Prognostic role of tumour-associated macrophages and regulatory T cells in EBV-positive and EBV-negative nasopharyngeal carcinoma

2017 ◽  
Vol 71 (3) ◽  
pp. 267-274 ◽  
Author(s):  
Marc L Ooft ◽  
Jolique A van Ipenburg ◽  
Maxime E Sanders ◽  
Mariette Kranendonk ◽  
Ingrid Hofland ◽  
...  
Keyword(s):  
T Cells ◽  
Nasopharyngeal Carcinoma ◽  
Regulatory T Cells ◽  
Cox Regression ◽  
Tumour Progression ◽  
Prognostic Significance ◽  
Foxp3 Expression ◽  
Tumour Microenvironment ◽  
Prognostic Role

AimsTumour-associated macrophages (TAMs) and regulatory T cells (Tregs) form a special niche supporting tumour progression, and both correlate with worse survival in head and neck cancers. However, the prognostic role of TAM and Tregs in nasopharyngeal carcinoma (NPC) is still unknown. Therefore, we determined differences in TAMs and Tregs in different NPC subtypes, and their prognostic significance.MethodsTissue of 91 NPCs was assessed for TAMs and Tregs by determination of CD68, CD163, CD206 and FOXP3 expression in the tumour microenvironment. Clinicopathological correlations were assessed using Pearson X2 test, Fisher’s exact test, analysis of variance and Mann-Whitney U test. Survival was analysed using Kaplan-Meier curves and Cox regression.ResultsCD68 and FOXP3 counts were higher in Epstein-Barr virus (EBV)-positive NPC, while CD68−/FOXP3−, CD163+/FOXP3− and CD206+/FOXP3− infiltrates were more common in EBV-negative NPC. In the whole NPC group, CD68−/FOXP3− correlated with worse overall survival (OS), and after multivariate analysis high FOXP3 count showed better OS (HR 0.352, 95% CI 0.128 to 0.968). No difference in M2 counts existed between EBV-positive and negative NPC.ConclusionsFOXP3, a Treg marker, seems to be an independent prognostic factor for better OS in the whole NPC group. Therefore, immune-based therapies targeting Tregs should be carefully evaluated. M2 spectrum macrophages are probably more prominent in EBV-negative NPC with also functional differences compared with EBV-positive NPC.

The Prognostic Role of CD68 and FoxP3 Expression in Primary Central Nervous System Lymphoma

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1457-1457 ◽  
Author(s):  
Soo-Jeong Kim ◽  
Hyunsung Park ◽  
Hyunsoo Cho ◽  
Yu Ri Kim ◽  
Jung Yeon Lee ◽  
...  
Keyword(s):  
Central Nervous System ◽  
T Cells ◽  
Nervous System ◽  
Multivariate Analysis ◽  
Central Nervous System Lymphoma ◽  
Foxp3 Expression ◽  
Prognostic Role ◽  
Median Level ◽  
The Difference

Abstract Background The prognostic role of CD68+ tumor-associated macrophages (TAMs) and tumor infiltrating T-cells including FoxP3+ regulatory T-cells (Tregs) has been extensively evaluated in areas of lymphoma research, however their expression and prognostic role have little been explored in primary central nervous system lymphoma (PCNSL). Therefore, we investigated CD68 and FoxP3 expression in tumor microenvironment of PCNSL and evaluated its prognostic role. Methods Seventy-six consecutive immunocompetent patients diagnosed with PCNSL between December 2004 and April 2015 treated homogenously with high-dose methotrexate (HD-MTX)-based chemotherapy as an initial treatment in Severance Hospital, Seoul, S. Korea and for whom archived formalin-fixed and paraffin-embedded (FFPE) tissue blocks for initial diagnosis were available were retrospectively identified. We studied CD68 and FoxP3 expression by immunohistochemical staining on FFPE biopsy specimen and evaluated correlations of their expression with obtained clinical data, treatment response depending on the upfront ASCT, and survival of the patients. The cut-off value for the expression of CD68+ TAMs and FoxP3+ Tregs were evaluated by the area under curve (AUC) of the receiver operating characteristic (ROC) curve for analysis purposes, and we established cut-offs of 55 cells/high power field (HPF) for CD68 and 15 cells/HPF for FoxP3. We stratified patients based on CD68 and FoxP3 expression according to the cut-off values we determined from the AUC. The overall survival (OS) and progression-free survival (PFS) were plotted using the Kaplan-Meier method and compared using the log-rank test. The Cox proportional-hazards regression model was used in both univariate and multivariate analyses. Results The median age of the patients was 57 (range 33-79) years. The median follow-up duration for survivors was 23.2 months (range, 2.4-128.5). Sixteen (21.1%) patients underwent upfront ASCT, after median 4 (range 2-4) cycles of HD-MTX based chemotherapy. The 2-year OS and PFS rates for all patients were 75.2% and 43.3%, respectively. The patients did not reach median OS, and the median PFS was 17.9 months (95% confidence interval [CI], 9.4-26.4). The median level of expression for CD68+ TAM/HPF was 25 (range, 5-80) and the median level of expression for FoxP3+ Tregs/HPF was 0 (range, 0-68). The difference in OS and PFS between the high and low CD68 groups was significant in the univariate (hazard ratio [HR] = 2.79, 95% CI: 0.97-8.03, P = 0.058 for OS, and HR = 2.17, 95% CI: 1.03-4.58, P = 0.043 for PFS), as well as in the multivariate analysis (HR = 3.71, 95% CI: 1.25-11.02, P = 0.018 for OS, and HR = 4.83, 95% CI: 1.91-12.27, P = 0.001 for PFS). The patients with high CD68 expression exhibited 2-year OS and PFS rates of 42.9%, and 10.0%, respectively, in comparison to 81.5%, and 50.7% for those with low CD68 expression (P = 0.048 for OS, and P = 0.035 for PFS) (Figure 1A, 1B). In a subgroup analysis of 60 patients who did not receive upfront ASCT, high CD68 expression was associated with inferior OS and PFS compared to low CD68 expression (P = 0.014 for OS, and P = 0.016 for PFS) (Figure 1C, 1D). The difference in OS and PFS between the high and low CD68 expression groups in the non-upfront ASCT subgroup (n = 60) was significant in the univariate (HR = 3.63, 95% CI: 1.21-10.88, P = 0.021 for OS, and HR = 2.60, 95% CI: 1.15-5.86, P = 0.021 for PFS) as well as in the multivariate analysis (HR = 4.05, 95% CI: 1.35-12.16, P = 0.013 for OS, and HR = 5.80, 95% CI: 2.25-14.95, P < 0.001 for PFS). However, the OS and PFS in the upfront ASCT cohort (n = 16) were similar between the high and low CD68 expression groups (P = 0.426 and P = 0.848, respectively) (Figure 1E, 1F). There were no differences in OS and PFS according to the expression level of FoxP3 in all patients as well as in subgroup of patients who did not receive upfront ASCT. Conclusion High level of CD68 expression in patients with PCNSL was significantly associated with inferior OS and PFS, especially in non-upfront ASCT treated subgroup of patients. FoxP3 expression level was also not associated with survival in this study. We suggest CD68 as a potential biomarker at initial PCNSL diagnosis and upfront ASCT consolidative strategy might improve survival in PCNSL patients by overcoming negative impact of high CD68 expression. Further validation studies are warranted. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

Prognostic potential of tumoral FOXP3 expression in relation with tumor-infiltrating regulatory T cells in human gastric cancers.

2013 ◽  
Vol 31 (15_suppl) ◽  
pp. e15036-e15036
Author(s):  
Jungho Suh ◽  
Wankyu EO ◽  
Si-Young Kim
Keyword(s):  
Gastric Cancer ◽  
Overall Survival ◽  
T Cells ◽  
Regulatory T Cells ◽  
Cancer Cells ◽  
Prognostic Marker ◽  
Prognostic Significance ◽  
Foxp3 Expression ◽  
Cancer Tissue ◽  
Human Gastric Cancer

e15036 Background: Expression of the transcription factor FOXP3 is crucial for the regulatory T cells (Tregs) that engage in the maintenance of immunological self-tolerance and immune homeostasis. Recently, expression of FOXP3 in cancer cells and its association with prognosis have been shown in clinical studies. For gastric cancer, however, prognostic significance of the tumoral FOXP3 expression and its relationship with Tregs remains unknown. We observed the tumoral FOXP3 and Tregs from the 118 gastric cancer patients who underwent surgery to explore its relationships with the prognosis. Methods: Tissue samples from 118 cases of gastric cancer were used for the present study. We investigated the tumoral expression of FOXP3 and Tregs count in human gastric cancer tissue by the use of immunohistochemical analysis using a tissue microarray to explore the relation with clinicopathological variables by retrospective manner. Results: FOXP3-positive cancer cells were observed in 62 of 118 (52.5%) patients. Positive Tregs (Tregs≥10/HPF) were observed in 66 of 118 (55.9%) patients. There was significant positive relationship between positive Tregs count and the tumoral FOXP3 expression (P=0.006).Positive tumoral FOXP3 expression was significantly related with the better disease free survival but not with the overall survival. But increased Tregs count was significantly related with the better overall survival (P<0.01, P<0.01, respectively). When we divide the patients into four groups by the FOXP3 expression and the Tregs count, FOXP3/Tregs(+/+) group showed the best overall survival followed by FOXP3/Tregs(-/+) group, FOXP3/Tregs(+/-,) and FOXP3/Tregs(-/-), respectively. And the survival difference between the FOXP3/Tregs(+/+)-FOXP3/Tregs(-/+) group and the FOXP3/Tregs(+/-)-FOXP3/Tregs(-/-)group became more prominent by the Tregs count. Conclusions: These results suggest that positive tumoral FOXP3 expression in relation with the high Treg count is a new prognostic marker in gastric cancer. The combination of tumoral FOXP3 and Tregs enhanced its statistical power more than separated as a prognostic marker.

Prognostic role of regulatory T cells in lymphoma: a systematic review and meta-analysis

2020 ◽  
Vol 146 (12) ◽  
pp. 3123-3135
Author(s):  
Fei Peng ◽  
You Qin ◽  
Shidai Mu ◽  
Jingwen Li ◽  
Lisha Ai ◽  
...  
Keyword(s):  
Systematic Review ◽  
T Cells ◽  
Regulatory T Cells ◽  
Meta Analysis ◽  
Prognostic Role

Stability of Foxp3 Expression Is a Critical Factor In the Ability of Regulatory T Cells to Mitigate Graft Versus Host Disease

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 731-731
Author(s):  
Amy Beres ◽  
Richard Komorowski ◽  
William R. Drobyski
Keyword(s):  
T Cells ◽  
T Cell ◽  
Regulatory T Cells ◽  
Critical Factor ◽  
Foxp3 Expression ◽  
Spleen Cells ◽  
Graft Versus Host

Abstract Abstract 731 Graft versus host disease (GVHD) is a proinflammatory T cell-mediated syndrome that is the major complication of allogeneic bone marrow transplantation (BMT). During the course of GVHD, there is a progressive loss of regulatory T cells (Tregs), leading to an imbalance between the effector and regulatory arms of the immune system. Tregs have been subdivided into two distinct subsets, termed natural and induced, which have overlapping yet unique characteristics. While the role of natural regulatory T cells (nTregs) in GVHD biology has been extensively examined, the role of induced regulatory T cells (iTregs) remains largely unknown. An attractive aspect of the latter cell population is that they can be differentiated in vitro from conventional T cells and expanded in large numbers making them a potential source for regulatory T cell therapy in vivo. To determine whether in vitro-expanded iTregs were able to suppress alloreactive donor T cell responses and to compare the efficacy of these cells relative to nTregs, studies were performed using an MHC-incompatible murine BMT model (B6[H−2b]−Balb/c[H−2d]). In initial studies, purified CD4+ Foxp3EGFP– T cells obtained from B6 Foxp3EGFP reporter mice were cultured with anti-CD3 and anti-CD28 antibodies in the presence of IL-2 and TGF-b. After three days in culture, approximately 60–70% of cells were Foxp3+, expressed GITR, CD25, and CD103, and were equally suppressive to nTregs in mixed lymphocyte cultures. To determine if iTregs were suppressive in vivo, lethally irradiated Balb/c mice were transplanted with either B6 BM alone, B6 BM and spleen cells, or B6 BM/spleen cells and in vitro-expanded iTregs. In contrast to in vitro results, adoptive transfer of iTregs failed to protect mice from lethal GVHD even when administered at high Treg: effector T cell ratios (5:1) and were much less effective than equivalent doses of nTregs at abrogating GVHD pathology. iTregs also had no additive effect when co-administered with nTregs. Notably, we observed that whereas transferred nTregs persisted for up to 60 days in transplanted animals, iTregs were undetectable after only 14 days in liver, lung, colon and spleen, indicating that reduced in vivo survival was a potential explanation for the lack of protection. Further examination, however, revealed that the inability to detect iTregs was primarily attributable to the loss of Foxp3 expression and the subsequent in vivo reversion of these cells to a proinflammatory phenotype characterized by the secretion of interferon-gamma. In prior studies (Chen et al, Blood, 2009), we demonstrated that blockade of IL-6 signaling augmented reconstitution of nTregs and reduced overall GVHD severity. To determine whether inhibition of IL-6 could stabilize Foxp3 expression and prevent phenotypic reversion of iTregs, lethally irradiated Balb/c recipients were transplanted with B6 BM and spleen cells along with in vitro-differentiated iTregs and then treated with either isotype control or anti-IL-6R-specific antibody. Analysis of cells obtained from spleen, liver, lung and colon revealed that blockade of IL-6 signaling did not prevent loss of Foxp3 expression or reversion of iTregs to a Th1 cytokine phenotype. While Tregs can be converted from conventional T cells in vitro, they can also be generated in vivo during inflammatory syndromes. We therefore examined whether in vivo induction of iTregs occurred during GVHD and the extent to which blockade of IL-6 signaling affected iTreg expansion and overall GVHD protection. To address this question, lethally irradiated Balb/c mice were transplanted with B6 Rag-1 BM cells and purified CD4+ Foxp3EGFP– T cells, and then treated with either anti-IL-6R or control antibody. We observed that in vivo conversion of Tregs was negligible in control animals (<1%), but that administration of anti-IL-6R antibody significantly increased the relative and absolute number of iTregs in GVHD target tissues with a commensurate reduction in overall pathological damage. Thus, blockade of IL-6 signaling was able to enhance reconstitution of iTregs in vivo, but had no discernible affect on adoptively transferred iTregs. In summary, these studies demonstrate that the stability of Foxp3 expression is a critical factor in the maintenance of transplantation tolerance and that instability of expression limits the utility of adoptively transferred iTregs as a source of cellular therapy for the abrogation of GVHD. Disclosures: No relevant conflicts of interest to declare.

Generation of Functional Regulatory T Cells From Umbilical Cord Blood Naïve T Cells: Potential Role of Hydroxymethylation in the Epigenetic Reprograming and Transcriptional Induction of FoxP3

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4835-4835
Author(s):  
Rodrigo Haddad ◽  
Josiane Lilian dos Santos Schiavinato ◽  
Felipe Saldanha Saldanha-Araújo ◽  
Priscila S Scheucher ◽  
Amélia G Araújo ◽  
...  
Keyword(s):  
T Cells ◽  
Regulatory T Cells ◽  
Cord Blood ◽  
Umbilical Cord ◽  
Umbilical Cord Blood ◽  
Foxp3 Expression ◽  
Methylation Pattern ◽  
Active Demethylation

Abstract Abstract 4835 The development and functionality of CD4+CD25hi regulatory T cells (Tregs) depends on stable FoxP3 expression, a central regulator of Treg differentiation. It is believed that this is accomplished by regulatory regions in the promoter and 3 evolutionarily conserved noncoding sequences, termed CNS1, CNS2 (or TSDR) and CNS3. The activation of TCR (with anti-CD2/3/28) in CD4+CD25− naïve T cells from PBMCs, in the presence of IL-2, TGF-β and atRA, induces the generation of Foxp3+ induced regulatory T cells (iTreg). While demethylation of 5mC residues in the CNS2 is associated with stable FoxP3 expression in nTregs, the epigenetic events involved in the regulation of FoxP3 in iTregs remains unexplored. Recently, the oxidation of 5-mC, originating hidroxymethylated 5-hmC residues, have been described as a key mechanism of active demethylation, with roles in biological processes, such as regulation of pluripotency and differentiation of hematopoietic stem cells. In contrast to PBMCs, in umbilical cord blood (UCB) T cells are mainly naïve making UCB an attractive source for the development of protocols for generation of iTregs. Here, we evaluated the iTregs generation from UCB naïve T cells. In addition, we compared the expression of FoxP3 on iTregs and on naturally occurring Tregs (nTregs) obtained from PBMCs. Also, we evaluated the methylation pattern of promoter and CNS2 and CNS3 in nTregs, fleshly isolated naïve T cells, activated naïve T cells (Teff), and iTregs. Finally, we evaluated the ability of iTregs, to suppress the proliferation of activated T cells, as compared to nTregs. For this, CD4+CD25-CD45RA+ naïve T cells were immunomagnetically isolated from UCB and activated with anti-human CD2/CD3/CD28 beads (1:2 beads:cell ratio) in the presence of IL-2 (50 U/ml) with (iTregs) or without (Teff) TGF-β (5 ng/ml) and atRA (100 nM) for 5 days. In parallel, PBMCs from 5 individuals were obtained for nTregs phenotypic characterization. CD4+ gated cells from iTregs and from PBMC were analyzed by flow cytometry for FoxP3 expression in the CD25+, CD25hi and CD25−population. nTregs (CD4+CD25+CD127−) were immunomagnetically isolated from PBMCs and CD4+CD25hi and CD4+CD25− populations were FACS-sorted from iTreg to observe the potential in regulate the proliferation of CD3+ T cells (CFSE staining). Finally, methylation pattern analysis of FoxP3 locus, including CNS2 and CNS3, were performed in naïve T cells, nTregs, iTreg and Teff. The mean percentage of FoxP3+ cells in CD4+CD25hi from iTreg was 98.5%, as compared to 82.4% in PBMCs. Interestingly, the percentage of FoxP3+ cells in total CD4+CD25+ was higher in cells from iTreg (97,3%) than on PBMCs (26,8%). Moreover, while the percentage of FoxP3+ cells in the CD4+CD25− population, was very low in PBMCs (2,8%), up to 55% of the cells derived from iTreg were FoxP3+. The immunossupression assay showed that, compared to activated CD3+ T cells cultured alone, nTregs (CD4+CD25+CD127−) decreased the proliferation of CD3+ T cells in 55%, while iTregs (CD4+CD25hi) decreased the proliferation in 46%. Interestingly, the CD4+CD25− population from iTreg (55% of FoxP3+ cells) also decreased the proliferation of CD3+ T cells, but to a lower extent (21%). Additionally, while naïve T cells and Teff presented low level of 5hmC in both segments evaluated of CNS2 (∼1%); upon in vitro induction, iTregs presented 5hmC levels comparable to that of nTregs (5–11% and 5% respectively), in line to FoxP3 expression. Furthermore, CNS3, which was found to be partially demethylated in naïve T cells and nTregs (45 and 50% respectively), presented even higher levels of demethylation upon activation in iTregs and nTregs (77 and 82% respectively). In summary, we show that functional Foxp3+CD4+CD25hi T cells can be generated in vitro from UCB naïve T cells. Additionally, our results indicate that active demethylation of CNS2 occurs in a TGF-β and atRA-dependent manner during iTregs generation. Moreover, the partial demethylation of CNS3 observed in naïve T cells and nTregs, and the increased demethylation promoted by activation (in Teff and iTreg), is consistent with the role of CNS3 as a pioneer element that initiates FoxP3 transcription. Our results contribute to the understanding of the epigenetic mechanisms underlying the differentiation of Tregs and may help in the development of protocols for the generation of functional iTregs for future therapeutic applications. Support: FAPESP, CNPq. Disclosures: No relevant conflicts of interest to declare.

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