Expression Of ICOS Research Articles

Regulatory T Cells in Ovarian Cancer Are Characterized by a Highly Activated Phenotype Distinct from that in Melanoma.

Purpose: Regulatory T (Treg) cells expressing the transcription factor FOXP3 are essential for the maintenance of immunologic self-tolerance but play a detrimental role in most cancers due to their ability to suppress antitumor immunity. The phenotype of human circulating Treg cells has been extensively studied, but less is known about tumor-infiltrating Treg cells. We studied the phenotype and function of tumor-infiltrating Treg cells in ovarian cancer and melanoma to identify potential Treg cell-associated molecules that can be targeted by tumor immunotherapies.Experimental Design: The phenotype of intratumoral and circulating Treg cells was analyzed by multicolor flow cytometry, mass cytometry, RNA-seq, and functional assays.Results: Treg cells isolated from ovarian tumors displayed a distinct cell surface phenotype with increased expression of a number of receptors associated with TCR engagement, including PD-1, 4-1BB, and ICOS. Higher PD-1 and 4-1BB expression was associated with increased responsiveness to further TCR stimulation and increased suppressive capacity, respectively. Transcriptomic and mass cytometry analyses revealed the presence of Treg cell subpopulations and further supported a highly activated state specifically in ovarian tumors. In comparison, Treg cells infiltrating melanomas displayed lower FOXP3, PD-1, 4-1BB, and ICOS expression and were less potent suppressors of CD8 T-cell proliferation.Conclusions: The highly activated phenotype of ovarian tumor-infiltrating Treg cells may be a key component of an immunosuppressive tumor microenvironment. Receptors that are expressed by tumor-infiltrating Treg cells could be exploited for the design of novel combination tumor immunotherapies. Clin Cancer Res; 24(22); 5685-96. ©2018 AACR.

Follicular Helper T Cells in DiGeorge Syndrome.

DiGeorge syndrome is an immunodeficiency characterized by thymic dysplasia resulting in T cell lymphopenia. Most patients suffer from increased susceptibility to infections and heightened prevalence of autoimmune disorders, such as autoimmune thrombocytopenia. B cells in DiGeorge syndrome show impaired maturation, with low switched-memory B cells and a wide spectrum of antibody deficiencies or dysgammaglobulinemia, presumably due to impaired germinal center responses. We set out to evaluate circulating follicular helper T cells (cTFHs) in DiGeorge syndrome, as markers of T–B interaction in the germinal centers in a cohort of 17 patients with partial DiGeorge and 21 healthy controls of similar age. cTFHs were characterized as CXCR5+CD45RA− CD4+ T cells using flow cytometry. We verify previous findings that the population of memory CD4+ T cells is relatively increased in diGeorge patients, corresponding to low naïve T cells and impaired T cell production in the thymus. The population of CXCR5+ memory CD4+ T cells (cTFHs) was significantly expanded in patients with DiGeorge syndrome, but only healthy controls and not DiGeorge syndrome patients showed gradual increase of CXCR5 expression on cTFHs with age. We did not observe correlation between cTFHs and serum IgG levels or population of switched memory B cells. There was no difference in cTFH numbers between DiGeorge patients with/without thrombocytopenia and with/without allergy. Interestingly, we show strong decline of PD1 expression on cTFHs in the first 5 years of life in DiGeorge patients and healthy controls, and gradual increase of PD1 and ICOS expression on CD4− T cells in healthy controls later in life. Thus, here, we show that patients with DiGeorge syndrome have elevated numbers of cTFHs, which, however, do not correlate with autoimmunity, allergy, or production of immunoglobulins. This relative expansion of cTFH cells may be a result of impaired T cell development in patients with thymic dysplasia.

Abstract 1678: Integrated genomics and histology based studies of triple negative breast cancer identify ICOS as potential target for therapeutic intervention

Abstract ICOS (Inducible CO-Stimulator of T cells) is a co-stimulatory molecule expressed primarily on T lymphocytes. ICOS was prioritized as a target of interest based on nonclinical and clinical data that identified ICOS as a potentially key molecule in providing optimal anti-tumor benefit following anti-CTLA-4 therapy. We are developing JTX-2011, an ICOS agonist antibody that is designed to generate an anti-tumor immune response through stimulation of T effector cells and preferential reduction of intratumoral T regulatory (Treg) cells. In preclinical mouse tumor models, efficacy of an ICOS agonist was greatest in tumors with the highest levels of intra-tumoral ICOS, suggesting a potential predictive biomarker approach for clinical development. In assessing ICOS expression across multiple tumor types at both the RNA and protein level, we have identified triple negative breast cancer (TNBC) as a potential indication for an ICOS-targeted immunotherapy approach. Integrated analysis of RNA, DNA and clinical data from the Cancer Genome Atlas (TCGA) was performed to understand the context in which ICOS is expressed. Additionally, ICOS levels were assessed by IHC in human tumor samples from an orthogonal data set. IHC and RNA analyses revealed a dynamic range of ICOS expression across indications and identified a subpopulation of breast cancer tumors enriched for high ICOS expression. Further analysis of both IHC and RNA data sets revealed that the triple negative subtype has higher enrichment of ICOS expression than other breast cancer subtypes. ICOS levels were correlated to gene signatures of immune infiltrate as well as other clinical attributes and molecular markers. There was a correlation between ICOS, ICOS signature, PD-L1 and IFNγ signatures. We then assessed TNBC samples obtained pre and post-neoadjuvant treatment to further understand the impact of chemotherapy on the tumor microenvironment. This included analysis of ICOS and PD-L1 protein expression as well as assessment of tumor-infiltrating immune cell subsets. While the distribution of certain immune cell subsets differed in pre and post-treatment samples, the expression of ICOS remained consistent. Based on these data, a TNBC cohort, enriched for the ICOS IHC biomarker, is included in the Phase 2 portion of the ICONIC study that is designed to assess the potential for a combination of JTX-2011 with a PD-1 checkpoint in this difficult to treat patient population. Citation Format: Heather A. Hirsch, Tong Zi, Rachel Fontana, Yun Wu, Jason Reeves, Alexander Needham, Edward Stack, David Lee, Emma Lees, Deborah A. Law, Elizabeth Trehu, Elizabeth A. Mittendorf. Integrated genomics and histology based studies of triple negative breast cancer identify ICOS as potential target for therapeutic intervention [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 1678.

Abstract 2792: The combination of immune checkpoint blockers with the anti-ICOS KY1044 antibody results in a strong tumor response

Abstract The last few years have shown a strong paradigm shift in cancer therapies with the approval of antibodies targeting immune checkpoints (CTLA-4, PD-1 and PD-L1). These immune checkpoint blockers (ICBs) are associated with a strong and long-lasting response in patients suffering from different malignancies. However, there is still a high proportion of patients showing intrinsic or acquired resistance to ICBs. Although the mechanisms associated with the lack of immune response are multiple, the presence of highly anti-inflammatory regulatory T cells (TRegs) in the tumour microenvironment (TME) is known to negatively affect the response to these therapies. Similarly, high TReg levels in the TME have been associated with poor prognosis in several cancers. Together, this highlights the potential of targeting and depleting TRegs to enhance anti-tumour responses. The Inducible T-cell costimulator (ICOS/CD278) is related to the CD28 superfamily and is induced when T cells get activated. ICOS expression levels vary in different immune cell subtypes and in different tissues. In preclinical mouse tumour models, TRegs (CD4+/FOXP3+) constitutively express ICOS on their surface and the expression of ICOS on TRegs is significantly higher than that on effector T cells (TEffs). In addition, ICOS expression on TRegs is higher in the TME than in the blood or spleen, which makes it a strong candidate for preferential depletion of TRegs in tumours. By immunizing Kymice™ in which the endogenous Icos gene has been knocked out, we identified a novel, fully human antibody called KY1044 that cross-reacts with mouse ICOS. KY1044 is an anti-ICOS subclass G1 kappa monoclonal antibody that selectively binds to dimeric ICOS (Fc fusion) with an affinity of less than 2nM. Using in vitro and in vivo approaches we demonstrate that KY1044 has a dual mechanism of action: (1) it promotes the preferential depletion of intratumoural ICOShigh TRegs resulting in an increase in the TEff:TReg ratio in the TME; and (2) it stimulates ICOSLow TEff cells. Using the mouse effector enabled version of KY1044 (mIgG2a) we confirm, using syngeneic models, a strong anti-tumour efficacy as monotherapy or in combination with surrogates of ICBs. We also demonstrated a tumour antigen specific immunity, as highlighted by the rejection of the original tumour cells in animals cured of the disease and re-challenged by the same cell line. Noteworthy, Pharmacodynamic studies demonstrate long-term depletion of TRegs and a significant increase in the TEff:TReg ratio in response to KY1044. In summary, our data demonstrate that targeting ICOS with KY1044 is a valid approach for manipulating the immune system and for inducing a strong anti-tumour response in several indications. The data presented here also warrant the assessment of KY1044 in cancer patients in a clinical trial. Citation Format: Richard C. Sainson, Matthew McCourt, Anil Thotakura, Nahida Parveen, Miha Kismac, Gwenoline Borhis, Joana Carvalho, Tracey Myers, Robert Rowlands, Hanif Ali, Hannah Craig, Vivian Wong, Qi Liang, Volker Germaschewski. The combination of immune checkpoint blockers with the anti-ICOS KY1044 antibody results in a strong tumor response [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 2792.

Abstract 3570: Adoptive transfer of tumor antigen-specific CTLs requires anti-CTLA-4 and anti-PD-1 to drive tumor eradication

Abstract Adoptive transfer of tumor-reactive T cells (ACT) has led to significant clinical responses in hematologic malignancies. However, its efficacy in solid tumors has been dismal, likely due to the immunosuppression imposed by the tumor microenvironment (TME) of solid tumors. A proven effective strategy to revert the immunosuppressive TME is immune checkpoint blockades (ICBs), including anti-CTLA-4 and anti-PD-1/PD-L1, which have been recently approved by FDA for cancer therapy. Here, using the orthotopic B16-F10 melanoma model, we combine ACT of PMEL-1 Tg+ CTLs that recognize the endogenous melanoma antigen gp100 with dual blockade of CTLA-4 and PD-1, the highly pursued effective combinatorial ICB, to treat established tumors. Although ACT alone transiently suppresses tumor growth, complete tumor regression requires additional dual anti-CTLA-4 and anti-PD-1 blockade. Mechanistically, we demonstrate that the ICOS-Eomes-IFN-γαμμα axis, but not the ICOS-T-bet-IFN-γαμμα axis, in CTLs mediates the therapeutic effects of ACT and ICBs. Our data provide direct evidence that combining ACT of CTLs with ICBs confers long-term protection to tumor challenges, which largely depends on CTL-intrinsic expression of ICOS. Citation Format: Lewis Zhichang Shi, Jianjun Gao, Luis Vence, Jorge Brando, James Allison, Padmanee Sharma. Adoptive transfer of tumor antigen-specific CTLs requires anti-CTLA-4 and anti-PD-1 to drive tumor eradication [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 3570.

Low-Dose Interleukin-2 Therapy Activates Circulating T Follicular Regulatory Cells and Suppresses Circulating T Follicular Helper Cells in Patients with Chronic Gvhd

Introduction: T follicular helper (TFH) and T follicular regulatory (TFR) cells play important roles in the regulation of B-cell immunity. While TFH promote B cell functions in the germinal center (GC), TFR function as negative regulators of the GC response. Previous studies in murine models established that TFH and GC B cells are required for the development of chronic graft-versus-host disease (cGVHD). We previously reported that circulating TFH (cTFH) were more functionally activated in patients with active cGVHD compared with patients with no cGVHD. Low-dose IL-2 therapy has been shown to selectively expand CD4Treg and improve cGVHD symptoms. In the current study, we examined the effects of IL-2 therapy on cTFH and circulating TFR (cTFR) in patients with steroid resistant cGVHD.Methods: Single cell mass cytomtery (CyTOF) was performed on cryopreserved peripheral blood mononuclear cells (PBMC) from healthy donors and 17 adult patients with active cGVHD receiving daily low-dose IL-2 therapy (Koreth et al. Blood 2016). A panel of 35 metal-tagged monoclonal antibodies was used to simultaneously examine the phenotypic and functional effects of low-dose IL-2 on lymphocyte populations in vitro and in vivo. The analytic panel included 22 cell surface markers to identify distinct lymphocyte subsets and 13 intracellular markers to measure functional status and activation of specific signaling pathways. Before staining for surface and intracellular antigens, serial samples from individual patients were barcoded to ensure uniformity of analysis. viSNE was used to visualize of high-dimensional data on a two-dimensional map and quantify single cell mass cytometry data.Results: In PBMC from healthy donors, expression of CD25 (IL-2Rα), CD95, CTLA-4, BLIMP-1 and GITR was higher in cTFR compared with cTFH. To examine the response to IL-2 in vitro, PBMC from healthy donors were stimulated with IL-2 for 15 minutes (Figure 1A). At low IL-2 concentrations (1 to 10 IU/mL), phospho-STAT5 (p-STAT5) was selectively activated in cTFR compared with cTFH. At high IL-2 concentrations (100 to 1,000 IU/mL), p-STAT5 was activated in both cTFR and cTFH. To examine the response to IL-2 in vivo, we used mass cytometry to examine serial PBMC samples from cGVHD patients receiving daily low dose IL-2 therapy (1x106 IU/M2/day). Selective expansion of cTFR was noted after 1 week of treatment and cTFR expansion remained stable for the 12 week duration of therapy. Expanded cTFR increased expression of p-STAT5, FoxP3, BCL6, HLA-DR (Figure 1B) and CD25, CD95, CTLA-4, ICOS, Ki67 and Helios 1 week after starting IL-2. cTFR:cTFH ratio increased rapidly after starting low dose IL-2 and paralleled the increased Treg:Tcon ratio (Figure 1C). Activated TFH and TFR can be identified by expression of ICOS and PD-1. The expansion of ICOS+PD-1+ cTFR was evident after 1 week of IL-2 and remained elevated at the end of therapy. In contrast, ICOS+PD-1+ cTFH increased 1 week after starting IL-2 therapy but subsequently decreased and fell below baseline 6 and 12 weeks after starting IL-2 (Figure 1D). Activated ICOS+PD-1+ cTFR expressed higher levels of p-STAT5, BCL-6, FoxP3, HLA-DR and CD25 during low dose IL-2 therapy. In contrast, these functional markers were not increased in ICOS+PD-1+ cTFH during IL-2 therapy (Figure 1B).Conclusion: Single cell mass cytometry analysis revealed that daily low dose IL-2 therapy induces selective activation and increased expression of functional proteins in ICOS+PD-1+ cTFR. In contrast, activated ICOS+PD-1+ cTFH were suppressed during IL-2 therapy. The selective activation of cTFR and suppression of cTFH provide a mechanism whereby low dose IL-2 therapy can promote B cell tolerance as well as T cell tolerance in patients with cGVHD. [Display omitted] DisclosuresForcade:Neovii: Other: Travel grant. Koreth:Amgen Inc.: Consultancy; Prometheus Labs: Research Funding; Kadmon Corp: Membership on an entity's Board of Directors or advisory committees; Millennium Pharmaceuticals: Research Funding; Takeda Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Nikiforow:Kite Therapeutics: Membership on an entity's Board of Directors or advisory committees. Armand:Infinity: Consultancy; Bristol-Myers Squibb: Consultancy, Research Funding; Otsuka: Research Funding; Tensha: Research Funding; Sequenta/Adaptive: Research Funding; Genmab: Consultancy; Affimed: Research Funding; Sigma Tau: Research Funding; Merck & Co., Inc.: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Roche: Research Funding. Cutler:Bristol-Myers Squibb: Consultancy; Pfizer: Consultancy; Kite: Consultancy; Pharmacyclics: Consultancy; Incyte: Consultancy; Astellas: Consultancy.

The prognostic landscape of tumor-infiltrating immune cell and immunomodulators in lung cancer

Tumor-infiltrating immune cells are closely associated with clinical outcome. However, immunohistochemistry-based analysis of tumor infiltrates can be misleading as the representative marker of an immune subpopulation might be expressed in other cell types. In this study, based on a metagene approach (known as CIBERSORT) and an online databse, The Cancer Immunome Atlas (https://tcia.at/), we comprehensively analyzed the tumor-infiltrating immune cells present in lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC). A total of 22 types of both adaptive and innate tumor-infiltrating immune cells were evaluated in LUAD (n=492) and LUSC (n=488). As a result, tumors lacking memory B cells or with increased number of M0 macrophages were associated with the poor prognosis in LUAD at early clinical stage. In LUSC, T follicular helper cells were associated with favorable outcome, while increased number of neutrophils predicted a poor outcome. Moreover, Kaplan-Meier analysis of the prognostic value of immune checkpoint molecules revealed that expression of ICOS was positively correlated the clinical outcome of patients with LUAD. Collectively, our data suggest that tumor-infiltrating immune cells in lung cancer are likely to be important determinants of both prognosis and response to immunotherapies.

Differential Requirements for Tcf1 Long Isoforms in CD8+ and CD4+ T Cell Responses to Acute Viral Infection.

In response to acute viral infection, activated naive T cells give rise to effector T cells that clear the pathogen and memory T cells that persist long-term and provide heightened protection. T cell factor 1 (Tcf1) is essential for several of these differentiation processes. Tcf1 is expressed in multiple isoforms, with all isoforms sharing the same HDAC and DNA-binding domains and the long isoforms containing a unique N-terminal β-catenin-interacting domain. In this study, we specifically ablated Tcf1 long isoforms in mice, while retaining expression of Tcf1 short isoforms. During CD8+ T cell responses, Tcf1 long isoforms were dispensable for generating cytotoxic CD8+ effector T cells and maintaining memory CD8+ T cell pool size, but they contributed to optimal maturation of central memory CD8+ T cells and their optimal secondary expansion in a recall response. In contrast, Tcf1 long isoforms were required for differentiation of T follicular helper (TFH) cells, but not TH1 effectors, elicited by viral infection. Although Tcf1 short isoforms adequately supported Bcl6 and ICOS expression in TFH cells, Tcf1 long isoforms remained important for suppressing the expression of Blimp1 and TH1-associated genes and for positively regulating Id3 to restrain germinal center TFH cell differentiation. Furthermore, formation of memory TH1 and memory TFH cells strongly depended on Tcf1 long isoforms. These data reveal that Tcf1 long and short isoforms have distinct, yet complementary, functions and may represent an evolutionarily conserved means to ensure proper programming of CD8+ and CD4+ T cell responses to viral infection.

Abstract 1469: Integrated analysis of microRNA, mRNA, and protein expression utilizing MultiOmyxTM and NanoStringTM from formalin-fixed paraffin-embedded, lung, head and neck, breast, and melanoma tumors

Abstract Cancer is characterized as a loss of normal cellular regulation, due to accumulation of mutations and disruption of complex biological pathways. MicroRNAs (miRNAs) regulation of co-stimulatory and immune checkpoint pathways have been implicated as one of the potential mechanisms for cancer evasion in immuno-oncology. It is estimated that 30% of all mRNA expression may be regulated by miRNAs, and some are either oncogenic or tumor suppressive. Complexity of miRNA regulation highlights the need for integrated assays, providing direct correlation between miRNA and mRNA, and protein expression. From a single 4 µm FFPE section, MultiOmyxTM hyperplexed immunofluorescent assay (demonstrated to stain up to 60 protein biomarkers) is utilized to measure CD3, CD4, CD8, CD16, CD56, Granzyme B, FoxP3, ICOS, OX40, OX40L, PD1, PDL1, HLA-DR, and Ki67 protein expression. From an adjacent 10 µm section, NanoStringTM nCounter PanCancer Immune Profiling Panel and Human v3 miRNA expression panel were utilized to comprehensively profile the expression of 770 mRNA and 800 miRNA. Integrating MultiOmyx and NanoString technologies, the current study measured miRNA, mRNA, and protein expression in lung, head and neck, breast, and melanoma samples. For each indication, three samples were selected from a larger sample set, based on high protein expression of lymphocytes and macrophage markers (CD3, CD4, CD8, CD56, CD16), co-stimulator markers (ICOS, OX40), and immune checkpoint markers (PD1, PD-L1). Protein expression results indicate positive correlation between expression of ICOS and OX40 with higher infiltration of Thelper (CD3+CD4+), Tcytotoxic (CD3+CD8+), and effector T cells (CD3+CD8+Granzyme B). NanoString normalized mRNA counts for the protein biomarkers profiled indicate that all markers except for HLA-DR belong to low expressers group with counts ranging from 20-700. Comparison of protein expression to mRNA counts revealed inconsistencies in modulated markers (PD1, PD-L1, ICOS, OX40) which are attributed to differences in population of cells between the two sections. Direct assessment of up regulation of miRNA and down regulation of target mRNA could not be made for miRNAs reported in literature as negative regulator of PD-L1 (miR-34a, 34b, 34c), PD1 (miR-28, miR-107), FoxP3 (miR-210, miR-24, miR-31), and ICOS (miR-101). Analysis of multiple miRNAs (combinatorial targeting) mapped in context to mRNA, and their respective protein expression will be presented from lung, head and neck, breast, and melanoma cancer samples. Citation Format: Qingyan Au, RaghavKrishna Padmanabhan, Nam Tran, Lakshmi Chandramohan, Nicholas Hoe. Integrated analysis of microRNA, mRNA, and protein expression utilizing MultiOmyxTM and NanoStringTM from formalin-fixed paraffin-embedded, lung, head and neck, breast, and melanoma tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1469. doi:10.1158/1538-7445.AM2017-1469

Abstract SY03-02: Preclinical assessment of JTX-2011, an agonist antibody targeting ICOS, supports evaluation in ICONIC clinical trial

Abstract ICOS (the inducible T-cell co-stimulator) is a co-stimulatory molecule expressed on the surface of T cells and a member of the CD28 family, which includes clinically validated targets of cancer immunotherapies, such as PD-1 and CTLA-4. Clinical data identified ICOS as a potentially key molecule in providing optimal antitumor benefit following anti-CTLA-4 therapy. We have developed a species cross-reactive humanized IgG1 agonist antibody, JTX-2011, that binds ICOS and is designed to induce an antitumor immune response. Our preclinical data suggest that JTX-2011 functions through a dual mechanism of action, by stimulating T effector cells (Teff) and depleting intratumoral T regulatory cells (Tregs). The ICOS antibody is efficacious as a single agent in mouse syngeneic tumor models and demonstrates enhanced activity when administered in combination with anti-PD-1. Single-agent activity in the preclinical models appears to correlate with ICOS expression, with greater efficacy observed in tumor models that exhibit a higher percentage of ICOS-expressing immune cell infiltrate. An integrated expression analysis of human tumors identified non-small cell lung cancer (NSCLC) and head and neck squamous cell carcinoma (HNSCC) as indications with higher percentages of ICOS-expressing cell infiltrate. Preclinical studies performed in rodent and monkeys evaluated safety, pharmacokinetics, and pharmacodynamics of JTX-2011 to inform the first in-human study. The ICONIC phase I/II clinical trial is currently ongoing for evaluation of JTX-2011 alone or in combination with the anti-PD-1 antibody Nivolumab in patients with advanced solid tumors and incorporates a patient enrichment strategy design based on the preclinical and translational findings. Citation Format: Jennifer S. Michaelson, Christopher Harvey, Kutlu Elpek, Ellen Duong, Lindsey Shallberg, Matthew Wallace, Robert Mabry, Jenny Shu, Amit Deshpande, Tong Zi, Stephen Sazinsky, Joshua Apgar, Barbara Mounho-Zamora, Michael Briskin, Elizabeth Trehu, Jason Reeves, Heather Hirsch, Sriram Sathyanarayanan, Deborah Law. Preclinical assessment of JTX-2011, an agonist antibody targeting ICOS, supports evaluation in ICONIC clinical trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr SY03-02. doi:10.1158/1538-7445.AM2017-SY03-02

A phase I study of sequential ipilimumab in the definitive treatment of node positive cervical cancer: GOG 9929.

5526 Background: The outcome of lymph node positive (LN) cervical cancer (CC) with chemoradiation (CRT) is dismal, especially with involved para-aortic nodes (PAN). The anti-CTLA-4 immune checkpoint inhibitor ipilimumab (ipi) holds promise. We report the safety, tolerability, and efficacy in this GOG phase I study examining sequential ipi after CRT for CC. Methods: Patients (pts) with LN CC were treated with 6 weekly doses of cisplatin (40 mg/m2) and extended field radiation (RT). 2-6 weeks after RT, if there was no progression of disease, sequential ipi was given at the following dose levels: dose level 1: 3mg/kg, level 2: 10mg/kg, and an expansion cohort of 10mg/kg. The primary endpoints (endpts) were the maximum tolerated dose (MTD), and dose-limiting toxicities (DLT) of adjuvant ipi. Secondary endpt included the 1-yr disease free survival (DFS). Translational endpts included the effect of CRT on enumeration and subsets of T-cells, and CTLA4, PD-1 and ICOS expression. Results: 34 pts were enrolled, and 19 pts are evaluable for the endpts: 14 pts went off study to reasons unrelated to the study drug, 1 pt continues in her DLT evaluable period. Of the evaluable pts, all had pelvic LN, with 25% PAN. All pts completed CRT, 90% had 4 cycles of ipi, and the other 10% had 2 cycles of ipi. The ipi MTD was 10 mg/kg. There were 3 pts (16%) with acute grade 3 toxicity (lipase, ↓ANC, rash) which self-resolved. Most of the acute toxicities were grade 1-2 GI distress, rash, endocrinopathies. There were no minor or major RT quality deviations. With a median follow up of 12 months, there were no major late toxicities reported, with a 1-year DFS of 74%. There was no difference in CD4+- and CD8+- T cell levels nor CTLA-4 expression with sequential ipi. CRT itself increased ICOS and PD-1 expression. Conclusions: This study is the first to describe the safety of immunotherapy sequencing with definitive CRT in CC. Our data suggests that immunotherapy is tolerable and shows possible activity in this population with a historical dismal prognosis with standard therapy. CRT increased ICOS and PD-1 expression which was sustained with ipi, illustrative of immune modulation targets for future clinical trials and radioimmunotherapy combinations. Clinical trial information: NCT01711515.

A phase I trial of ipilimumab (ipi) in patients (pts) with myelodysplastic syndromes (MDS) after hypomethylating agent (HMAs) failure.

7010 Background: Pts with HR-MDS after HMA failure have a poor overall survival (OS) of < 6 months. Immune escape is associated with resistance to HMAs in MDS. We hypothesized that CTLA-4 blockade in these pts would be tolerable and lead to clinical responses. Methods: This investigator-initiated, CTEP-sponsored, multi-center phase 1b study enrolled pts after failure of HMAs. In dose-escalation, ipi monotherapy was given at 2 dose levels (DL): 3 and 10mg/kg. Four doses (every 3 weeks) were administered followed by a maintenance phase (4 doses every 3 months) for non-porgressors. Toxicities and responses were evaluated with CTCAE4 and IWG2006 criteria, respectively. OS was estimated using the Kaplan-Meier method. The impact on T-cells were studied by flow cytometry and TCR sequencing. Results: 29 pts from 7 centers were enrolled. Mean age (SD) was 67 (8) years. Most had IPSS high/int-2 (55%), 45% had int-1. Three of 6 pts in DL1 and 4 of 5 pts in DL2 experienced grade [G]2-4 immune-related adverse events [IRAEs] that were reversible with drug discontinuation or systemic steroids. The DL1 (3mg/kg) was expanded with no G2-4 IRAEs reported in the 18 additional pts. A total of 15 deaths occurred due to disease progression or other complications but none attributed to ipilimumab. In total, 52% received all 4 induction doses, and 24% received ≥1 maintenance dose. Best objective responses were 2 marrow complete responses (mCR, 7%). Prolonged stable disease (PSD) for ≥46 weeks occurred in 6 pts (21%) and for ≥54 weeks in 3 pts (10%). Five pts (17%) subsequently underwent allogeneic transplantation (alloSCT) without evidence of excessive toxicity. Median OS (censoring at alloSCT) was 294 days (95%CI, 240-671+) and 400 days (95%CI, 240-671+) for those who received maintenance (n = 7). Pts who achieved PSD and mCR had significantly increased expression of ICOS, a marker of T-cell activation. Conclusions: Immune checkpoint blockade with ipi is tolerable and can lead to PSD/mCR in a proportion of pts. However, ipi monotherapy efficacy is limited after HMA failure and combination-based approaches should be considered. Increased frequencies of ICOS expression might predict clinical benefit. Clinical trial information: NCT01757639.

Type 1 regulatory T cells (Tr1) homeostasis and function in aging

Abstract Type 1 regulatory T cells (Tr1) are a unique population of CD4+ Foxp3− cells that express high levels of IL-10, and have been defined based on their expression of CD49b and LAG-3. Despite the critical roles played by Tr1 cells in controlling T cell responses in autoimmunity and infection, the mechanisms underlying the homeostasis of Tr1 cells remain unclear. Here, we investigated the homeostasis, phenotype and function of Tr1 cells with age. We found that Tr1 cells accumulated dramatically with age. Further, Tr1 cells produced more IL-10 per cell compared to Foxp3+ Treg. While aged Tr1 cells expressed significant levels of c-Maf and Egr-2, two of the Tr1 defining transcription factors, they largely lacked expression of CD49b and were heterogeneous in LAG-3 and ICOS expression. Despite this heterogeneity, LAG-3 identified a population of aged Tr1 cells that potently inhibited T-cell proliferation in a partially IL-10-dependent manner in vitro and controlled colitis severity in vivo. Additional flow cytometric analysis revealed that roughly half of the IL-10 producing cells co-produced IFN-γ. Further, single cell mRNA-seq analysis revealed that IL-10+IFN-γ− cells had a TGF-beta signature, while IL-10+IFN-γ+ cells had a STAT4 signature. Strikingly, IL-6 was critical for overall Tr1 cell accumulation, as aged IL-6 KO mice showed a profound reduction in Tr1 cells and their expression of c-Maf. Combined, these data show that IL-6 promotes Tr1 accumulation with age and provide a new insight into a novel feedback loop whereby an inflammatory cytokine drives accrual of an anti-inflammatory cell population.

The role of B7 family costimulatory molecules and indoleamine 2,3-dioxygenase in primary Sjögren's syndrome and systemic sclerosis.

B7 costimulatory molecules are present on antigen-presenting cells (APCs) and influence intracellular expression of indoleamine 2,3-dioxygenase (IDO), a molecule with important immunoregulatory functions. We determined the frequency of activated (CD11b+) monocytes expressing B7-1, B7-2, B7-H1, and B7-H2 molecules, and that of CD3+ and CD4+ T cells expressing the corresponding CD28, CTLA-4, PD-1, and ICOS receptors in peripheral blood samples of 20 healthy adults and 9 SSc and 15 pSS patients using flow cytometry. We also examined the intracellular expression of IDO. The expression of CD28 was lower in both SSc and pSS patients. The frequency of CTLA-4 was increased in pSS. The expression of ICOS, a stimulator of T cell activation, was elevated in pSS, but not in SSc, while that of its corresponding costimulatory molecule, B7-H2, was strongly decreased in SSc compared to controls. The frequency of PD-1 expressing T lymphocytes was decreased in both pSS and SSc. The frequency of IDO-expressing APCs, as well as intracellular IDO content in T cells was higher in pSS than in controls. Our investigation identified a number of differences in B7 costimulation between SSc and pSS patients which may play a role in the distinct pathogenesis and clinical features of these autoimmune disorders.

IFNAR1-Signalling Obstructs ICOS-mediated Humoral Immunity during Non-lethal Blood-Stage Plasmodium Infection.

Parasite-specific antibodies protect against blood-stage Plasmodium infection. However, in malaria-endemic regions, it takes many months for naturally-exposed individuals to develop robust humoral immunity. Explanations for this have focused on antigenic variation by Plasmodium, but have considered less whether host production of parasite-specific antibody is sub-optimal. In particular, it is unclear whether host immune factors might limit antibody responses. Here, we explored the effect of Type I Interferon signalling via IFNAR1 on CD4+ T-cell and B-cell responses in two non-lethal murine models of malaria, P. chabaudi chabaudi AS (PcAS) and P. yoelii 17XNL (Py17XNL) infection. Firstly, we demonstrated that CD4+ T-cells and ICOS-signalling were crucial for generating germinal centre (GC) B-cells, plasmablasts and parasite-specific antibodies, and likewise that T follicular helper (Tfh) cell responses relied on B cells. Next, we found that IFNAR1-signalling impeded the resolution of non-lethal blood-stage infection, which was associated with impaired production of parasite-specific IgM and several IgG sub-classes. Consistent with this, GC B-cell formation, Ig-class switching, plasmablast and Tfh differentiation were all impaired by IFNAR1-signalling. IFNAR1-signalling proceeded via conventional dendritic cells, and acted early by limiting activation, proliferation and ICOS expression by CD4+ T-cells, by restricting the localization of activated CD4+ T-cells adjacent to and within B-cell areas of the spleen, and by simultaneously suppressing Th1 and Tfh responses. Finally, IFNAR1-deficiency accelerated humoral immune responses and parasite control by boosting ICOS-signalling. Thus, we provide evidence of a host innate cytokine response that impedes the onset of humoral immunity during experimental malaria.

Abstract A052: Characterization of immune regulatory molecules B7-H4, PD-L1, and ICOS in micro-satellite stable (MSS) and micro-satellite instable (MSI) endometrial tumors

Abstract Background: Endometrial cancer is the most frequent tumors of the female reproductive system. While 5-year survival rates are greater than 90% for patients with low histological grade endometrial tumors, survival rates drop for patients with high grade endometrial tumors. With the recent success in checkpoint targeting therapies, particularly in genomic instable tumors, we wanted to characterize the immune infiltrate in a cohort of endometrial tumors. B7-H4 and PD-L1 are known members of checkpoint inhibitory pathways that regulate T cell activity, and have been shown to play a role in promoting immunogenic tolerance in tumors. Both these receptors have been shown to be expressed by tumor cells and tumor-associated macrophages (TAM). ICOS (Inducible CO-Stimulator molecule), a member of the CD28 superfamily expressed primarily on T lymphocytes, has been identified as a potentially key molecule in providing optimal anti-tumor benefit following anti-CTLA-4 therapy. Preclinical data have confirmed that engagement of the ICOS pathway plays a crucial role in mediating anti-tumor responses to checkpoint inhibitors. Published reports have shown that PD-L1 expression is strongly associated with MSI status and can enrich for response to anti-PD-1 therapy. To understand the role of these molecules in mediating immune tolerance we profiled the expression of B7-H4, PD-L1, and ICOS in a cohort of low grade and high grade endometrial patients and correlated with MSI and MSS status. Methods: FFPE tissues from a cohort of 92 patients with endometrial cancer, collected at the Massachusetts General Hospital, were analyzed for expression of B7-H4, PD-L1, and ICOS using established immunohistochemistry (IHC) protocols. Microsatellite instability status was assessed for approximately 50 tumors using an IHC assay for expression of the mismatch repair (MMR) genes, MLH1, MSH2, MSH6 and PMS2. Additionally, ICOS expression on intratumoral T cells was evaluated using RNA sequencing data collected from Cancer Genome Atlas (TCGA). Results: Staining of endometrial cancer samples showed that while a majority of endometrial tumors expressed B7-H4, only a small subset expressed PD-L1. No significant overlap was observed in tumors expressing both inhibitory ligands suggesting that these mechanisms are mutually exclusive in the generation of immune tolerance process. The MSI status was determined by evaluating the expression of miss-match repair genes (MMR) by IHC as a surrogate for genomic stability. We observed that PD-L1 expression was strongly associated with microsatellite instable (MSI) tumors (∼40%) with little-to-no expression of PD-L1 observed in the microsatellite stable (MSS) patients. In both sub-sets, PD-L1 expression was observed predominantly on the infiltrating tumor cells. In contrast, B7-H4 was observed to be in both MSI and MSS sub-set of tumors, however higher levels of B7-H4 (2+/3+) were observed in MSI compared to the MSS subset. In addition to inhibitory ligands we evaluated expression of ICOS on the immune infiltrates. High infiltration of ICOS+ cells was observed primarily in the MSI subset. This is consistent with published literature showing high numbers of T cells are associated with the MSI phenotype. Similar data was observed with the POLE mutant tumors that also have high levels of mutations. Together, these data support development and use of B7-H4 or ICOS targeted therapeutics for treatment of MSI sub-set of endometrial tumors. Citation Format: Amit Deshpande, Whitfield B. Growdon, Heather Hirsch, Tong Zi, Chris Grange, Jason Reeves, Jennifer Stall, Bo Rueda, Sriram Sathyanarayanan. Characterization of immune regulatory molecules B7-H4, PD-L1, and ICOS in micro-satellite stable (MSS) and micro-satellite instable (MSI) endometrial tumors [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A052.

Abstract A053: Targeting ICOS improves immune-mediated control of tumor growth in humanized mice

Abstract Checkpoint blockade inhibitors are the most promising and effective strategy for T-cell mediated cancer immunotherapy of the past 20 years. Part of the anti-tumoral effect of these checkpoint inhibitors might be due to preferential targeting of regulatory T cells (Treg), essential for immune homeostasis. Here, we investigated whether the reported higher expression of ICOS on Treg than on regular T cells might be used as a flag to target human Treg to improve tumor-directed immune response. We report that a novel murine anti-human ICOS IgG1 mAb preferentially depleted Treg in NSG mice reconstituted with a human immune system from cord blood CD34+ cells, leading to an increased CD8 to Treg ratio. However, this was insufficient to affect growth of the breast cancer cell line MDA-MB-231 in humanized mice. We thus administered low dose cyclophosphamide that only moderately affected tumor growth to induce immunogenic cell death and improve tumor infiltration by human cells. Treatment of humanized mice with a combination of cyclophosphamide and the anti-ICOS mAb led to a marked reduction in tumor growth. Using mass cytometry (Cytof), we then analyzed the expression of 29 markers to quantify the main human and murine cell subsets in the tumor. We observed higher activation of CD8+ T cells in the combined-treatment group as judged by their higher expression of CD45RO and HLA-DR, relative to chemotherapy alone. Accordingly, depletion of CD8+ T cells with a mAb partly abolished the therapeutic effect of the combination, showing that these cells participated in tumor rejection. Notably, we also observed improved infiltration of human monocytes, human plasmacytoid dendritic cells and murine CD11b+CD11c+ cells in the tumor, suggesting that part of the effect of the combination therapy on tumor growth might proceed through these subsets. Altogether, our results demonstrate that ICOS, through its effect on regulatory T cells, release a brake in the immune response to tumors in humanized mice, and as such might be considered as a novel immune checkpoint inhibitor.> Note: This abstract was not presented at the conference. Citation Format: Aude Burlion, Aurélien Corneau, Gilles Marodon. Targeting ICOS improves immune-mediated control of tumor growth in humanized mice [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A053.

ICOS+ Foxp3+ TILs in gastric cancer are prognostic markers and effector regulatory T cells associated with Helicobacter pylori.

Regulatory T cells (Tregs) have an immunosuppressive role in the tumor microenvironment. Since effector Tregs (eTregs), which have highly suppressive functions, are located in a subpopulation of Foxp3+ CD4+ Tregs, the TCR-inducible costimulatory receptor (ICOS) was applied as a marker of eTregs that infiltrated gastric cancer tissue and the induction pathway of ICOS+ Foxp3+ cells was analyzed by flow cytometry and immunohistochemistry. In tumor-infiltrating lymphocytes (TILs), ICOS+ Foxp3+ CD4+ T cells were abundantly observed in the late stages of gastric cancer. ICOS+ CD4+ TILs exhibited the ability to produce IL-10, but not IFN-γ, TNF, or IL-17 and also to suppress the proliferation of CFSE-labeled responder CD8+ T cells. With the agonistic ICOS-L protein (rICOS-L Ig), ICOS+ Foxp3+ cells were efficiently induced from naive CD4+ T cells under a stimulation with TGF-β and CD3/CD28 mAbs. Furthermore, when A*0201 PBMCs were cultured with the CMV or Melan-A antigenic peptide and rICOS-L Ig, the induction of CMV or Melan-A tetramer-binding CD8+ T cells, respectively, was inhibited. The expression of ICOS in Foxp3+ cells was closely related to plasmacytoid dendritic cells (pDCs) and their expression of ICOS-L and TLR9 as well as Helicobacter pylori infection. Collectively, our results demonstrate the potential of ICOS as a promising target for direct Treg-targeting therapeutic agents for gastric cancer, and that of eradicating therapy for H. pylori as an indirect immune therapy for gastric cancer.

Activation of intrahepatic CD4+CXCR5+ T and CD19+ B cells is associated with viral clearance in a mouse model of acute hepatitis B virus infection.

The role of immunity in the pathogenesis of acute hepatitis B virus (HBV) infection is poorly understood. The purpose of this research was to define the intrahepatic immune factors responsible for viral clearance during acute HBV infection. The model of acute HBV infection was established by hydrodynamically transfecting mice with pCDNA3.1-HBV1.3 plasmids which contained a supergenomic HBV1.3-length transgene. The frequency of CD4+ CXCR5+ T cells, CD19+ B cells and their surface molecules in livers, spleens and peripheral blood were detected using flow cytometry. The lymphomononuclear cells isolated from the livers of transfected mice were further stimulated by HBc-derived peptides and then the frequency and cytokine secretion of HBV-specific CD4+CXCR5+ T cells were detected. We found that the frequency of CXCR5+ in CD4+ T cells was specifically increased; the expression of PD-1 was decreased while the expression of ICOS was increased on intrahepatic CD4+CXCR5+ T cells. Although the frequency of CD19+ B cells was not affected, the expression of PDL-1, ICOSL and IL-21R on B cells was increased in the livers of mice. The frequency of HBV-specific CD4+CXCR5+ T cells and the production of IL-21 by intrahepatic CD4+CXCR5+ T cells of mice with acute HBV infection were increased after stimulation. Furthermore, the expression of function-related molecules of intrahepatic CD4+CXCR5+ T, including Bcl-6, CXCR5, IL-6, IL-6R, IL-21 and IL-4 in the liver was increased during acute HBV infection. In conclusion, the activation of intrahepatic CD4+CXCR5+ T cells and B cells was associated with the clearance of HBV during acute infection.

Abstract 4139: TfhX13 cells link breast cancer immune suppression and adaptive memory

Abstract Background: Previously, we identified PD-1hiCD200hiCD4+ follicular helper T (Tfh) cells infiltrating human breast cancer (BC TIL) as principal producers of the B cell chemoattractant CXCL13 and correlated their presence with improved patient outcome. Our recent efforts have focused on understanding the relationship between Tfh, Th1 and Treg BC TIL, investigating the role(s) that CXCL13+CD4+ TIL play in tumor immunity and determining the condition(s) that favor their differentiation. Methods: Ten-color flow cytometry was used to characterize CD4+ TIL subpopulations in fresh tissues. Confocal microscopy was employed to study the in situ interaction between CXCL13+CD4+ T cells and B cells. CXCL13 was induced in normal PBMC in vitro. Results: Our data reveal that an exacerbated Treg response accompanies high CXCL13 expression within the activated CD4+ TIL compartment in BC. Th1 responses, using IFN-γ expression as a barometer, are limited. CXCL13+CD4+ TIL display both similar and distinct characteristics compared to their tonsillar counterparts. Tumor bed-localized CXCL13+ TIL, external to tertiary lymphoid structure (TLS) germinal centers (GC), are abundant in some tumors with extensive lymphoid infiltrates. Based on our observations, we designate the CXCL13+CD4+ TIL as TfhX13 cells. A combination of PD-1 and ICOS can clearly segregate the CD4+ TIL into three distinct subpopulations: PD-1loICOSlo, PD-1hiICOSint and PD-1intICOShi. The PD-1hiICOSint TIL are enriched in TfhX13 and Th1 cells while the PD-1intICOShi TIL are principally FoxP3hi Treg. A linear correlation is observed between these two TIL subpopulations in 90% of BC while the remaining 10% contain an unbalanced, higher level of PD-1intICOShi Treg. qRT-PCR data confirmed the significant positive prognostic value of CXCL13 gene expression with ICOS expression signaling the adverse effects of Treg cell dominance observed in some BC. TfhX13 TIL abundance appears to parallel both a GC and post-TLS memory B cell presence. Confocal microscopy revealed TfhX13 TIL interact directly with B cells and plasma cells, potentially guiding B cell migration through the tumor and promoting TLS formation with functional GC. Finally, we show that IL-2 deprivation is critical for inducing CXCL13 while TGF-β1 treatment reduces IFN-γ and increases FoxP3 expression in normal CD4+ T cells. Conclusion: IL-2 consumption by Treg cells was shown to be essential for murine Tfh cell development and a subsequent GC response. Supported by this observation, our data indicate that TfhX13 cell differentiation is an important element of the Tfh cell program and suggests their propagation in BC occurs in response to Treg accumulation. CXCL13 expression may activate adaptive memory responses dependent upon in situ B cell maturation and thereby initiate a secondary attempt at protective anti-tumor immunity in the face of Treg-mediated immune suppression. Citation Format: Chunyan Gu-Trantien, Edoardo Migliori, Denis Larsimont, Karen Willard-Gallo. TfhX13 cells link breast cancer immune suppression and adaptive memory. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4139.