor-near stromal areas were microdissected and gene expression levels of conventional and regulatory T cell markers were assessed by quantitative polymerase chain reaction. Also, the presence of infiltrating CD4+, CD8+, and FOXP3+ cells in tumor samples was assessed by immunohistochemistry. The relative proportion of conventional and regulatory T cells present in the tumor environment was assessed and found to be key to understand the importance that the immune system analysis has in the prognostics of NSCLC patients. The presence of CD8+ cells in the tumor compartment was associated with better outcome, whereas the presence of FOXP3+ cells was associated with worse overall survival. The negative prognostic value of combined biomarkers, indicating high levels of FOXP3 in the stroma and low levels of CD4 or CD8 in tumors, was observed at mRNA level and was validated by immunohistochemistry.In conclusion, the proportion of T helper and cytotoxic cells vs. regulatory T cells in different locations of the tumor microenvironment have opposite prognostic impacts in resected NSCLC.

Immunotoxin Against a Donor MHC Class II Molecule Induces Indefinite Survival of Murine Kidney Allografts.

Rejection of donor organs depends on the trafficking of donor passenger leukocytes to the secondary lymphoid organs of the recipient to elicit an immune response via the direct antigen presentation pathway. Therefore, the depletion of passenger leukocytes may be clinically applicable as a strategy to improve graft survival. Because major histocompatibility complex (MHC) class II+ cells are most efficient at inducing immune responses, selective depletion of this population from donor grafts may dampen the alloimmune response and prolong graft survival. In a fully MHC mismatched mouse kidney allograft model, we describe the synthesis of an immunotoxin, consisting of the F(ab′)2 fragment of a monoclonal antibody against the donor MHC class II molecule I‐Ak conjugated with the plant‐derived ribosomal inactivating protein gelonin. This anti–I‐Ak gelonin immunotoxin depletes I‐Ak expressing cells specifically in vitro and in vivo. When given to recipients of kidney allografts, it resulted in indefinite graft survival with normal graft function, presence of Foxp3+ cells within donor grafts, diminished donor‐specific antibody formation, and delayed rejection of subsequent donor‐type skin grafts. Strategies aimed at the donor arm of the immune system using agents such as immunotoxins may be a useful adjuvant to existing recipient‐orientated immunosuppression.