Tregs In Cancer Patients Research Articles

Tregs, gMDSCs, and levels of soluble MICA as prognostic biomarkers for combined NEO-201 and pembrolizumab.

2530 Background: The humanized IgG1 monoclonal antibody NEO-201 binds to Core 1 and/or extended Core 1 O-glycans expressed by several human solid and blood tumors, as well as neutrophils, and mediates killing of cancer cells, neutrophils, regulatory T cells (Tregs) and granulocytic myeloid-derived suppressor cells (gMDSCs) via ADCC and CDC. Resistance to PD-1/PD-L1 blockade may be due to the accumulation of Tregs and gMDSCs in the tumor microenvironment (TME). NEO-201 was proven to bind and reduce the amount of circulating Tregs in cancer patients. This supports the rationale of the ongoing phase II clinical trial (NCT03476681) evaluating the activity of NEO-201 with pembrolizumab in adults with solid tumors resistant to prior checkpoint inhibitors. Furthermore, elevated serum levels of soluble MICA (sMICA) have been correlated with decreased NK cell activity. Methods: Cancer patients were treated each cycle with 3 doses of NEO-201 1.5mg/kg every 2 weeks and pembrolizumab 400mg IV every 6 weeks and imaged for response every 2 cycles. PBMCs and serum from cancer patients pre- and at multiple time points post-treatment were used to evaluate the percentage of circulating gMDSCs and Tregs (flow cytometry) and sMICA levels (ELISA). Results: NEO-201 recognizes naïve Tregs (nTregs: CD3+/CD4+/CD45RA+/Foxp3low cells) but not effector Tregs (eTregs: CD3+/CD4+/CD45RA-/Foxp3high cells). NEO-201 also binds to gMDSCs, defined as HLA-DRneg/CD33+/CD15+/ CD14neg/CD66b+ cells. In patients with durable SD, a patient with HNSCC (SD >11 months) showed >50% reduction of nTregs and >90% of gMDSCs at C3D1 (beginning of cycle 3) compared to baseline. One patient with cervical cancer (SD >8 months) showed 40% reduction of nTregs at C3D1 compared to baseline, while gMDSCs trended down to baseline levels at C3D1 after initial increase. Conversely, we observed a general uptrend of nTregs and gMDSCs in patients with PD after treatment. Median serum levels of sMICA pre-treatment were 33-fold higher in patients with PD compared to patients with SD. Levels of sMICA remained elevated in patients with PD and low in patients with SD at all time points post-treatment. Conclusions: Depletion of circulating Tregs and gMDSCs may prevent their accumulation in the TME and enhance the efficacy of pembrolizumab in subjects with tumors resistant to checkpoint inhibitors. The decrease in circulating nTregs and gMDSCs after treatment with NEO-201 and pembrolizumab was associated with durable SD. High levels of sMICA can impair ADCC mediated by NEO-201, resulting in poor clinical response. Low levels of sMICA in patients with SD, together with the reduction of both circulating nTregs and gMDSCs, could be favorable prognostic markers for clinical benefit following treatment with NEO-201 and pembrolizumab. Ongoing enrollment in this clinical trial will validate these findings in larger cohorts. Clinical trial information: NCT03476681 .

Abstract 2716: The O-glycan epitope targeting anti-human carcinoma monoclonal antibody (mAb) NEO-201 can also target human regulatory T cells (Tregs)

Abstract Introduction:NEO-201 is a humanized IgG1 mAb that targets Core 1 and/or extended Core 1 O-glycans expressed by several human solid and blood tumors, as well as neutrophils, but it does not bind to most normal tissues and human immune cell subsets (B cells, CD4+ T cells, CD8+ T cells, NK cells, monocytes). Functional analysis revealed that NEO-201 mediates the killing of its target cells through ADCC and CDC. Previous study has shown that approximately 4.6% of CD4+ T cells express NEO-201 target antigen and that those CD4+ T cells have Tregs phenotype. In addition, in a recent published phase 1 study we observed that NEO-201 binds to CD4+/CD25+/, CD127−/, Foxp3+/, CD15s+ cells from peripheral blood mononuclear cells (PBMCs) from cancer patients with solid tumors. The purpose of this study was to further characterize the phenotype of the specific subset of CD4+ T cells expressing the NEO-201 target antigen. Experimental Design: Human PBMCs were obtained from 7 healthy donors (HD) collected at Osaka University and 6 cancer patients from our ongoing phase II clinical trial (Clinical Trial NCT03476681) evaluating the efficiency of the combination of NEO-201 with pembrolizumab in adults with solid tumors resistant to checkpoint inhibitors. Phenotypic analysis was performed by flow cytometry using NEO-201 and antibodies CD3, CD4, CD25, CD45RA, FoxP3, and CD15s. The same gating strategy was applied for both normal donors and patients to evaluate which fraction of CD4+ T cells is recognized by NEO-201. Fraction (Fr) 1 is naïve Treg, Fr ll is effector Treg, Fr lll is non-Treg, Fr lV is effector CD4 T cells and Fr V is naïve CD4 T cells. Results: Flow cytometry analysis of PBMCs revealed that NEO-201 recognizes naïve Tregs (nTregs: CD3+/CD4+/CD45RA+/Foxp3low cells) while it does not bind to effector Tregs (eTregs: CD3+/CD4+/CD45RA−/Foxp3high cells) in both HD and cancer patients. The % of nTregs in cancer patients was higher than HD. Preliminary results from the ongoing Phase II clinical trial showed that subjects with durable SD had a reduction of circulating nTregs after treatment with NEO-201 compared to baseline levels. Conclusions: NEO-201 binds to human Tregs with significantly higher % of binding to Fr l and a greater % of Tregs expressing NEO-201 target antigen in cancer patients compared to HD. It is conceivable that naïve Tregs in cancer patients express high levels of Core 1 O-glycans. Furthermore, when subjected to TCR stimulation, naïve Tregs undergo proliferation and differentiate into eTregs. eTregs can then infiltrate into tumor microenvironment (TME). These data suggest that depletion of circulating nTregs in cancer patients after NEO-201 treatment may prevent the differentiation of nTregs into eTregs and their accumulation in the TME. This study suggests the potential use of NEO-201 to reduce the Treg-mediated suppression of anticancer immunity. Citation Format: Atsushi Tanaka, Massimo Fantini, Philip M. Arlen, Christina M. Annunziata, Kwong Y. Tsang, Shimon Sakaguchi. The O-glycan epitope targeting anti-human carcinoma monoclonal antibody (mAb) NEO-201 can also target human regulatory T cells (Tregs) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2716.

Expression of costimulatory and inhibitory receptors in FoxP3+ regulatory T cells within the tumor microenvironment: Implications for combination immunotherapy approaches.

The unprecedented success of immune checkpoint inhibitors has given rise to a rapidly growing number of immuno-oncology agents undergoing preclinical and clinical development and an exponential increase in possible combinations. Defining a clear rationale for combinations by identifying synergies between immunomodulatory pathways has therefore become a high priority. Immunosuppressive regulatory T cells (Tregs) within the tumor microenvironment (TME) represent a major roadblock to endogenous and therapeutic tumor immunity. However, Tregs are also essential for the maintenance of immunological self-tolerance, and share many molecular pathways with conventional T cells including cytotoxic T cells, the primary mediators of tumor immunity. Hence the inability to specifically target and neutralize Tregs within the TME of cancer patients without globally compromising self-tolerance poses a significant challenge. Here we review recent advances in the characterization of tumor-infiltrating Tregs with a focus on costimulatory and inhibitory receptors. We discuss receptor expression patterns, their functional role in Treg biology and mechanistic insights gained from targeting these receptors in preclinical models to evaluate their potential as clinical targets. We further outline a framework of parameters that could be used to refine the assessment of Tregs in cancer patients and increase their value as predictive biomarkers. Finally, we propose modalities to integrate our increasing knowledge on Treg phenotype and function for the rational design of checkpoint inhibitor-based combination therapies. Such combinations have great potential for synergy, as they could concomitantly enhance cytotoxic T cells and inhibit Tregs within the TME, thereby increasing the efficacy of current cancer immunotherapies.

Coexistence of regulatory B cells and regulatory T cells in tumor-infiltrating lymphocyte aggregates is a prognostic factor in patients with breast cancer.

Tumors can acquire tolerance to tumor immunity and develop enhanced proliferation. Regulatory B cells (Bregs), whose role in immune tolerance is similar to that of regulatory T cells (Tregs), appear to be involved in tumor immunity. Recently, Bregs were found to induce Tregs against tumor immunity. However, the platform for the coexistence of Bregs and Tregs in cancer patients and its clinical significance remain unclear; thus, they were evaluated in breast cancer patients. In 489 breast cancer patients, CD25- and IL10-positive Bregs and Foxp3-positive Tregs were immunohistochemically evaluated in tumor-infiltrating lymphocyte aggregates (TIL aggregates) that consisted of CD19-positive B-cell follicles and CD3-positive T-cell parafollicles. Then the correlations of the localization and existence of these cells with metastasis-free survival (MFS) were evaluated in breast cancer patients. TIL aggregates were observed in marginal regions of tumors in breast cancer patients. In the TIL aggregates, the existence of Bregs was closely related to that of Tregs (p < 0.0001). On multivariate analysis, the coexistence of Bregs and Tregs in TIL aggregates was correlated with MFS in breast cancer patients (p = 0.007). Furthermore, MFS was significantly shorter for patients with the coexistence of Tregs and Bregs in TIL aggregates than in those with Tregs alone without Bregs (p = 0.0475). The present results suggest that Bregs are related to the induction of Tregs in TIL aggregates and the development of metastasis of breast cancer cells. Bregs are expected to be a new diagnostic and therapeutic target in breast cancer patients.

Abstract 2787: Generation of first-in-class anti-CD25 antibodies depleting Treg without interfering with IL2 signalling for cancer therapies

Abstract Regulatory T cells (Treg) are key players of the suppressive tumour microenvironment (TME). Their presence is correlated with a bad prognosis in multiple cancers, while a greater ratio of effector T cells (Teff) to Treg is associated with improved outcome. Studies demonstrating high expression of CD25 on Tregs but not Teff in human tumors have underscored its relevance as a target for Treg depletion. However, clinical trials conducted to target Treg in cancer patients with existing anti-CD25 (aCD25) antibodies have shown contradicting results, and no aCD25 antibody is in clinical development for this application. Importantly, aCD25 antibodies tested to date block IL2 signalling via CD25. We demonstrated (Solomon et al. AACR2018) that such blocking drastically reduces the therapeutic activity of aCD25 antibodies and that a single administration of a depleting antibody targeting CD25 but not blocking the IL2 signalling effectively depletes Treg, increases Teff activity and promotes eradication of established tumors in several mouse models of cancer. With this rationale in mind, we have generated a panel of fully human aCD25 IgG1 antibodies, which we characterized for their binding to human and cynomolgus monkey CD25. The antibodies were then screened for their impact on IL2 binding, using a sandwich binding assay on Octet, and on IL2 signalling, using a STAT5 phosphorylation assay. Antibodies were also tested for their ability to deplete CD25 positive cell lines and in vitro-derived human Treg in ADCC and ADCP assays. Finally, the impact of selected antibodies on Treg within the TME and on Teff responses was evaluated in human samples. Among the antibodies binding to human and cynomolgus CD25, we have selected a panel interfering with neither IL2 binding to CD25 nor IL2 signalling. Interestingly, epitope binning assays demonstrated that none of the selected antibodies bind to epitopes overlapping with those of the existing clinical antibodies, Daclizumab and Basiliximab. Among the non-IL2 blocking antibodies, those showing the maximum target cell lysis via ADCC and highest phagocytosis via ADCP represent potential lead clinical candidates. Most interestingly, contrary to the existing clinical aCD25 antibodies, which block IL2 signalling, our candidates do not inhibit Teff proliferation and function, confirming the specificity of action toward Treg and the importance of preserving IL2 signalling. Finally, we have shown that our clinical candidates efficiently deplete Treg in the TME. We present the first anti-human CD25 antibodies selected for their capacity to deplete human Treg while preserving IL2 signalling and activity of Teff. These antibodies provide a novel therapeutic approach to alleviate immune suppression in the TME, which would provide an ideal combination partner for existing standard of care and IO treatments, and could potentially be used as a monotherapy. Citation Format: Josephine C. Salimu, Mark Brown, Pascal Merchiers, Beatriz Goyenechea, Kevin Moulder, Robert Dejonge, Aghiles Boughetane, Isabelle Solomon, Frederick Arce Vargas, Karl S. Peggs, Anne Goubier, Sergio A. Quezada. Generation of first-in-class anti-CD25 antibodies depleting Treg without interfering with IL2 signalling for cancer therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2787.

Abstract 3850: FOXP3 dimerization by stapled alpha-helical peptides alters regulatory T cell function

Abstract Regulatory T cells (Tregs) are critical for the maintenance of immune tolerance and control of immune responses to foreign agents. Tregs depend upon the transcriptional protein FOXP3 for their ontogeny and genetic orchestration of their suppressive mechanisms. While T cells have the ability to target tumor-associated antigens from many cancer subtypes, these T cells are often suppressed in patients with immunologically “silenced” tumors. The tolerizing function of Tregs can limit effective anti-tumor T cell-mediated killing, even in the presence of immunotherapies, including checkpoint inhibition, and their presence predicts reduced survival in patients with a myriad of cancers. Currently, there exists no means of depleting Tregs in patients without also targeting activated effector T cells. In an effort to overcome this, we have specifically inhibited Tregs by targeting FOXP3 through molecular mimicry using peptides designed to inhibit FOXP3 homodimerization. Homodimerization of FOXP3 is mediated by an antiparallel coiled-coil interaction between the leucine-zipper domains of two FOXP3 proteins. A series of single and double hydrocarbon-stapled alpha-helical (SAH) peptides were synthesized using the leucine zipper dimerization domain (DD) interface of FOXP3 (SAH-FOXP3DDs) as a molecular template. SAH-FOXP3DDs, but not native peptides, specifically bind FOXP3's leucine zipper domain with nanomolar affinity. Unlike stapled peptide point mutant controls, lead SAH-FOXP3DDs also showed dose-dependent inhibition of FOXP3 binding to cognate DNA. Lead SAH-FOXP3DDs are cell permeable and nontoxic to T cells as measured by absence of non-specific LDH-release and apoptosis induction following treatment. Flow cytometric analysis of treated Tregs measured changes in expression of a number of Treg/T cell - associated proteins including increases in FOXP3 and CD127 and decreases in CD25, while no changes were observed in treated conventional CD4+ T cells (Tcons) or with cells treated with point-mutant control peptides. In addition, treated Tregs demonstrated mRNA expression changes of FOXP3-regulated genes including Foxp3, CD127, IL2 and Ctla. Treatment of freshly isolated and in vitro expanded Tregs with lead SAH-FOXP3DD compounds inhibited Treg-mediated suppression of Tcon proliferation in response to CD3/CD28 activation, while no effects were measured with a point-mutant control. We believe that SAH-FOXP3DDs will be powerful molecular probes to further our understanding of FOXP3 transcriptional control. Additionally, they may be a foundation for prototype therapeutics to target Tregs in cancer patients in an effort to amplify the endogenous anti-tumor immune response alone or in combination with other cancer immunotherapies. Citation Format: Rachel Eclov, Marie Fefferman, Ravand Samaeekia, James L. LaBelle. FOXP3 dimerization by stapled alpha-helical peptides alters regulatory T cell function [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3850.

Canonical Wnt signaling in Treg contributes to colon cancer

Abstract Regulatory T-cells (Tregs) play a dual role in colon cancer, promoting immune tolerance and suppressing inflammation. Two types of Tregs infiltrate the gut. Thymic derived Helios+CD4+Foxp3+ Tregs have high affinity for self-antigens, including tumor-antigens and protect the tumor, while Helios−CD4+Foxp3+ extra-thymic Tregs that are generated in the gut have microbial specificity and suppress harmful inflammation. In colon cancer, there is a preferential expansion of a population of Helios−CD4+Foxp3+RORγt+ Tregs with enhanced T-cell suppressive properties and pro-inflammatory properties that promote tumor growth. We showed that this population of Tregs in cancer patients expresses elevated levels of β-catenin. To test the role of canonical Wnt signaling in the generation of pro-inflammatory Tregs, we stabilized β-catenin or deleted its DNA binding partner TCF-1 in Tregs. In both instances expression of RORγt was upregulated. While the stabilization of β-catenin led to loss of Treg functions, ablation of TCF-1 reproduced the phenotype and functional characteristics of Tregs in colon cancer. The TCF-1 deficient Tregs were pro-inflammatory, potent T-cell suppressive, and tumor promoting.

The role of regulatory T cells in cancer immunology.

Regulatory T cells (Treg) are generally considered to be significant contributors to tumor escape from the host immune system. Emerging evidence suggests, however, that in some human cancers, Treg are necessary to control chronic inflammation, prevent tissue damage, and limit inflammation-associated cancer development. The dual role of Treg in cancer and underpinnings of Treg diversity are not well understood. This review attempts to provide insights into the importance of Treg subsets in cancer development and its progression. It also considers the role of Treg as potential biomarkers of clinical outcome in cancer. The strategies for monitoring Treg in cancer patients are discussed as is the need for caution in the use of therapies which indiscriminately ablate Treg. A greater understanding of molecular pathways operating in various tumor microenvironments is necessary for defining the Treg impact on cancer and for selecting immunotherapies targeting Treg.

Abstract 1291: Tumor-infiltrating FOXP3+ T-regulatory (Treg) cells in early-stage human lung cancer exhibit enhanced suppressive function when compared to blood or lymph node (LN) Treg cells

Abstract Aim: Advances in immunotherapy require a detailed understanding of host immunity. Data from murine models indicate solid tumors of various types can recruit Tregs and/or promote iTreg development, and thereby curtail antitumor responses. However, clinical data have been restricted to either evaluation of Treg numbers in tumors, or studies of PBMC Treg in patients with cancers. Surprisingly, no studies of Treg function have ever been reported using Tregs isolated from tumors themselves. Methods: Using our recently reported technique, we isolated Tregs within 20 mins of resection from 13 lung cancer patients, stages Ia-IIIa, and 2 control non-cancer patients (diaphragmatic hernia and lung granuloma). The mean age of the cancer patients (10 males, 3 females) was 65.4±2.2 years; 7 patients had adenocarcinoma, 3 had squamous cell carcinoma, and the remainder had large cell neuroendocrine carcinoma, colon adenocarcinoma and high-grade carcinoma. The mean size of their tumors was 4.0±0.8 cm, and 1 of the 13 patients developed metastases during the follow-up period. We compared the suppressive function of Tregs isolated from each patient's freshly resected tumor, LN and blood, using cryopreserved healthy donor PBMC aliquots as responders, and area-under-curve (AUC) analysis. We controlled for comparable CD4+ and FOXP3+ purity in all isolated Treg samples, and cell viability, excluding from further analysis all data from non-comparable FOXP3+ subsets. Thus, we were able to avoid artifacts arising from different cell conditions (e.g. use of autologous responder cells) or from unequal FOXP3+ content in Treg isolates. Results: Tumor-infiltrating Tregs had significantly enhanced suppressive function when compared with Tregs isolated from other locations in the same patient. Thus, tumor Tregs were significantly more suppressive than lung LN Tregs (p = 0.0003), and significantly more suppressive than blood Tregs (p = 0.03). Tregs isolated from LN in close proximity to tumors had comparable suppressive function to Tregs isolated from distant LN (p&amp;gt;0.05), and blood Tregs had comparable suppressive function to that of LN Tregs (p&amp;gt;0.05). Additional analyses showed that the enhanced suppressive function of tumor Treg was not due to Treg-induced killing/apoptosis of responder cells, cytokine and/or growth factor deprivation, or soluble factors produced by tumor Treg cells. Conclusions: We demonstrate for the first time that tumor-infiltrating human lung cancer Treg have significantly increased suppressive function. This finding is important for the development of effective anti-tumor therapy. While we are continuing to study the basis for such differences, our data suggest that studies of peripheral blood Tregs in cancer patients may be inadequate and inefficient approaches for understanding the fundamental mechanisms of limited host antitumor immune responses. Citation Format: Tatiana Akimova, Evgeniy B. Eruslanov, Pratik S. Bhojnagarwala, Jon G. Quatromoni, Jacqueline Morgen, Sunil Singhal, Steven M. Albelda, Wayne W. Hancock. Tumor-infiltrating FOXP3+ T-regulatory (Treg) cells in early-stage human lung cancer exhibit enhanced suppressive function when compared to blood or lymph node (LN) Treg cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1291. doi:10.1158/1538-7445.AM2015-1291

FoxP3+Helios+, but not FoxP3+Helios-, Tregs express GARP and LAP, representing the expanded subpopulation in pancreatic cancer patients (TUM2P.911)

Abstract Recent evidences show that Tregs negatively impact anti-tumour immunity and cancer immunotherapy. We, and others, reported that Tregs are expanded in peripheral blood and tumour microenvironment of cancer patients, correlating with poor prognosis and reduced survival. Our work has further established that Treg infiltration of tumors is correlated with a lack of patients’ responsiveness to therapy. Recent observations indicate that Tregs could be phenotypically and functionally different in cancer settings. However, molecules contributing to their enhanced immunosuppressive activity are yet to be elucidated. We recently found that the majority of peripheral and tumor-infiltrating FoxP3+ Tregs expresses Helios, an Ikaros family transcription factor. Interestingly, FoxP3+Helios+ Treg subset is significantly higher in cancer patients, compared to healthy individuals. Herein we show that markers of activated Tregs [latency associated peptide (LAP) and glycoprotein A repetitions predominant (GARP, or LRRC32)] are mainly expressed on FoxP3+Helios+ Treg subset, but not on FoxP3+Helios- Treg subset. Additionally, FoxP3+Helios+GARP+LAP+ activated Tregs represent the expanded subset in pancreatic cancer patients, compared to controls. Taken together, these observations confirm that the expanded FoxP3+Helios+ Tregs in cancer patients represent the activated Treg subset, with more suppressive characteristics, that could be targeted by specific protocols.

Human hepatocellular carcinoma-infiltrating CD4+CD69+Foxp3− regulatory T cell suppresses T cell response via membrane-bound TGF-β1

Tumors can recruit, induce, and expand regulatory T cells (Tregs) to suppress antitumor immune responses for survival and progression. The complicated tumor-related Treg subsets and their functional mechanisms are not fully addressed yet. We have previously identified a novel CD4⁺CD69⁺Foxp3⁻ Treg subset in tumor-bearing mice, which suppresses CD4 T cell response via membrane-bound transforming growth factor beta 1 (mTGF-β1) and then promotes tumor progression. In hepatocellular carcinoma patients, here, we identified tumor-infiltrating human CD4⁺CD69⁺ Tregs which represent ~67.2 % of tumor-infiltrating CD4 T cells that is significantly higher than conventional CD4⁺CD25⁺Foxp3⁺ Tregs. They expressed mTGF-β1, PD-1, and CTLA-4, but not CD25 or Foxp3, and only produced a little interleukin (IL)-10 and TGF-β1. More importantly, they significantly suppressed CD4 T cell response via mTGF-β1 in vitro. Furthermore, the percentage of these CD4⁺CD69⁺ Tregs in tumor tissue was significantly correlated with tumor progression, which is more pronounced at the late stage of cancer patients. Thus, we have identified a tumor-induced new population of human CD4⁺CD69⁺ Tregs in cancer patients with phenotype of CD25⁻Foxp3⁻mTGF-β1⁺CTLA-4⁺PD-1⁺, and these Tregs can suppress antitumor immune response via mTGF-β1. Our results not only enrich the family of Treg subsets, providing new mechanistic insight to tumor-induced immune suppression in human, but also suggest a potential target for cancer immunotherapy. CD4⁺CD69⁺Foxp3⁻ regulatory T cells were identified in hepatocellular carcinoma patients. These Treg cells inhibit T cell response via membrane-bound TGF-β. The percentage of these cells was significantly correlated with tumor progression. The percentage of these cells was higher than conventional CD4⁺CD25⁺Foxp3⁺ Tregs. These Treg cells not only exist in tumor-bearing mice, but also in cancer patients.

1314 - Assessment of the Predictive/ Prognostic Value of the Myeloid-Derived Suppressor Cells (MDSC) and Regulatory T Cells (TREGS) in Non-Small Cell Lung Cancer (NSCLC). Preliminary Results

ABSTRACT Background The circulating MDSCs and Tregs in cancer patients suppress immune system. This study is investigating the expression of the MDSCs and Tregs in the peripheral blood of NSCLC patients and their correlation with the clinical outcome of the 1st line chemotherapy. Methods 62 chemotherapy naive patients (57 males) with stage IIIB/ IV NSCLC have been enrolled in this study, so far; median age 67. Peripheral blood was collected prior to treatment. 19 healthy, aged-matched donors (12 males) were used as controls. The distinct MDSC subpopulations [A (monocytic): CD11b+CD14+CD15-CD33+CD13+IL-4R+Linlow/-HLA-DR-; B (monocytic): CD11b+CD14+CD15+CD33+CD13+IL-4R+Linlow/-HLA-DR- and C (granulocytic): CD11b+CD14-CD15+CD33+ CD13+IL-4R+)], CD4+ Tregs (CD4+CD25+high CD127low FoxP3+CD39+CD13+) and CD8+ Tregs (CD3+CD8+CD25+ CD45RO+CD13+CCR7+FoxP3+CD39+) were determined by using flow cytometry. A comparison of the overall survival (OS) and the progression-free survival (PFS) according to the frequency of the MDSC and Tregs was performed (high expression defined as the percentage of cells above the 75% percentile of the controls). Results The levels of Tregs prior to treatment did not differ from the controls'. Patients with progression (PD) during the 1st line treatment had significantly elevated percentage of CD4+ (24.6 ± 8.5) and CD8+ (0.7± 0.2) Tregs at baseline compared to those with no PD (3.7± 1.8, p = 0.04; 0.1± 0.05, p= 0.03, respectively). In contrast, MDSCs were significantly increased (A: 3.8 ±0.7; β: 2.5 ± 0.5 and C: 10.8 ± 2.3) in patients compared to controls (0.8 ± 0.4, p = 0.001; 0.5 ± 0.2, p= 0.01, and 2.7 ± 1.3, p= 0.05, respectively) but that difference was not associated with response to treatment. Patients with normal CD8+ Tregs levels at baseline had higher OS and PFS compared to those with high levels (13.2 mo vs 7.9 mo, p= 0.02 and 13.1 mo vs 3.7 mo, p= 0.003, respectively). Conclusion The MDSCs are elevated in NSCLC. The increased expression of CD4+ καi CD8+ Tregs negatively correlated with the treatment outcome, indicating that CD4+ and CD8+ Tregs could be a potential predictive/prognostic biomarker. The study is still opened to accrual and more mature data will be presented at the meeting. Disclosure All authors have declared no conflicts of interest.

Differential Effects of Denileukin Diftitox IL-2 Immunotoxin on NK and Regulatory T Cells in Nonhuman Primates

Denileukin diftitox (DD), a fusion protein comprising IL-2 and diphtheria toxin, was initially expected to enhance antitumor immunity by selectively eliminating regulatory T cells (Tregs) displaying the high-affinity IL-2R (α-β-γ trimers). Although DD was shown to deplete some Tregs in primates, its effects on NK cells (CD16(+)CD8(+)NKG2A(+)CD3(-)), which constitutively express the intermediate-affinity IL-2R (β-γ dimers) and play a critical role in antitumor immunity, are still unknown. To address this question, cynomolgus monkeys were injected i.v. with two doses of DD (8 or 18 μg/kg). This treatment resulted in a rapid, but short-term, reduction in detectable peripheral blood resting Tregs (CD4(+)CD45RA(+)Foxp3(+)) and a transient increase in the number of activated Tregs (CD4(+)CD45RA(-)Foxp3(high)), followed by their partial depletion (50-60%). In contrast, all NK cells were deleted immediately and durably after DD administration. This difference was not due to a higher binding or internalization of DD by NK cells compared with Tregs. Coadministration of DD with IL-15, which binds to IL-2Rβ-γ, abrogated DD-induced NK cell deletion in vitro and in vivo, whereas it did not affect Treg elimination. Taken together, these results show that DD exerts a potent cytotoxic effect on NK cells, a phenomenon that might impair its antitumoral properties. However, coadministration of IL-15 with DD could alleviate this problem by selectively protecting potentially oncolytic NK cells, while allowing the depletion of immunosuppressive Tregs in cancer patients.

T-Regulatory Cells: Key Players in Tumor Immune Escape and Angiogenesis

T-regulatory cells (Tregs) are found infiltrating tumors in a vast array of tumor types, and tumor-infiltrating Tregs are often associated with a poor clinical outcome. Tregs are potent immunosuppressive cells of the immune system that promote progression of cancer through their ability to limit antitumor immunity and promote angiogenesis. Here, we discuss the ways in which Tregs suppress the antitumor immune response and elaborate on our recent discovery that Tregs make significant direct contributions to tumor angiogenesis. Further, we highlight several current therapies aimed at eliminating Tregs in cancer patients. Given the multifaceted role of Tregs in cancer, a greater understanding of their functions will ultimately strengthen future therapies.

Tremelimumab (anti-CTLA4) mediates immune responses mainly by direct activation of T effector cells rather than by affecting T regulatory cells

Cytotoxic T Lymphocyte Antigen 4 (CTLA4) blockade has shown antitumor activity against common cancers. However, the exact mechanism of immune mediation by anti-CTLA4 remains to be elucidated. Further understanding of how CTLA4 blockade with tremelimumab mediates immune responses may allow a more effective selection of responsive patients. Our results show that tremelimumab enhanced the proliferative response of T effector cells (Teff) upon TCR stimulation, and abrogated Treg suppressive ability. In the presence of tremelimumab, frequencies of IL-2-secreting CD4(+) T cells and IFN-γ-secreting CD4(+) and CD8(+) T cells were increased in response to polyclonal activation and tumor antigens. Importantly, Treg frequency was not reduced in the presence of tremelimumab, and expanded Tregs in cancer patients treated with tremelimumab expressed FoxP3 with no IL-2 release, confirming them as bona fide Tregs. Taken together, this data indicates that tremelimumab induces immune responses mainly by direct activation of Teff rather than by affecting Tregs.

T regulatory cells in cancer: recent advances and therapeutic potential

Importance of the field: The active suppression of immune responses against tumor is a major barrier to the likely success of cancer immunotherapy. There is now compelling evidence implicating T regulatory cells (Tregs) as being key players driving immune suppression. Elevated frequencies of Tregs within the peripheral circulation and tumor microenvironment of cancer patients correlate with poor prognosis and reduced survival. Understanding the mechanism of Treg elevation is critical for the development of new approaches aiming to modulate the frequency and function of Tregs to enhance the efficacy of cancer immune-based therapies.Areas covered in this review: This review focuses on current knowledge concerning Tregs in cancer and discusses putative mechanisms which underlie the expansion of Tregs in cancer patients. Additionally, we review current strategies to deplete/suppress Treg activity, the limitations of these strategies and future perspective for improving their efficacy.What the reader will gain: An insight of the current aspects concerning Treg subsets in cancer and an overview of recent advances in the identification of Treg-specific markers, in addition to the potential strategies to target Tregs for enhancing antitumor immunity.Take home message: Mechanisms by which Treg functions modulate the immune response to tumors are becoming further understood. However, specific markers to tumor-specific/induced Tregs are yet to be clearly identified, which is a major limitation in optimizing strategies to specifically target Tregs in cancer. Despite this, strategies aimed at modulating Tregs in patients are providing some early encouraging results supporting the overall concept and indicating that further studies are clearly warranted.

Antigen-specific Tregs control T cell responses against a limited repertoire of tumor antigens in patients with colorectal carcinoma.

Spontaneous antitumor T cell responses in cancer patients are strongly controlled by Tregs, and increased numbers of tumor-infiltrating Tregs correlate with reduced survival. However, the tumor antigens recognized by Tregs in cancer patients and the impact of these cells on tumor-specific T cell responses have not been systematically characterized. Here we used a broad panel of long synthetic peptides of defined tumor antigens and normal tissue antigens to exploit a newly developed method to identify and compare ex vivo the antigen specificities of Tregs with those of effector/memory T cells in peripheral blood of colorectal cancer patients and healthy subjects. Tregs in tumor patients were highly specific for a distinct set of only a few tumor antigens, suggesting that Tregs exert T cell suppression in an antigen-selective manner. Tumor-specific effector T cells were detectable in the majority of colorectal cancer patients but not in healthy individuals. We detected differences in the repertoires of antigens recognized by Tregs and effector/memory T cells in the majority of colorectal cancer patients. In addition, only effector/memory T cell responses against antigens recognized by Tregs strongly increased after Treg depletion. The selection of antigens according to preexisting T cell responses may improve the efficacy of future immunotherapies for cancer and autoimmune disease.

Clinical Use of Anti‐CD25 Antibody Daclizumab to Enhance Immune Responses to Tumor Antigen Vaccination by Targeting Regulatory T cells

CD4(+) regulatory T cells frustrate productive tumor immune surveillance and represent an obstacle for cancer immunotherapy. In mice, anti-CD25 antibody is an effective method of depleting CD25(+) FOXP3(+) T regulatory cells (Tregs) in vivo and enhancing cancer immunity. Here, we propose the use of the anti-CD25 monoclonal antibody daclizumab for the depletion of Tregs in cancer patients. In early results from an ongoing clinical trial, a single intravenous infusion of daclizumab in patients with metastatic breast cancer is associated with a marked and prolonged elimination of CD25(+) FOXP3(+) Tregs in peripheral blood. When a cancer antigen peptide vaccine is administered during the daclizumab-induced Treg nadir, effective generation of cytotoxic T lymphocytes has been observed, including those specific for neo-antigens, such as cytomegalovirus peptide used as an immunological control. If confirmed in additional patients, these observations suggest that daclizumab may be an effective and available therapeutic agent for Treg modulation in patients with cancer.

The Wilms' Tumor Antigen Is a Novel Target for Human CD4+ Regulatory T Cells: Implications for Immunotherapy

Compelling evidences indicate a key role for regulatory T cells (T(reg)) on the host response to cancer. The Wilms' tumor antigen (WT1) is overexpressed in several human leukemias and thus considered as promising target for development of leukemia vaccine. However, recent studies indicated that the generation of effective WT1-specific cytotoxic T cells can be largely affected by the presence of T(regs). We have generated T-cell lines and clones that specifically recognized a WT1-84 (RYFKLSHLQMHSRKH) peptide in an HLA-DRB1*0402-restricted manner. Importantly, they recognized HLA-DRB1*04-matched fresh leukemic cells expressing the WT1 antigen. These clones exerted a T helper 2 cytokine profile, had a CD4(+)CD25(+)Foxp3(+)GITR(+)CD127(-) T(reg) phenotype, and significantly inhibited the proliferative activity of allogeneic T cells independently of cell contact. Priming of alloreactive T cells in the presence of T(regs) strongly inhibited the expansion of natural killer (NK), NK T, and CD8(+) T cells and had an inhibitory effect on NK/NK T cytotoxic activity but not on CD8(+) T cells. Furthermore, priming of T cells with the WT1-126 HLA-A0201-restricted peptide in the presence of T(regs) strongly inhibited the induction of anti-WT1-126 CD8(+) CTL responses as evidenced by both very low cytotoxic activity and IFN-gamma production. Moreover, these T(reg) clones specifically produced granzyme B and selectively induced apoptosis in WT1-84-pulsed autologous antigen-presenting cells but not in apoptotic-resistant DR4-matched leukemic cells. Importantly, we have also detected anti-WT1-84 interleukin-5(+)/granzyme B(+)/Foxp3(+) CD4(+) T(regs) in five of eight HLA-DR4(+) acute myeloid leukemia patients. Collectively, our in vitro and in vivo findings strongly suggest important implications for the clinical manipulation of T(regs) in cancer patients.

Regulatory T Cells in Cancer Biology: A Possible New Target for Biochemical Therapies

CD4+ CD25+ T cells are essential for maintenance of self-tolerance and therefore have been referred to as regulatory T cells (Treg). Experimental tumor models revealed that Treg are potent inhibitors of an anti-tumor immune response. Treg are expanded in human cancer. Currently, a variety of strategies for the induction of a specific anti-tumor immune response are tested in preclinical and clinical settings. Biochemical strategies modifying and/or depleting Treg in cancer patients for an enhancement of vaccine-based therapeutic concepts will be discussed in detail in this review.