Cell Inhibitory Signals Research Articles

The CD6 interactome orchestrates ligand-independent T cell inhibitory signaling

BackgroundT-cell membrane scaffold proteins are pivotal in T cell function, acting as versatile signaling hubs. While CD6 forms a large intracellular signalosome, it is distinguished from typical scaffolds like LAT or PAG by possessing a substantial ectodomain that binds CD166, a well-characterized ligand expressed on most antigen-presenting cells (APC), through the third domain (d3) of the extracellular region. Although the intact form of CD6 is the most abundant in T cells, an isoform lacking d3 (CD6∆d3) is transiently expressed on activated T cells. Still, the precise character of the signaling transduced by CD6, whether costimulatory or inhibitory, and the influence of its ectodomain on these activities are unclear.MethodsWe expressed CD6 variants with extracellular deletions or cytosolic mutations in Jurkat cells containing eGFP reporters for NF-κB and NF-AT transcription factor activation. Cell activation was assessed by eGFP flow cytometry following Jurkat cell engagement with superantigen-presenting Raji cells. Using imaging flow cytometry, we evaluated the impact of the CD6-CD166 pair on cell adhesiveness during the antigen-dependent and -independent priming of T cells. We also examined the role of extracellular or cytosolic sequences on CD6 translocation to the immunological synapse, using immunofluorescence-based imaging.ResultsOur investigation dissecting the functions of the extracellular and cytosolic regions of CD6 revealed that CD6 was trafficked to the immunological synapse and exerted tonic inhibition wholly dependent on its cytosolic tail. Surprisingly, however, translocation to the synapse occurred independently of the extracellular d3 and of engagement to CD166. On the other hand, CD6 binding to CD166 significantly increased T cell:APC adhesion. However, this activity was most evident in the absence of APC priming with superantigen, and thus, in the absence of TCR engagement.ConclusionsOur study identifies CD6 as a novel ‘on/off’ scaffold-receptor capable of modulating responsiveness in two ways. Firstly, and independently of ligand binding, it establishes signaling thresholds through tonic inhibition, functioning as a membrane-bound scaffold. Secondly, CD6 has the capacity for alternative splicing-dependent variable ligand engagement, modulating its checkpoint-like activity.

Abstract A096: Blockade of LAIR1-collagen interaction enhances the therapeutic activity of BiTE-secreting T cells in ovarian cancer

Abstract Introduction: The tumor microenvironment (TME), abundant with collagen produced by cancer associated fibroblasts (CAFs), plays a critical role in evading the immune system and limiting the efficacy of cell therapies. High levels of collagen and CAF expression correlate with malignant potential, decreased T cell accumulation, and overall unfavorable clinical outcomes in ovarian cancer (OC). Finding ways to address this immunosuppressive TME is therefore critical to improving therapeutic options. We recently reported on the therapeutic potential of adoptively transferred T cells engineered to secrete a folate receptor alpha (FRα)-targeted bispecific T cell engager (FR-B T cells) to target a tumor antigen commonly overexpressed in OC. Transcriptional profiling following ACT revealed that effector-like FR-B T cells (FR-BE T cells) upregulated genes associated with extracellular matrix interaction, suggesting interaction with the tumor stroma may limit antitumor immunity by FR-B T cells. Our goal was to improve the effectiveness of FR-B T cells by inhibiting LAIR-1, a widely present immune checkpoint receptor that sends T cell inhibitory signals after binding to collagen-like domains, leading to T cell exhaustion. We hypothesized that interrupting the LAIR1-collagen pathway using a LAIR1 decoy (NC410, supplied by NextCure Inc.) could amplify the FR-BE T cell effector function, thereby improving the response. Methods: We utilized in vitro co-culture assays to understand the phenotype and mechanistic changes occurring at the level of the T cell/tumor interface when exposed to collagen, as well as effects of LAIR 1 pathway blockade with the addition of NC410. In vivo murine models were used to determine disease progression and overall survival (OS) following treatment with bispecific T cell engager-armed T cells ± LAIR1 blockade. Results: Phenotypic analysis of FR-B T cells revealed elevated LAIR-1 expression on FR-BE T cells. FRα+ OC lysis by FR-BE T cells was diminished in the presence of collagen, suggesting LAIR-1 signaling can suppress tumor attack. In vitro, the addition of NC410 enhanced the activity of FR-BE T cells when exposed to collagen, improving both FRα+ target cell lysis and partially restoring the production of IFN-γ by FR-BE T cells. In vivo, the combination of NC410 with FR-BE T cells led to improved tumor control compared to FR-BE T cell therapy alone. Conclusions: Our results highlight understanding the interactions between infiltrating immune cells and the immunosuppressive TME to better design combination therapies that improve duration and magnitude of immune attack on tumors. By blocking T cell/stromal interactions, FR-B T cell therapy can be further enhanced, creating a promising opportunity to improve the impact of adoptive cell therapy in OC. Citation Format: Sarah Werner, Nicole Gaulin, Jessie Chiello, Theja Giridharan, Anm Nazmul Khan, Sora Suzuki, Suzanne Hess, Sol Langermann, Emese Zsiros, Brahm H. Segal, A.J. Robert McGray. Blockade of LAIR1-collagen interaction enhances the therapeutic activity of BiTE-secreting T cells in ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr A096.

Fabrication of Nanoscale Arrays to Study the Effect of Ligand Arrangement on Inhibitory Signaling in NK Cells.

Molecular scale nanopatterns of bioactive molecules have been used to study the effect of transmembrane receptor arrangement on a variety of cell types, including immune cells and their immune response in particular. However, state-of-the-art fabrication approaches have thus far enabled the production of patterns with control over one receptor type only. Herein, we describe a protocol to fabricate arrays for the molecular scale control of the segregation between activating and inhibitory receptors in NK cells. We used this platform to study how ligand segregation regulates NK cell inhibitory signaling and function. The arrays are based on patterns of nanodots of two metals, selectively functionalized with activating and inhibitory ligands. Due to the versatility of our functionalization approach, this protocol can be applied to configurate virtually any combination of extracellular ligands into controlled multifunctional arrays.

Abstract A034: Cancer and immunology: The homeostasis dance

Abstract The human body is made up of not only tissues and blood, but also present are microbiota on surfaces of the skin, mucosal membrane of the intestine, urogenital, lungs, and mouth, to form ecological niches. Some of these niches have been well studied, while some are yet to be elucidated due to the unknown presence of these microbiota in such system. T cell response by immune system is one step of the cancer-immunity cycle, that maintains the prevention of autoimmunity. Cancer cells have T cell inhibitory signals including PD-L1 which have been identified for the development of new immunotherapy that are specifically responsible for hindering immune effector inhibition, thereby reinvigorating, and enhancing pre-existing anticancer immune response. Previous activity in immune therapies have always considered suppressive factors in the tumor microenvironment without consideration to other factors such as the genetic basis of the immune system. Attention to the immune system has always been on the response to the pathogens, or the threatening foreign target, but not on the genes responsible for regulating the immune system. The immune system is a concert of interactions between existing microbes and host. One of the major tools of cellular interaction is epigenetics. Epigenetic information regulates differentiation and development, thus can impact on pathological condition. Therefore, epigenetics interaction between microbes and host is vital in cancer development and treatment. Diagnostic and prognosis will be impacted by the understanding of the interactions between microbes and host, thereby informing on precision medicine. Citation Format: Bene Akromaa Ekine-Afolabi. Cancer and immunology: The homeostasis dance [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer; 2022 Oct 1-4; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_1):Abstract nr A034.

Cancer and Immunology-the Homeostasis Dance

The human body is made up of not only tissues and blood, but also microbiota on the surfaces of the skin, mucosal membrane of the intestine, urogenital, lungs, and mouth, to form ecological niches. Some of these niches have been well studied, while some are understudied, due to the unknown presence of these microbiota in such systems. The T cell response by the immune system is one step of the cancer-immunity cycle, that maintains the prevention of autoimmunity. Cancer cells have T cell inhibitory signals, including programmed death-ligand-1, which have been identified for the development of new immunotherapies that are specifically responsible for hindering immune effector inhibition, thereby reinvigorating, and enhancing pre-existing anticancer immune response. Previous activity in immune therapies has always considered suppressive factors in the tumour microenvironment without consideration to other factors such as the genetic basis of the immune system. Attention to the immune system has always been on the response to the pathogens, or the threatening foreign target, but not on the genes responsible for regulating the immune system. The immune system is a concert of interactions between existing microbes and host. One of the major tools of cellular interaction is epigenetics. Epigenetic information regulates differentiation and development, thus can impact on pathological condition. Therefore, it is vital to understand the resident parties (constituents) in an ecosystem, the basic system behind the ecosystem and epigenetics interaction within the ecosystem (microbes and host) is vital in cancer development and treatment.

PRDM1 Is Sufficient for Inducing Human Primary T Cell Hyporesponsiveness and Implicates Low Gvhd Occurrence after Allo-HSCT

T cell hyporesponsiveness is crucial for functional immune system and prevent the damage induced by alloreactive T cells in autoimmune pathology and transplantation. As one of the most important regulators during B cell development, PRDM1 (also known as BLIMP-1) has been demonstrated its essential role for maintaining T cell hyporesponsiveness and homeostasis, evidenced by Prdm1 deficient mice accumulating activated T cells and developing multiorgan inflammatory disease. However, the mechanism of PRDM1 regulating T cell hyporesponsiveness is still ambiguous.In this study, we took advantage of multiomics technologies and systemic report the central role of PRDM1 in inducing human primary T cell hyporesponsiveness. Firstly, we overexpressed PRDM1 in human primary T cells and found increased ratio of CD4 +CD25 +FOXP3 + Treg cell subsets and increased IL-4 secretion. In parallel, inhibited PRDM1 expression level in human T cells decreased ratio of Treg cells and secretion of IL-4. Meanwhile, transcriptome analyses revealed that overexpressed PRDM1 enriched negative regulation of cell proliferation signaling pathway and resulted in a global reduction in IL-2 and inflammatory response signaling pathways. Furthermore, overexpressed PRDM1 in primary T cells upregulated several negative regulators of T cell function like EOMES, KLF2, LILRB1, KLRB1 and CD244, indicating a pioneer role of PRDM1 in inducing T cell hyporesponsive. To further investigate the regulation role of T cell hyporesponsiveness of PRDM1, we performed CUT&Tag and ATAC-seq in PRDM1 overexpressed primary T cells. CUT&Tag analysis showed PRDM1 could directly upregulated T cell hyporesponsiveness related gene such as KLF2, CD244 and KLRD1. Importantly, we observed consistent changes of IL-2, central regulator of T cell activation, in PRDM1 overexpressed T cell from ATAC-seq, CUT&Tag and RNA-seq data. We found PRDM1 could binding to IL-2 locus and decreased the chromatin accessibility of IL-2, consequently downregulated the expression level of IL-2 in human primary T cells. Moreover, altered open chromatin regions (OCRs) in PRDM1 overexpressed T cells enriched the similar transcription factors (TFs) with PRDM1 binding sites, indicating PRDM1 might be a pioneer TF in T cell hyporesponsiveness. These results demonstrated PRDM1 is sufficient for inducing T cell hyporesponsiveness in human primary T cells.To further validate the coexpression relationship between PRDM1 and Treg cell central TF FOXP3, we upregulated PRDM1 expression level on Jurkat T cells lines. The results also showed that elevated FOXP3 both in mRNA and protein level accompanied with upregulated PRDM1 expression level. To analyze the mechanism of PRDM1 regulating FOXP3 expression level, CUT&Tag data analyses showed that PRDM1 might upregulated FOXP3 by directly binding to the enhancer region of upstream of FOXP3 locus. Meanwhile, PRDM1 indirectly upregulated FOXP3 by upregulated KLF2, evidenced by inhibiting KLF2 in PRDM1 overexpressed primary T cells downregulated FOXP3 expression level. To further investigate the clinical implication of PRDM1 inducing T cell hyporesponsiveness, we detected the relationship of PRDM1 expression level and acute graft-versus-host disease(aGVHD) occurrence after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Our results showed that patients with aGVHD (n=7) exhibited lower PRDM1 expression level than those without aGVHD in the same period after HSCT (n=7). Furthermore, we detected the expression level of PRDM1 in CD4 + T cell and CD8 + T cells from bone marrow allografts (BM) or peripheral blood allografts (PB) and followed up the occurrence of GVHD after HSCT for 2 years (n=18). There are low expression levels of PRDM1 in CD4 + T cells both from BM or PB grafts corelated with aGVHD occurrence in patients after allo-HSCT compared with those without aGVHD occurrence. In conclusion, our study provides the global regulatory model of PRDM1 in human primary T cell. We introduced PRDM1 as a sufficient regulator in T cell hyporesponsiveness induction, which is altering the chromatin accessibility and directly upregulated T cell inhibitory signals and downregulated T cell activated signals. The negative relationship between PRDM1 expression level with GVHD occurrence indicated it might be a potential biomarker for indicating HSCT prognosis. DisclosuresNo relevant conflicts of interest to declare.

Targeting Siglec-7: A Novel Immunotherapeutic Approach to Potentiate the Cytotoxic Functions of Natural Killer Cells Against Multiple Myeloma

Background: MultipleMyeloma (MM) is a malignant plasma cell disorder, accounting for approximately 10% of all haematological malignancies. The intrinsic ability of the malignant cells to evade the host immune system is one of the key hallmarks of the disease, and is associated with poor overall survival of MM patients. The immune-suppressive functions of sialylated glycans, which are abundantly expressed on the surface of myeloma cell, has not previously been addressed. We hypothesize that hypersialylation of MM cells is a key mechanism enabling evasion of cytotoxic Natural Killer (NK) cells within the MM bone-marrow (BM) niche. Furthermore, we hypothesize that disrupting the immune-suppressive interactions between sialylated glycans on MM cells, and their cognate sialic acid-binding lectin (Siglec) receptors on NK cells could pave the way for the development of novel immunotherapeutic strategies for treating MM patients.Methods and Results: Using a recombinant Siglec-7 Fc chimera, we observed that Siglec-7 ligands (Siglec-7L) are highly expressed (>85%) across a panel of MM cell lines (RPMI 8226, H929 and MM1S). Furthermore, Siglec-7L expression was also observed on CD38+ CD138+ primary MM cells isolated from BM aspirate of MM patient. To determine if Siglec-7L expression on MM cells plays a role in immune-evasion, we next analysed the cognate Siglec-7L receptor (Siglec-7R) on primary BM NK cells from MM patients (n=5). Interestingly, immunophenotyping analysis revealed that CD3- NKp46+ NK cells circulating within the BM of MM patients have significant expression of Siglec-7R on their cell surface (78 ±27%).To investigate the immune-suppressive effects of Siglec-7L and Siglec-7R interaction in dampening the cytotoxic functions of NK cells, we treated MM cell lines RPMI-8226, H929 and MM1S with a sialyltransferase inhibitor (SIA) (3Fax-Peracetyl Neu5Ac; dose-200µM) for 5 days. As expected, this treatment resulted in a complete eradication of Siglec-7L from the surface of MM cells. Subsequently, SIA pre-treated cells were co-cultured with the human cytotoxic NK cell line KHYG-1 at E:T ratios of 1:1, 2.5:1 and 5:1 for 12 hours. Flow cytometry based analysis of cell viability using Annexin V and Sytox Green revealed that abolishing the expression of sialylated glycans from the cell surface of MM cells results in a 1.3-1.6 fold increase (p<0.05) in NK cell induced cell death in MM cell lines RPMI 8226 and H929 (n=3) (Figure 1). However, a significant increase in NK cell mediated killing was not observed for MM1S cells (n=3) pre-treated with SIA, suggesting a role of “classical” NK cell inhibitory signals in NK cell anti-tumor immunity in the case of MM1S.Furthermore to validate our observations we knocked-down (KD) Siglec-7R in KHYG-1 cells using an siRNA approach. KHYG-1 has relatively lower expression of Siglec-7R (20-25%), as compared to peripheral blood NK cells (>90%), and other NK cell lines such as NK-92 (>60%). siRNA targeting Siglec-7R reduced the expression of Siglec-7R from 25% in the scrambled siRNA control to 15% in the Siglec-7R siRNA experimental condition. Upon co-culture of the scrambled siRNA or Siglec-7R siRNA KD KHYG-1 cells with RPMI-8226 at E:T ratio of 0.25:1 and 0.5:1, we observed a 7% increase in NK cell mediated cytotoxicity towards the myeloma cells (n=2) .Conclusion: We observed that sialylated Siglec-7L is abundantly expressed on the surface of MM cells. The cognate receptors of the ligands are significantly expressed on primary NK cells circulating within the BM of MM patients. Functional assays revealed that Siglec-7L and Siglec-7R receptor interactions may have significant immune-suppressive effects on the cytotoxicity of NK cells. Thus, we can leverage the findings of this study to develop NK cell based cellular therapies to target Siglec-7 ligand- receptor interaction in MM. This could be achieved by knocking down Siglec-7R in the setting of an autologous or allogeneic blood derived NK cell therapy. A more efficient approach, both therapeutically and logistically, would be the use of Siglec-7R deficient NK cell lines, such as KHYG-1 or NK-92 for the treatment of patients with MM.Conflict Of Interest: Michael O'Dwyer: Founder, Board of Directors, Equity in Onkimmune. Subhashis Sarkar is supported by a fellowship from Onkimmune. [Display omitted] DisclosuresSarkar:Onkimmune: Other: Fellowship Funding. O'Dwyer:Onkimmune Ltd: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

Combined anti-PD-1 and anti-CTLA-4 checkpoint blockade: Treatment of melanoma and immune mechanisms of action.

Cytotoxic T-lymphocyte associated protein-4 (CTLA-4) and the Programmed Death Receptor 1 (PD-1) are immune checkpoint molecules that are well-established targets of antibody immunotherapies for the management of malignant melanoma. The monoclonal antibodies, Ipilimumab, Pembrolizumab, and Nivolumab, designed to interfere with T cell inhibitory signals to activate immune responses against tumors, were originally approved as monotherapy. Treatment with a combination of immune checkpoint inhibitors may improve outcomes compared to monotherapy in certain patient groups and these clinical benefits may be derived from unique immune mechanisms of action. However, treatment with checkpoint inhibitor combinations also present significant clinical challenges and increased rates of immune-related adverse events. In this review, we discuss the potential mechanisms attributed to single and combined checkpoint inhibitor immunotherapies and clinical experience with their use.

Abstract B87: NK cells lose their ability to control MHC-I-deficient tumor cells outside circulation

Abstract A common tumor immune-evasion mechanism is downregulation of major histocompatibility complex Class-I (MHC-I) that enables malignant cell escape from cytotoxic T-cell responses. This strategy also contributes to nonresponsiveness of tumors to T-cell targeted immune-checkpoint blockade. MHC-I is an antigen-presenting molecule that also functions as an inhibitory ligand for natural killer (NK) cells. Thus, loss of MHC-I should render cells susceptible to NK cell-mediated cytotoxicity. However, this does not always occur. Previously it was demonstrated that NK cells were able to strongly reject MHC-I-deficient bone marrow cells in the circulation, yet they failed to reject MHC-I-deficient organ grafts, suggesting restricted NK cell cytotoxicity in tissues. We hypothesized that a similar NK cell tolerance to MHC-I-deficient cells occurs in the context of solid cancers. We compared NK cell recruitment and activation using WT and MHC-I-deficient B16F10 melanoma models. We demonstrated that NK cells eradicate circulating MHC-I−/− B16F10 cells, effectively preventing tumor metastasis to the lungs. In contrast, NK cells failed to clear MHC-I−/− B16F10 tumors when implanted subcutaneously, nor was the growth of these tumors affected by NK cell depletion. Interestingly, spontaneous lymph node (LN) metastasis from MHC-I-deficient solid tumors was fully abrogated in an NK cell-dependent manner, indicating functional inhibition of NK cells in peripheral tissues, but not in circulation or draining lymphatics. In conclusion, we demonstrate NK cell tolerance to MHC-I-deficient tumor cells in tissues, but not in circulation, which suggests the presence of non-MHC-I NK cell-inhibitory signals in solid organs. Our research uncovers a fundamental aspect of NK cell biology and will open up the possibility of new NK cell-based therapeutic strategies to control solid tumors that escape cytotoxic CD8+ T-cell responses by downregulating MHC-I. Citation Format: Marta Requesens, Mark Bunting, Maulik Vyas, Shawn Demehri. NK cells lose their ability to control MHC-I-deficient tumor cells outside circulation [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B87.

Epigenetic profiling of tumor infiltrating lymphocytes

Blocking T cell inhibitory signals, or “checkpoint blockade”, has provided unprecedented clinical results for therapy of cancer (1). Antibodies that block negative interactions between programmed death-1 (PD-1) and programmed death-ligand 1 (PD-L1) allow activation of T cells that recognize tumor antigens and promote immune destruction of the tumor. To determine the activities of T cells of tumor infiltrating lymphocytes (TILs), and to determine which patients will most effectively respond to checkpoint blockade therapies, Schietinger and colleagues developed a novel inducible autochthonous tumor in AST × CreER T2 mice. In this system, they successfully analyzed TIL activity and chromatin remodeling during tumor development (2,3).

Abstract 1662: Development and validation of humanized mice models implanted with patient derived colorectal cancer xenografts

Abstract Introduction: Agents that reverse T cell inhibitory signals have reinvigorated the strategy of cancer immunotherapy and are leading to robust clinical responses. In colorectal cancer (CRC), objective responses to single-agent therapy with anti-PD-1/PD-L1 antibodies have largely been restricted to the small proportion of CRC patients with microsatellite unstable (MSI-high) disease. Although there are additional subsets of advanced CRC patients that are responsive to immune checkpoint inhibition in combination with tumor-targeted agents, preclinical models have been hampered by their immune-compromised status. In order to gain a better biological understanding of the context of immune responses and facilitate preclinical evaluation of combination strategies incorporating cancer immunotherapy, we developed a “hematopoietic” humanized mouse model utilizing patient-derived CRC xenografts with the intent of leveraging this model for the development of rational combinations. Methods: BRG.NODSirpalfa newborn pups were humanized through transplantation of 1x105 CD34+ cells purified from umbilical cord blood. Mice were evaluated for chimerism at 8 and 12 weeks. At 16 weeks, tumor tissue from established PDX models was implanted on the right and left flanks of humanized mice. When the average tumor size reached a volume of ~150-300 mm3, the mice were randomized into vehicle or nivolumab (30 mg/kg twice weekly i.p.) treatment groups according to %chimerism. Mice were monitored for signs of toxicity and tumor size was evaluated twice weekly by caliper measurements (tumor volume= (length × width2) × 0.52). At the end of the treatment, mice were euthanized while sera, lymph nodes, spleen, bone marrow and tumors were collected for immunological assessment. Results: As preliminary proof-of-concept, we successfully established 3 humanized CRC PDX models and one breast cancer cell line (MDA-MB-231). In one of the CRC (MSI-high) and in the breast model we observed tumor growth inhibition in the treated groups vs control. We detected differences in PD-1 expression among treated versus control mice, with lower expression in the nivolumab-treated groups. We also observed an increased number of TILs, CD8+ T cells and greater numbers of T cells in the lymph nodes of treated mice, suggesting T cell expansion. Mice were highly chimeric with high TILs whereas responder tumors exhibited an increase of CD44 high IFN+ T cells, high CD8% and a higher effector memory% (HLADR+, CD45RO+). Conclusions: Humanized PDX models were successfully established and tumor engraftment occurred in all humanized mice with nivolumab-treated mice demonstrating the development of lymph nodes that were populated by activated T cells. These preliminary results demonstrate that human immunity and PD-1 expressing T cells exist in these models and provide the basis for planned immunotherapy combination studies. Citation Format: Anna Capasso, Julie Lang, Todd M. Pitts, Sarah Lindsey Davis, Christopher Lieu, Scott Kopetz, Stacey Bagby, Paul Francoeur, John J. Tentler, Jill Slansky, Roberta Pelanda, Gail Eckhardt. Development and validation of humanized mice models implanted with patient derived colorectal cancer xenografts [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 1662. doi:10.1158/1538-7445.AM2017-1662

Anti-CD20 Therapy Acts via FcγRIIIA to Diminish Responsiveness of Human Natural Killer Cells.

Natural killer (NK) immune cells mediate antibody-dependent cellular cytotoxicity (ADCC) by aggregating FcγRIIIA/CD16, contributing significantly to the therapeutic effect of CD20 monoclonal antibodies (mAb). In this study, we show that CD16 ligation on primary human NK cells by the anti-CD20 mAb rituximab or ofatumumab stably impairs the spontaneous cytotoxic response attributable to cross-tolerance of several unrelated NK-activating receptors (including NKG2D, DNAM-1, NKp46, and 2B4). Similar effects were obtained from NK cells isolated from patients with chronic lymphocytic leukemia in an autologous setting. NK cells rendered hyporesponsive in this manner were deficient in the ability of these cross-tolerized receptors to phosphorylate effector signaling molecules critical for NK cytotoxicity, including SLP-76, PLCγ2, and Vav1. These effects were associated with long-lasting recruitment of the tyrosine phosphatase SHP-1 to the CD16 receptor complex. Notably, pharmacologic inhibition of SHP-1 with sodium stibogluconate counteracted CD20 mAb-induced NK hyporesponsiveness, unveiling an unrecognized role for CD16 as a bifunctional receptor capable of engendering long-lasting NK cell inhibitory signals. Our work defines a novel mechanism of immune exhaustion induced by CD20 mAb in human NK cells, with potentially negative implications in CD20 mAb-treated patients where NK cells are partly responsible for clinical efficacy.

Cancer Dormancy: A Regulatory Role for Endogenous Immunity in Establishing and Maintaining the Tumor Dormant State.

The significant contribution of host immunity in early tumorigenesis has been recently recognized as a result of our better understanding of the molecular pathways regulating tumor cell biology and tumor-lymphocyte interactions. Emerging evidence suggests that disseminated dormant tumor cells derived from primary tumors before or after immune surveillance, are responsible for subsequent metastases. Recent trends from the field of onco-immunology suggest that efficiently stimulating endogenous anticancer immunity is a prerequisite for the successful outcome of conventional cancer therapies. Harnessing the immune system to achieve clinical efficacy is realistic in the context of conventional therapies resulting in immunogenic cell death and/or immunostimulatory side effects. Targeted therapies designed to target oncogenic pathways in tumor cells can also positively regulate the endogenous immune response and tumor microenvironment. Identification of T cell inhibitory signals has prompted the development of immune checkpoint inhibitors, which specifically hinder immune effector inhibition, reinvigorating and potentially expanding the preexisting anticancer immune response. This anticancer immunity can be amplified in the setting of immunotherapies, mostly in the form of vaccines, which boost naturally occurring T cell clones specifically recognizing tumor antigens. Thus, a promising anticancer therapy will aim to activate patients’ naturally occurring anticancer immunity either to eliminate residual tumor cells or to prolong dormancy in disseminated tumor cells. Such an endogenous anticancer immunity plays a significant role for controlling the balance between dormant tumor cells and tumor escape, and restraining metastases. In this review, we mean to suggest that anticancer therapies aiming to stimulate the endogenous antitumor responses provide the concept of the therapeutic management of cancer.

Long noncoding RNA derived from CD244 signaling epigenetically controls CD8+ T-cell immune responses in tuberculosis infection

Molecular mechanisms for T-cell immune responses modulated by T cell-inhibitory molecules during tuberculosis (TB) infection remain unclear. Here, we show that active human TB infection up-regulates CD244 and CD244 signaling-associated molecules in CD8(+) T cells and that blockade of CD244 signaling enhances production of IFN-γ and TNF-α. CD244 expression/signaling in TB correlates with high levels of a long noncoding RNA (lncRNA)-BC050410 [named as lncRNA-AS-GSTT1(1-72) or lncRNA-CD244] in the CD244(+)CD8(+) T-cell subpopulation. CD244 signaling drives lncRNA-CD244 expression via sustaining a permissive chromatin state in the lncRNA-CD244 locus. By recruiting polycomb protein enhancer of zeste homolog 2 (EZH2) to infg/tnfa promoters, lncRNA-CD244 mediates H3K27 trimethylation at infg/tnfa loci toward repressive chromatin states and inhibits IFN-γ/TNF-α expression in CD8(+) T cells. Such inhibition can be reversed by knock down of lncRNA-CD244. Interestingly, adoptive transfer of lncRNA-CD244-depressed CD8(+) T cells to Mycobacterium tuberculosis (MTB)-infected mice reduced MTB infection and TB pathology compared with lncRNA-CD244-expressed controls. Thus, this work uncovers previously unidentified mechanisms in which T cell-inhibitory signaling and lncRNAs regulate T-cell responses and host defense against TB infection.

HIV-Mediated Phosphatidylinositol 3-Kinase/Serine–Threonine Kinase Activation in APCs Leads to Programmed Death-1 Ligand Upregulation and Suppression of HIV-Specific CD8 T Cells

Recent evidence demonstrates that HIV-1 infection leads to the attenuation of cellular immune responses, which has been correlated with the increased expression of programmed death (PD)-1 on virus-specific CD8(+) T cells. PD-1 is induced upon T cell activation, and its prolonged expression facilitates CD8(+) T cell inhibitory signals when bound to its B7 family ligands, PD-ligand (L)1/2, which are expressed on APCs. Importantly, early reports demonstrated that blockade of the PD-1/PD-L interaction by Abs may help to counter the development of immune exhaustion driven by HIV viral persistence. To better understand the regulation of the PD-1 pathway during HIV infection, we examined the ability of the virus to induce PD-L expression on macrophages and dendritic cells. We found a direct relationship between the infection of APCs and the expression of PD-L1 in which virus-mediated upregulation induced a state of nonresponsiveness in uninfected HIV-specific T cells. Furthermore, this exhaustion phenotype was revitalized by the blockade of PD-L1, after which T cells regained their capacity for proliferation and the secretion of proinflammatory cytokines IFN-γ, IL-2, and IL-12 upon restimulation. In addition, we identify a critical role for the PI3K/serine-threonine kinase signaling pathway in PD-L1 upregulation of APCs by HIV, because inhibition of these intracellular signal transducer enzymes significantly reduced PD-L1 induction by infection. These data identify a novel mechanism by which HIV exploits the immunosuppressive PD-1 pathway and suggest a new role for virus-infected cells in the local corruption of immune responses required for viral suppression.

SAP binds to CD22 and regulates B cell inhibitory signaling and calcium flux

The signaling lymphocyte activation molecule (SLAM)-associated protein (SAP or SH2D1A) is an important regulator of immune function which, when mutated or deleted, causes the X-linked lymphoproliferative syndrome (XLP). Because B cell lymphoma is a major phenotype of XLP, it is important to understand the function of SAP in B cells. Here we report that SAP is expressed endogenously in mouse splenic B cells, is inducibly expressed in the human BJAB cells, and co-localizes and interacts with CD22. We also show that SAP binding to the inhibitory immunoreceptor CD22 regulates calcium mobilization in B cells. Moreover, forced expression of SAP leads to constitutive CD22 tyrosine phosphorylation and decreased Ca 2+ response in B cells. Biochemical analysis reveals that, in response to IgM cross-linking, the phosphorylation of Syk, Blnk, or PLCγ2 and their interactions with one another were either diminished or completely abolished in SAP-expressing cells compared to cells that lack SAP. Collectively our work identifies a novel role for SAP in B cells and extends its function to inhibitory immunoreceptor signaling and calcium mobilization.

Enhancing cancer vaccines with immunomodulators

Harnessing the immune system to control cancer has been a challenge for cancer immunotherapists for many years. However, while specific immune responses to tumour-associated antigenic targets have been successfully induced in some patients, these responses have not always been sufficient to reproducibly and consistently mediate useful anti-tumour clinical activity. Many checks and balances have been incorporated into the immune response by nature to prevent or reduce the likelihood of autoimmunity or exaggerated protective inflammatory responses. Tolerance to self-antigens expressed on tumours is a major limitation in generating functional anti-tumour responses. In recent years, many of the pathways that mediate this tolerance have been identified, and reagents that can be used to manipulate these pathways have been clinically evaluated. These include: (a) pathways to activate professional antigen presenting cells, such as through Toll-like receptors, growth factors, such as GM-CSF, and the CD40 pathway; (b) use of cytokines, such as IL-2, IL-12, and Interferon alpha to enhance immune activation; and (c) pathways that inhibit T cell inhibitory signals, or Tregs. This article reviews clinical trials that have evaluated these approaches, and highlights promising combination vaccine/immunomodulator combination treatments based upon published clinical trial results.

Reciprocal age related change in natural killer cell receptors for MHC class I

Natural killer (NK) cells are essential for healthy aging. NK cell activation is controlled by MHC class I-specific CD94/NKG2 receptors and killer immunoglobulin-like receptors (KIR). To assess NK cytotoxic function in isolation from MHC receptor engagement, we measured the ability of purified NK cells to kill mouse P815 target cells in the presence of anti-CD16 mAb. CD16-mediated cytotoxicity did not change with age, indicating that NK activation and cytotoxic granule release remained functional. We then investigated MHC class I receptor expression on NK cells. There was an age related decrease in CD94 and NKG2A expression and a reciprocal age related increase in KIR expression. NKG2A expression also declined with age on CD56 + T cells. CD94/NKG2A receptor function was proportional to expression, indicating that NK cell inhibitory signaling pathways were intact. NKG2A and KIR expression were complementary, suggesting that CD94/NKG2A function could substitute for inhibitory KIR function during polyclonal NK cell development in both young and elderly adults. The distinct roles of CD94/NKG2A and KIR receptors suggest that shifting MHC class I receptor expression patterns reflect age related changes in NK cell and CD56 + T cell turnover and function in vivo.

CD40L AND CD28 BLOCKADE INDUCES REGULATORY MECHANISM AND INHIBITS CHRONIC ALLOGRAFT REJECTION

P1037 Aims: Blockade of individual T cell costimulatory pathways may result in long-term allograft survival but tolerance is not obtained since chronic rejection is observed. To obtain transplantation tolerance, blockade of more than one costimulatory pathway is needed but the importance of different pathways and the optimal reagents to be used have not been fully defined. Methods: Heart transplantation was performed between MHC mismatched rats. CD40-CD154 interactions were blocked by gene transfer of CD40Ig. We interfered with CD28 costimulatory signals by administering an anti-CD28 Mab, which in contrast to CTLA4Ig, preserves T cell inhibitory signals through CTLA4. Results: CD40Ig treatment resulted in long-term (>120d, n=19) heart allograft survival and development of chronic rejection in all grafts whereas acute rejection occurred in the non-coding adenovirus-treated (7.6±1.6d, n=9) group. Treatment with anti-CD28 Mab resulted in a moderate but significant prolongation of allograft survival (17.4±1.7d, n=10). Cotreatment with both CD40Ig and anti-CD28Mab resulted in long term allograft survival (>120d, n=7) and a significant reduction in the percentage and degree of vessels with chronic rejection lesions, with 3/7 grafts completely free of lesions and therefore tolerant. Splenocytes from cotreated tolerant recipients, but not those with chronic rejection (CD40Ig or cotreated), showed completely suppressed MLR responses against 1st but not 3rd party donor cells. T cells purified from splenocytes of tolerant cotreated recipients showed normal MLR responses indicating that among total splenocytes, non T cells inhibited the proliferation of T cells. Splenocytes from tolerant recipients but not those with chronic rejection lesions inhibited naïve MLR, indicating the presence of active tolerogenic mechanisms. Transfer of splenocytes from CD40Ig-treated recipients to naïve sublethaly irradiated rats conferred long term allograft survival (>120d, n=4 vs. 17 d, n=2 in controls). In contrast, transfer of splenocytes from CD40Ig+anti-CD28 cotreated rats, either tolerant or with chronic rejection lesions, did not prolong allograft survival (17d, n=3). Conclusions: The association of CD40Ig with anti-CD28 Mab resulted in decreased chronic rejection lesions and tolerogenic mechanisms different to those seen with CD40Ig.

CD80 and CD86 C domains play an important role in receptor binding and co-stimulatory properties.

CD80 and CD86 expressed on the surface of antigen-presenting cells interact with cytotoxic T lymphocyte antigen-4 [CTLA-4 (CD152)] expressed on activated T cells and mediate critical T cell inhibitory signals. CD80 and CD86 are type I glycoproteins, and are made up of two extracellular (EC) Ig-like domains-a transmembrane region and a cytoplasmic tail. The N-terminal (V domain) and membrane-proximal (C) domains share homology with the variable region (V) and the constant region (C) of Ig respectively. Co-crystallographic structures of both CD80 and CD86 bound to CTLA-4 indicate that there is no direct interaction of the C domain of either CD80 or CD86 with the CTLA-4. In contrast, previous mutagenesis studies have identified specific amino acids within the C domain of CD80 that are critical for CTLA-4 binding. To further understand the importance of C domains in the functioning of CD80 and CD86, we constructed chimeric human CD80 and CD86 molecules by swapping their respective C domains, and tested their ability to stimulate T cells. A Chinese hamster ovary (CHO) cell line expressing CD86 activated murine T cells. In contrast, CHO cells expressing either CD80 or a chimeric construct of the CD86 V domain and the CD80 C domain showed a significantly reduced activation. Our studies further demonstrated that the decreased activation by cells expressing the CD80 or a chimera containing CD80 C domain is most likely due to enhanced CTLA-4 binding. From these results we conclude that C domains play a critical, albeit indirect, role in determining CTLA-4 binding affinities and co-stimulatory properties.