Abstract LB-139: In situ Treg immunomodulation at a single tumor site with CpG and immune checkpoint antibodies cures metastatic disease
Aurélien MarabelleHolbrook E. KohrtJoshua BrodyJames A. TorchiaRanjani RajapaksaRichard LuongGang ZhouHyam I. LevitskyVictor TseRonald Levy
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Abstract Background: CD4+CD25+FOXP3+ regulatory T-cells (Tregs) infiltrate all tumor sites and play a central role in maintaining immune tolerance to cancers. Scientific question: Is the local immunomodulation of Tregs from a single tumor site sufficient to trigger a systemic anti-tumor immune response able to eradicate distant tumor sites ≤ Results: We found that tumor infiltrating Tregs preferentially express CTLA4 (CD152) and OX40 (CD134) compared to their counterparts in the blood and other lymphoid organs, both in mice and in human lymphomas. We show in a murine lymphoma model that OX40 and CTLA4 upregulation occurs specifically on Tregs directed against tumor antigens. Injections of low doses of anti-CTLA4 and anti-OX40 together with CpG, a TLR-9 agonist, directly into a single tumor site depletes the Tregs from the injected tumor but not from distant ones. This immunomodulation subsequently triggers an anti-tumor immune response able to cure mice with established disseminated disease. This triple combination is uniquely required as neither CpG alone nor mAbs without CpG are effective. Significance: immunomodulatory antibodies are currently under clinical development for cancer therapy. Their major toxicity is the triggering of auto-immune diseases. We show here that after injections of very little doses of these antibodies with CpG at one tumor site, their serum levels become undetectable. However, these doses are sufficient to trigger a systemic anti-tumor response able to eradicate distant sites. Impact: we recently have published positive results of intra-tumoral CpG in patients with follicular Lymphoma (Brody, Levy, et al. JCO, 2010). Anti-CTLA4 has just been approved by the FDA/EMEA in patients with metastatic melanoma. Anti-Ox40 antibodies are currently being tested in phase I/II clinical trials. Therefore, the combination described here can be tested in patients with injectable sites of lymphoma. Together, these results are in favor of a paradigm shift in cancer therapy where the immune system is targeted rather than the tumor itself. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-139. doi:1538-7445.AM2012-LB-139Keywords:
CpG Oligodeoxynucleotide
Objective To investigate the immune stimulation activity of CpG oligodeoxynucleotides to different species and develop CpG oligodeoxynucleotides that possess broad-spectrum immune stimulation activity. Method Chickens and mice were respectively vaccinated with CpG oligodeoxynucleotides or synergistically with different antigens, and then titers of specific antibody in the blood serums of chickens and mice were detected with ELISA. Results CpG oligodeoxynucleotides could induce the generation of antigen specific antibody in chickens and mice, and the level of antibodies in groups which use CpG ODN as adjuvant is higher than that in the control group (P0.05). Conclusion CpG ODN that we made restrictively possesses broad-spectrum immune stimulation activity.
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Purpose of review Foxp3 is the transcription factor that induces the regulatory T cell phenotype. This review will examine issues around Foxp3 induction and function as well as clinical data on tolerance and rejection. Recent findings Recent findings have included identification of the signals that drive naive T lymphocytes to express Foxp3 in the thymus and the signals peripherally that induce non-Foxp3 expressing T cells to express FOXP3. Further, the identification of the downstream targets of Foxp3 both by analysis of Foxp3 expressing cells and by analysis of gene promoters that bind Foxp3 has provided new insights into its function. Whereas Foxp3 T regulatory cells (Tregs) are associated with tolerance in a variety of animal transplant models, the human data show expansion of Foxp3 Tregs associated with rejection, though Tregs are also found in transplants in patients with mixed chimerism-induced tolerance. Further, there is a significant difference in the effect of the different immunosuppressive medications on Treg function and expansion that may be important in developing strategies to enhance Tregs in human trials. Conclusion Foxp3 CD4 T cells are frequently associated with rejection; however, this does not preclude their protective role and importance in tolerance induction.
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瞄准：学习在包含 unmethylated deoxycytidyl-deoxyguanosine (CpG ) 的 oligodeoxynucleo 潮(ODN ) 的主要结构之间的关系在老鼠怒气房间的二核甙酸主题和他们的 immunostimulatory 活动。方法：有不同主要结构的一系列 CpG ODN 被综合。他们刺激老鼠怒气房间增长的能力被决定由[3H ] 胸腺嘧啶核甙加入试金。Cytokine (interleukin [IL ]-6, IL-12，和 IFN-alpha ) CpG ODN 导致的分泌物系列被 ELISA 估计。到 activate 天赋杀手房间的 CpG ODN 的能力被标准 4 h (51 ) 评估 Cr 版本试金。流动血细胞计数被利用在多样的 immunocytes 上检验各种各样的淋巴细胞表面分子的表情。有效 CpG ODN 为鼠科， ODN1826，被放作为修正和积极控制的模板。结果：有不同序列和作文的 CpG ODN 的 immunostimulatory 活动显著地变化了，在特性并且在程度。它为由简单地增加功能的 hexameric CpG 主题数字改进 ODN1826 的 immunostimulatory 活动是无用的，修改 CpG 主题的地点，或改变在 multi-CpG 主题之间的距离。然而，自我补足的回文的增加在第 3'-e，然而并非第 5'-e CpG ODN 组织，在它的活动的兴奋显著改进。几设计 ODN 与 ODN1826 相比有优异全面 immunostimulatory 性质。结论：CpG ODN 的 immunostimulatory 活动与它的主要结构相关。简单地改变 CpG 主题数字，空格，或距离，为支持 immunostimulatory 活动是无用的。CpG ODN 的第 3'-e 回文结构与提高的 immunostimulatory 活动被联系。
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Abstract NTregs exhibit higher homeostatic proliferation (HP) than Tconv which may provide a competitive advantage during Treg adoptive immunotherapy. Increased Treg HP could be 2° to a self-reactive TCR repertoire and/or Foxp3-mediated increase in survival/fitness. To address this, CFSE-labeled CD4 populations from Foxp3-reporter mice, were compared 10d after transfer into wt mice. We generated Foxp3+ iTregs by activating Tconv and adding TGFβ, or transducing with Foxp3-retrovirus (RV). iTregs did not increase HP over Tconv controls (no TGFβ; RV-vector). Thus, Foxp3 itself is not sufficient to augment HP. To determine the role of Foxp3, we examined GFP+ nTreg from Filig mice (F-nTreg; Foxp3 ~10% wt levels due to unstable mRNA). F-nTreg exhibited reduced HP (10%) vs. wt nTregs (50%). Foxp3-RV transduction increased HP of F-nTregs, while RV-control had no effect (40 vs. 12%). Thus, Foxp3 is necessary for high HP by nTreg. We also examined role of Foxp3 during thymic ontogeny, irradiated Thy1.1 hosts received Filig (Thy1.2) bone marrow that was transduced with Foxp3-RV vs. RV-control. Surprisingly, in this setting, Tconv cells (no endogenous Foxp3) constitutively expressing Foxp3, exhibited increased HP vs. Foxp3- RV-controls (32 vs. 12%). Thus, Foxp3 increases HP in nTreg and when expressed during thymic ontogeny but not in mature Tconv. Our findings provide new insight into the role of Foxp3 expression in Treg homeostasis and highlight additional distinctions between iTreg and nTreg.
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AIM:To establish a set of reasonable and convenient experimental system to provide a screening method for the development of novel immunoregulatory oligodeoxynucleotides.METHODS:The human PBMCs were stimulated by CpG ODN and/or immunoregulatory ODN.The cell proliferation and anti-viral activity of the supernatant induced by CpG ODN were examined by thymidine incorporation and anti-viral bioassay to evaluate the immunoregulatory activity of candidate ODN.The experimental conditions were also optimized.RESULTS:A screening method on which A151,a positive immunoregulatory ODN,inhibited the proliferation and anti-viral activity of the CpG ODN-induced human PBMCs was successfully established.CONCLUSION:The successful establishment of CpG ODN based screening method lays the foundations for further development of novel immunoregulatory oligodeoxynucleotides.
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Unmethylated cytosine-phosphate-guanosine (CpG) oligodeoxynucleotides (ODNs) are recognized by Toll-like receptor 9 (TLR9) found in antigen-presenting cells and B cells and can activate the immune system. Using CpG ODNs as an adjuvant has been found to be effective for treating infectious diseases, cancers, and allergies. Because natural ODNs with only a phosphodiester backbone are easily degraded by nuclease (deoxyribonuclease [DNase]) in serum, CpG ODNs with a phosphorothioate backbone have been studied for clinical application. CpG ODNs with a phosphorothioate backbone have raised concern regarding undesirable side effects; however, several CpG ODNs with only a phosphodiester backbone have been reported to be stable in serum and to show an immunostimulatory effect. In recent years, research has been conducted on delivery systems for CpG ODNs using nanoparticles (NPs). The advantages of NP-based delivery of CpG ODN include (1) it can protect CpG ODN from DNase, (2) it can retain CpG ODN inside the body for a long period of time, (3) it can improve the cellular uptake efficiency of CpG ODN, and (4) it can deliver CpG ODN to the target tissues. Because the target cells of CpG ODN are cells of the immune system and TLR9, the receptor of CpG ODN is localized in endolysosomes, CpG ODN delivery systems are required to have qualities different from other nucleic acid drugs such as antisense DNA and small interfering RNA. Studies until now have reported various NPs as carriers for CpG ODN delivery. This review presents DNase-resistant CpG ODNs with various structures and their immunostimulatory effects and also focuses on delivery systems of CpG ODNs that utilize NPs. Because CpG ODNs interact with TLR9 and activate both the innate and the adaptive immune system, the application of CpG ODNs for the treatment of cancers, infectious diseases, and allergies holds great promise.
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Abstract CD4 + FOXP3 + regulatory T (Treg) cells are essential for maintaining immunological self-tolerance. Treg cell development and function depend on the transcription factor FOXP3, which is present in several distinct isoforms due to alternative splicing. Despite the importance of FOXP3 in the proper maintenance of Treg cells, the regulation and functional consequences of FOXP3 isoform expression remains poorly understood. Here, we show that in human Treg cells IL-1β promotes excision of FOXP3 exon 7. FOXP3 is not only expressed by Treg cells but is also transiently expressed when naïve T cells differentiate into Th17 cells. Forced splicing of FOXP3 into FOXP3Δ2Δ7 strongly favored Th17 differentiation in vitro . We also found that patients with Crohn’s disease express increased levels of FOXP3 transcripts lacking exon 7, which correlate with disease severity and IL-17 production. Our results demonstrate that alternative splicing of FOXP3 modulates T cell differentiation. These results highlight the importance of characterizing FOXP3 expression on an isoform basis and suggest that immune responses may be manipulated by modulating the expression of FOXP3 isoforms, which has broad implications for the treatment of autoimmune diseases.
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Synthetic oligodeoxynucleotides(ODN) containing unmethylated CpG motifs mimic the immunostimulatory activity of bacterial DNA.CpG ODN directly stimulate a variety of immunological effects.CpG ODN is used as vaccine adjuvants,where they increase the vaccine-specific immune responses and the mucosal immune responses.Ongoing clinical studies indicate that CpG ODN are safe and well-tolerated when administered as adjuvants to animals.
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