CTL Killing Research Articles

Activation-induced thrombospondin-4 works with thrombospondin-1 to build cytotoxic supramolecular attack particles

Cytotoxic attack particles released by CTLs and NK cells include diverse phospholipid membrane and glycoprotein encapsulated entities that contribute to target cell killing. Supramolecular attack particles (SMAPs) are one type of particle characterized by a cytotoxic core enriched in granzymes and perforin surrounded by a proteinaceous shell including thrombospondin (TSP)-1. TSP-4 was also detected in bulk analysis of SMAPs released by CTLs; however, it has not been investigated whether TSP-4 contributes to distinct SMAP types or the same SMAP type as TSP-1 and, if in the same type of SMAP, whether TSP-4 and TSP-1 cooperate or compete. Here, we observed that TSP-4 expression increased upon CD8+ T cell activation while, surprisingly, TSP-1 was down-regulated. Correlative Light and Electron Microscopy and Stimulated Emission Depletion microscopy localized TSP-4 and TSP-1 in SMAP-containing multicore granules. Superresolution dSTORM revealed that TSP-4 and TSP-1 are usually enriched in the same SMAPs while particles with single-positive shells are rare. Retention Using Selective Hooks assays showed that TSP-4 localizes to the lytic granules faster than TSP-1 and promotes its accumulation therein. TSP-4 contributed to direct CTL-mediated killing, as previously shown for TSP-1. TSP-4 and TSP-1 were both required for latent SMAP-mediated cell killing, in which released SMAPs kill targets after removal of the CTLs. Of note, we found that chronic lymphocytic leukemia (CLL) cell culture supernatants suppressed expression of TSP-4 in CTL and latent SMAP-mediated killing. These results identify TSP-4 as a functionally important component of SMAPs and suggest that SMAPs may be targeted for immune suppression by CLL.

Abstract LB125: Tri-specific T cell engager targeting of selective surface cancer-selective ligands recruits both T cells and APC targeting of cancer neoantigens of tumor-initiating stem-like cells

Abstract Chemotherapy does not sufficiently eliminate tumor-initiating cells (TICs) of hepatocellular carcinoma (HCC). To remove TICs and achieve personalized cancer therapy, dendritic cells (DC) were vaccinated against tumor germline mutations, but metastatic cancers recurred in many cases. Emerging data suggest that nanoparticle-based siRNA delivered targeting of stemness transcription factor NANOG (pluripotency transcription factor) potentiates anti-tumor immunity. We hypothesized that pre-treatment of selective TIC inhibitors targeting stemness genes sensitized the efficacy of immunotherapy targeting of liver TICs. Novel TIC-targeting immunoglobulin, variable chain fragments were identified by phage-display library screening. The top single chain camelid antibody (scFv) candidates were identified and individually cloned into mammalian expression vectors to test binding to TICs. A Tri-specific T cell engager (TriTE) was constructed to connect TIC-targeting scFv with anti-dendritic cell (DC) scFv (anti-CD14) and CD3. Such scFv vectors were tested for target cell lysis. Candidate ligands of anti-TIC ScFvs present on the tumor cell surface were identified through a series of bioinformatic analysis., The latter provided the preconditions for one of our aims to identify the target, tumor surface ligand(s) of anti-TIC ScFv to better understand the mechanism of TriTE action upon a tumor cell. IP-Mass spectrometry analyses demonstrated that two target surface antigens, TBCE and CEP170, along with their respective mRNAs were highly expressed in CD133+ TIC Huh7 cells when compared to CD133- non-TIC Huh7 cells. This accounted for specific TriTE binding to TIC cells, but not to CD133(-) Huh7 cells or primary hepatocytes. These data demonstrated that these unique tumor antigens, primarily arising during neoplastic transformation, elicited T and/or monocyte immunity capable of protecting the host from cancer progression. These conjunctive with PD1 antibody therapeutic modality leads to the personalized immune/chemotherapy of HCC patients. Human monocyte-derived DCs, TIC and T cells were incubated with TriTE to measure IFNγ production during CTL killing effects against TICs. TriTE treatment significantly reduced tumor growth of PDX tissues in NSG-SG3 mice with humanized immunity. Other novel surface antigens on TIC were identified by mass spectroscopy analyses. These data demonstrated that the presence of unique tumor antigens, primarily arising during neoplastic transformation, which elicited T and/or myeloid cell immunity capable of protecting the host from cancer progression. Self-adapting antigen-targeting machinery with Trite therapy is adaptable and fine-tuning next-generation immunotherapy. Citation Format: Wei Ma, Nuan Wang, Hye Yeon Choi, Lin Ziying, Nicolette Fajardo, Royce Ilaga, Risa Machida, Zhou Lin, Ambika Ramrakhiani, Yvonne Y. Chen, Linda Sher, Stanley M. Tahara, Keigo Machida. Tri-specific T cell engager targeting of selective surface cancer-selective ligands recruits both T cells and APC targeting of cancer neoantigens of tumor-initiating stem-like cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB125.

Abstract 3966: NK receptors enable TCR-mediated CTL recognition and killing of cancer cells expressing non-mutated tumor-associated antigens

Abstract Background: Responsiveness or ignorance of antigens presented to naïve T cells by dendritic cells (DC) involves the CD28-driven “signal 2”, but it is unclear how activated CTLs respond to the same non-mutated tumor-associated antigens (TAAs) presented by cancer versus healthy cells to avoid autoimmunity. Since CD8+ T cells express activating NK receptors (NKRs) and NKR ligands are commonly expressed by cancer cells but not healthy tissues, we evaluated the crosstalk between NKRs and TCR during recognition of cancer cells by DC-primed TAA-specific CTLs. Methods: Associations between DNAM-1, NKG2D, CTL markers, and patients’ survival was analyzed using TCGA. In vitro sensitization was used to induce human TAA-specific CTLs by TAA-loaded DCs. The roles of DNAM-1 and NKG2D in CTL recognition and killing were tested using IFN-γ ELISpot, conjugate formation, degranulation, and cytotoxicity assays. The mechanisms and molecular effects of NKR-mediated costimulation were analyzed using IsoPlexis, calcium flux, western blot, and RNA sequencing. Double-transduction of T cells with TCR and NKR constructs or pre-treatment of cancer cells by chemotherapy was used to manipulate the levels of NKRs on T cells or their ligands on cancer cells. Results: TCGA analysis revealed that DNAM-1 and NKG2D are strongly associated with intratumoral CD8+ T cells rather than NK cells, being critical for the long-term survival of melanoma patients. Human DC-primed CTLs significantly upregulated DNAM-1 and NKG2D compared to naïve CD8+ T cells, but retained strict dependence on TCR in cancer cell recognition. Unexpectedly, blockade of DNAM-1 and NKG2D prevented the TCR-mediated CTL recognition and killing of cancer cells expressing low-TAA levels, but were redundant in the recognition of high-TAA-expressing cancer cells. DNAM-1, and to a lesser extent NKG2D, lowered TCR activation threshold and enhanced proximal TCR signaling and CTL polyfunctionality. NKR overexpression in TCR-transgenic T cells or chemotherapy-driven elevation of NKR ligands on cancer cells allowed effective recognition and killing of weakly immunogenic cancer cells. Conclusions: Reduced TCR activation threshold in the presence of NKR costimulation enables CTL activation by low levels of cognate MHC I-peptide complexes on cancer cells. Our data helps explain the ability of non-mutated “self” antigens to mediate tumor rejection in the absence of autoimmunity, and provides new tools to enhance the effectiveness of cancer therapies. Citation Format: Bowen Dong, Nataša Obermajer, Takemasa Tsuji, Junko Matsuzaki, Cindy Bonura, Henry Withers, Mark Long, Colin Chavel, Scott H. Olejniczak, Hans Minderman, Robert P. Edwards, Walter J. Storkus, Pedro Romero, Pawel Kalinski. NK receptors enable TCR-mediated CTL recognition and killing of cancer cells expressing non-mutated tumor-associated antigens [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3966.

Abstract LB036: IRF8 regulates tumor cell sensitivity to intrinsic ferroptosis by repressing p53

Abstract Tumor cell resistance to ferroptosis, which is a recently discovered CTL induced cell death pathway, remains incompletely understood. We report here that interferon regulatory factor 8 (IRF8) functions as a regulator of tumor cell intrinsic ferroptosis. Genome-wide gene expression profiling identified ferroptosis pathway as an IRF8-regulated pathway in tumor cells. IRF8.KO tumor cells are resistant to intrinsic ferroptosis induction and also exhibit decreased ferroptosis in response to tumor specific CTL killing. Loss of IRF8 increases p53 express in tumor cells and knocking out Tp53 in IRF8.KO cells restored tumor cell sensitivity to intrinsic ferroptosis induction. We were able to show that treating tumor-bearing mice with IRF8-encoding plasmid NTC9385R-mIRF8 encapsulated in a DOTAP-cholesterol lipid nanoparticle significantly reduced tumor growth and increased the percentage of dead cells in the tumor. Our data therefore determine that IRF8 represses p53 expression to maintain tumor cell sensitivity to intrinsic ferroptosis. Citation Format: Dakota Poschel, Mercy Kehinde-Ige, John Klement, Dafeng Yang, Alyssa Merting, Natasha Savage, Huidong Shi, Kebin Liu. IRF8 regulates tumor cell sensitivity to intrinsic ferroptosis by repressing p53 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB036.

Recombinant MUC1-MBP fusion protein combined with CpG2006 vaccine induces antigen-specific CTL responses through cDC1-mediated cross-priming mainly regulated by type I IFN signaling in mice

In this study, we explored the initiation and regulation mechanism of antigen-specific CTL responses induced by a novel cancer vaccine containing recombinant human mucin1-maltose-binding protein fusion protein (MUC1-MBP) and CpG2006. First, DC subsets were analyzed by flow cytometry in vivo and in vitro. After vaccination, the proportion and maturation of cDC1s in mouse dLNs were upregulated, and the proportion of cDC2s and pDCs was also increased. In vitro studies on vaccine components showed similar changs, which may mainly depend on the activity of CpG2006. Subsequently, the regulatory effect of type Ⅰ IFN signaling on CTL triggering was confirmed through co-culture of sorted DC subsets and T cells and subsequent CTL activity experiments. CTL killing activity exhibited a 61.9% decrease once type I IFN signaling was blocked. Further analysis showed that blocking IFNAR1 on cDC1s but not on CTLs resulted in significant defects in CTL killing activity. Collectively, M-M combined with CpG2006 vaccine promotes MUC1-specific CTL responses by increasing the cDC1 activity in mice, and this is mainly regulated by type Ⅰ IFN signaling in cDC1s.

MHC Class II Ubiquitination Regulates Dendritic Cell Function and Immunity.

MHC class II (MHC II) Ag presentation by dendritic cells (DCs) is critical for CD4+ T cell immunity. Cell surface levels of MHC II loaded with peptide is controlled by ubiquitination. In this study, we have examined how MHC II ubiquitination impacts immunity using MHC IIKRKI/KI mice expressing mutant MHC II molecules that are unable to be ubiquitinated. Numbers of conventional DC (cDC) 1, cDC2 and plasmacytoid DCs were significantly reduced in MHC IIKRKI/KI spleen, with the remaining MHC IIKRKI/KI DCs expressing an altered surface phenotype. Whereas Ag uptake, endosomal pH, and cathepsin protease activity were unaltered, MHC IIKRKI/KI cDC1 produced increased inflammatory cytokines and possessed defects in Ag proteolysis. Immunization of MHC IIKRKI/KI mice identified impairments in MHC II and MHC class I presentation of soluble, cell-associated and/or DC-targeted OVA via mAb specific for DC surface receptor Clec9A (anti-Clec9A-OVA mAb). Reduced T cell responses and impaired CTL killing was observed in MHC IIKRKI/KI mice following immunization with cell-associated and anti-Clec9A-OVA. Immunization of MHC IIKRKI/KI mice failed to elicit follicular Th cell responses and generated barely detectable Ab to anti-Clec9A mAb-targeted Ag. In summary, MHC II ubiquitination in DCs impacts the homeostasis, phenotype, cytokine production, and Ag proteolysis by DCs with consequences for Ag presentation and T cell and Ab-mediated immunity.

Mitochondrial translation is required for sustained killing by cytotoxic T cells.

T cell receptor activation of naïve CD8+ T lymphocytes initiates their maturation into effector cytotoxic T lymphocytes (CTLs), which can kill cancer and virally infected cells. Although CTLs show an increased reliance on glycolysis upon acquisition of effector function, we found an essential requirement for mitochondria in target cell–killing. Acute mitochondrial depletion in USP30 (ubiquitin carboxyl-terminal hydrolase 30)–deficient CTLs markedly diminished killing capacity, although motility, signaling, and secretion were all intact. Unexpectedly, the mitochondrial requirement was linked to mitochondrial translation, inhibition of which impaired CTL killing. Impaired mitochondrial translation triggered attenuated cytosolic translation, precluded replenishment of secreted killing effectors, and reduced the capacity of CTLs to carry out sustained killing. Thus, mitochondria emerge as a previously unappreciated homeostatic regulator of protein translation required for serial CTL killing.

The ABCs of ovarian cancer immunology: IgA, B cells, and CTLs.

Intratumoral B cells in ovarian cancers produce IgA, which binds tumor antigens and inhibit tumor growth through myeloid cell-dependent mechanisms or by neutralization of secreted factors, and by antigen-nonspecific binding to poly-Ig receptor on cancer cells leading to transcytosis, which sensitizes the cells for CTL killing.

Doxorubicin/CpG self-assembled nanoparticles prodrug and dendritic cells co-laden hydrogel for cancer chemo-assisted immunotherapy

Conversion of combination cancer therapy from immuno-assisted chemotherapy to chemo-assisted immunotherapy would be a promotion of strategies to highlight the crucial role of immunotherapy. How to improve the efficiency of immunotherapy and reduce the side effect of chemotherapy is the focus of optimization for chemo-assisted immunotherapy. Herein, we designed a doxorubicin/CpG self-assembled nanoparticles prodrug and dendritic cells (DC) co-laden hydrogel system for cancer chemo-assisted immunotherapy. Importantly, DC-based immunotherapy afforded a feasible compensatory treatment for insufficient endogenous DC in vivo by implanting exogenous DC. The pH sensitive nano-prodrug system reduced the side effects of DOX and induced tumor cells to generate tumor antigens. The interaction of tumor antigens and adjuvants with DC could promote the level of antigen presentation, thereby enhancing the immune response. Moreover, the hydrogel scaffold provided a better growth microenvironment to maintain DC viability besides as a carrier for drug and adjuvant. By the merging of doxorubicin-stimulated tumor antigens, DC and adjuvants, they induced a strong CTL killing effect, remarkably increased the infiltration of effector T cells, alleviated the intratumoral immunosuppressive microenvironment and maximized the immune effect for improving the chemo-assisted immunotherapy. In addition, fluorescence imaging tracking of immune cells, chemotherapeutic drugs, and immune adjuvants revealed their in vivo process for efficient therapy, which could afford guidance for the optimization of biomaterials-mediated chemo-assisted immunotherapy.

Hydrogel/Nanoadjuvant-Mediated Combined Cell Vaccines for Cancer Immunotherapy

Cancer immunotherapy aims to effectively eliminate the residual tumor cells and prevent tumor recurrence and metastasis. The combined cell vaccines of tumor whole cell and dendritic cells (DC) provide an effective individualized immunotherapy for malignant tumors. It is a key issue how to improve the immunogenicity of tumor whole cell vaccine and maintain DC activity for antigen presentation in vivo. We proposed an innovative strategy called “biomaterial-mediated combined cell vaccines for immunotherapy”, which combined tumor cell vaccine and DC vaccine by cyclodextrin-polyethylene glycol hydrogel and CpG nanoadjuvant. The nanoadjuvant promoted antigen presentation level and amplified immune eliciting potency by co-delivery of antigen and adjuvant. The hydrogel scaffold provided a better growth microenvironment for injected exogenous DC and recruited endogenous DC to maintain their viability and play their synergistic action. Based on effective antigen presentation from the combination of tumor antigen, adjuvant and DC, the combined cell vaccines induced strong CTL killing effect to continuously stimulate the body's specific anti-tumor immunity. The combined hydrogel/ nanoadjuvant system merged immunogenically dying tumor cells, DC and adjuvants, which remarkably increased the infiltration of effector T cells, alleviated the intratumoral immunosuppressive microenvironment and maximized the immune effect of the vaccines for improving the cancer immunotherapy.

Abstract 5543: TAK1 deficiency in tumor cells enhances sensitivity to CTL-mediated killing via TNF-α

Abstract The clinical successes achieved by different immunotherapies have resulted in a paradigm shift in treatment modalities. Despite these significant advances, not all patients benefit from the use of these therapies, creating a need to develop additional approaches to enhance and broaden their clinical application. To identify genes whose products can increase or decrease the sensitivity of tumor cells to the immune system, we used a CTL assay to screen a whole genomic CRISPR library. We co-culture a mouse cell line, ID8, expressing a model antigen (Ova) with transgenic CD8 T cells (OT-I) recognizing this antigen. A set of controls that enhance or decrease CTL activity behaved as expected. Comparison of the CRISPR score identified several hits that increased or decreased the sensitivity of the tumor cells to CTL killing. Subsets of these hits belong to two pathways involved in CTL-mediated killing: the IFN-γ and the TNF-α signaling pathways. We evaluated which of these hits would be amenable to therapeutic modulation, and decided to focus on the kinase TAK1 for confirmation and validation studies. A TAK1 deficient cell line was more sensitive to CTL killing, which was prevented by expression of TAK1, confirming the role of TAK1 in this process. A TAK1 gene carrying an inactivation mutation K63W did not rescue the effects of TAK1 KO, indicating that TAK1 enzymatic activity was necessary. Several pathways mediate CTL killing: Perforin/Granzyme B, IFN-γ, TNF-α, Fas & TRAIL pathways. To determine TAK1 MOA, we studied the effects of a Perforin/Granzyme B inhibitor CMA. CMA inhibited CTL activity in a dose-dependent manner on WT cells, but did not inhibit CTL activity on TAK1 deficient cells, indicating TAK1 effects are independent of this pathway. We then tested the sensitivity of TAK1 KO cells to TNF-α. TAK1 KO cells were more sensitive to TNF-α mediated killing, and similar results were observed with several additional cell lines (MC38, EMT6, KP). TNF-α can activate the JNK, p38, and NF-κB pathways, and the apoptosis extrinsic pathway to regulate cell growth and cell death. Kinetics studies monitoring pathway activity upon TNF-α stimulation showed that TAK1 KO cell lines induced cFLIP degradation before observing PARP cleavage, and that the NF-κB pathway, which has been observed to mediate cFLIP synthesis, was not activated. We proceeded to evaluate the effects of TAK1 deficiency in a mouse syngeneic model. TAK1 deficiency resulted in reduced growth and increased survival in the MC38 in vivo model. In summary, by screening a CRISPR library against a CTL assay, we identified TAK1 as a novel potential target for immunotherapies. TAK1 deficiency enhances CTL killing and results in decreased tumor growth and increased survival in vivo. This results support the development of TAK1 inhibitors to enhance the anti-tumor action of the immune system. Citation Format: Juan J. Miret, Troy A. Luster, Patrick Lizotte, Min Wu, Sarah Nzikoba, Luke Taus J. Taus, Prafulla C. Gokhale, Paul Kirschmeier, David Barbie, Cloud P. Paweletz. TAK1 deficiency in tumor cells enhances sensitivity to CTL-mediated killing via TNF-α [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5543.

Combination of CpG Oligodeoxynucleotide and Anti-4-1BB Antibody in the Treatment of Multiple Hepatocellular Carcinoma in Mice.

BackgroundTo investigate the effect of topical application of CpG oligodeoxynucleotide (CpG-ODN) combined with anti-4-1BB antibodies on mouse HCC multiple tumor-bearing models and the degree of improvement of anti-tumor immune response in mice.Materials and MethodsWe inoculated each BALB/c male mouse subcutaneously with one tumor in the axillae of the four limbs and divided them into four groups. We only selected the tumor-bearing part of the left lower limb for drug treatment. We measured the tumor-bearing volume of mice in each group. Then, we tested the organ coefficients of mice, the concentrations of IL-12 and IFN-γ in peripheral blood, the ratio of spleen Tregs and CD8+T cells, the spleen CTL killing activity, and the survival time of mice.ResultsWe found that the tumor-bearing volume decreased significantly after the combination of CpG-ODN and anti-4-1BB antibody (P<0.001). The organ coefficients of treated mice were not significantly different from normal mice (P>0.05). The concentration of IL-12 and IFN-in serum and the ratio of CD8+T cells in spleen were increased, while the ratio of spleen Tregs was decreased. CTL activity of spleen was increased. The survival time of mice was significantly prolonged (P<0.001).ConclusionThe treatment programme combining CpG-ODN with an anti-4-1BB antibody can significantly reduce tumor growth at the treatment site, slow the growth rate of metastases and improve host prognosis.

Allogenic mouse cell vaccine inhibits lung cancer progression by inhibiting angiogenesis

ABSTRACT Aim: This research investigated the therapeutic effect of an allogeneic mouse brain microvascular endothelial cell vaccine on lung cancer and further elucidated its potential anti-angiogenic mechanism. Materials & methods: The immune effect of the allogeneic bEnd.3 vaccine and DC vaccine loaded with bEnd.3 antigen on the subcutaneous transplantation of Lewis lung cancer (LLC) was assessed by ELISA, the CCK test and the CTL killing test. The mechanism was preliminarily revealed by immunohistochemistry and immunoblot analysis. Results: This study revealed that tumor volume was decreased (p < .01) and the survival was prolonged significantly (p < .05) by the bEnd.3 vaccine in subcutaneous LLC transplantation in the vaccine prevention group. In contrast, both tumor volume in the serum therapeutic group and survival of bEnd.3 vaccine were not significantly different from those of the control group (p > .05). Importantly, tumor volume and survival of the T lymphocyte therapeutic group were decreased and prolonged (p < .05). In addition, both tumor volume and survival of DC vaccine loaded with bEnd.3 in the vaccine prevention group were decreased and prolonged significantly (p < .01). Furthermore, bEnd.3 vaccine and DC vaccine loaded with bEnd.3 both produced the activity of killing bEnd.3 target cells in vitro.The reason may induce the immune mice to produce anti-VEGFR-II, anti-endoglin and anti-integrin αν antibodies to have an anti-angiogenesis function. Conclusion: The allogeneic mouse bEnd.3 cell vaccine can block angiogenesis and prevent the development of lung cancer transplantation tumors.

BCL-2 Antagonism Sensitizes CTL-Resistant HIV Reservoirs to Elimination Ex Vivo

Curing HIV infection will require the elimination of a reservoir of infected CD4+ T-cells that persists despite HIV-specific CTL responses. While viral latency is a critical factor in this persistence, recent evidence also suggests a role for intrinsic resistance of reservoir-harboring cells to CTL killing. We explored the hypothesis that this resistance is mediated by BCL-2 family proteins, which can antagonize CTL-induced apoptosis. We show that the reactivatable HIV reservoir is disproportionately present in BCL-2hi CD4+ T-cells, which are relatively resistant to CTL. BCL-2/BCL-XL antagonists were sufficient for inducing the elimination of HIV-infected cells from a primary-cell model of latency, but did not drive reductions in ex vivo viral reservoirs when tested either alone or with a latency reversing agent (LRA). The triple combination of LRAs, HIV-specific T-cells, and a BCL-2 antagonist uniquely enabled depletions in ex vivo viral reservoirs, providing rationale for novel therapeutic approaches targeting HIV cure.

Evaluating CD8+ T Cell Responses In Vitro.

The 51Cr-release assay described in the 1960s has been for decades the gold standard cytolytic assay but for safety reasons is no longer in use in many laboratories. Whereas other radioactive tests were later on described, they never ousted the 51Cr-release assay. More thorough understanding of CTL biology and killing pathways has more recently resulted in the design of reliable nonradioactive tests to analyze CD8+ T cell responses. Allowing for real-time evaluation of target cell viability, some of them are particularly attractive and are described here together with traditional assays.

Abstract 2783: Empowering therapeutic monoclonal antibodies with IFN-alpha for cancer immunotherapy

Abstract Type 1 IFNs stimulates secretion of IP-10 (CXCL10) which is a critical chemokine to recruit effector T cells to the tumor microenvironment and IP-10 knockout mice exhibit a phenotype with compromised effector T cell generation and trafficking. Type 1 IFNs also induces MHC class 1 upregulation on tumor cells which can enhance anti-tumor CD8 T cell effector response in the tumor microenvironment. Although type 1 IFNs show great promise in potentiating anti-tumor immune response, systemic delivery of type 1 IFNs is associated with toxicity thereby limiting clinical application. In this study, we fused tumor targeting antibodies with IFN-alpha and showed that the fusion proteins can be produced with high yields and purity. IFN fusions selectively induced IP-10 secretion from antigen positive tumor cells, which was critical in recruiting the effector T cells to the tumor microenvironment. Further, we establish a real time in vitro antigen specific CTL killing assay using IncuCyte Zoom and tested the anti-tumor efficacy of anti-PDL1-IFN-alpha fusion using this system. We found anti-PDL1-IFN-alpha as low as 10 pM exhibits potent activity in potentiating OT1 CD8 T cells killing against OVA expressing tumor cells, while control IFN fusion did not exhibit any activity in the same experiment. Furthermore, IFN-alpha fusion antibody was well tolerated in vivo and demonstrated anti-tumor efficacy in an anti-PD-L1 resistant syngeneic mouse tumor model. Our data supports the hypothesis of targeting type 1 IFN to the tumor microenvironment may enhance effector T cell responses in non-inflamed tumors. Citation Format: Jun Guo, Yu Xiao, Ramesh B. Lyer, Marc R. Lake, Uri S. Ladror, Xin Lu, Bill Pappano, John E. Harlan, Medha J. Tomlinson, Gail T. Bukofzer, Cherrie K. Donawho, Alexander R. Shoemaker, Tzu-Hsuan Huang. Empowering therapeutic monoclonal antibodies with IFN-alpha for cancer immunotherapy [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 2783.

Abstract 4725: Blockade of cyclin D-CDK4/6 sensitizes immune-refractory tumor cells to tumor antigen-specific T cell-mediated cytotoxicity by repressing SCP3-NANOG axis

Abstract Antitumor immune drives tumor cell to acquire immune-refractory phenotypes. Previously, we demonstrated tumor antigen-specific T cells edited them resistant to CTL killing and enriched NANOGhigh cancer stem cell-like (CSC-like) cells. In this study, in an effort to elucidate targetable pathways of immune-edited tumor cells with the multiaggressiveness, we show synaptonemal complex protein 3 (SCP3), a member of Cor1 family, which is overexpressed in the immune-edited cells, upregulates NANOG by hyperactivating cyclin D1-CDK4/6 axis. The SCP3-cyclin D1-CDK4/6 axis was preserved across various types of human cancer and, notably, negatively correlated with the progression-free survival of cervical cancer patients. Moreover, targeting CDK4/6 with its inhibitor, palbociclib, succeeded in reversing the multiaggressive phenotypes of immune-edited tumor cells, and led to long-term control of the disease. Collectively, our findings establish a firm molecular link of multiaggressiveness among SCP3, NANOG, cyclin D1, and CDK4/6, and identify CDK4/6 inhibitors as an actionable drug for controlling SCP3high immune-refractory cancer. Citation Format: Se Jin Oh, Tae Woo Kim. Blockade of cyclin D-CDK4/6 sensitizes immune-refractory tumor cells to tumor antigen-specific T cell-mediated cytotoxicity by repressing SCP3-NANOG axis [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 4725.

Both perforin and FasL are required for optimal CD8 T cell control of autoreactive B cells and autoantibody production in parent-into-F1 lupus mice

To test the relative roles of perforin (pfp) vs. FasL in CTL control of autoreactive B cell expansion, we used the parent-into-F1 model of murine graft-vs.–host disease in which donor CD8 CTL prevent lupus like disease by eliminating activated autoreactive B cells. F1 mice receiving either pfp or FasL defective donor T cells exhibited an intermediate short-term phenotype. Pairing of purified normal CD4 T cells with either pfp or FasL defective CD8 T cell subsets resulted in impaired host B cell elimination and mild lupus like disease that was roughly equivalent in the two experimental groups. Thus, in addition to major roles in tumor and intracellular pathogen control, pfp mediated CD8 CTL killing plays a significant role in controlling autoreactive B cell expansion and lupus downregulation that is comparable to that mediated by FasL killing. Importantly, both pathways are required for optimal elimination of activated autoreactive B cells.

Targeting Cyclin D-CDK4/6 Sensitizes Immune-Refractory Cancer by Blocking the SCP3-NANOG Axis.

Immunoediting caused by antitumor immunity drives tumor cells to acquire refractory phenotypes. We demonstrated previously that tumor antigen-specific T cells edit these cells such that they become resistant to CTL killing and enrich NANOGhigh cancer stem cell-like cells. In this study, we show that synaptonemal complex protein 3 (SCP3), a member of the Cor1 family, is overexpressed in immunoedited cells and upregulates NANOG by hyperactivating the cyclin D1-CDK4/6 axis. The SCP3-cyclin D1-CDK4/6 axis was preserved across various types of human cancer and correlated negatively with progression-free survival of cervical cancer patients. Targeting CDK4/6 with the inhibitor palbociclib reversed multiaggressive phenotypes of SCP3high immunoedited tumor cells and led to long-term control of the disease. Collectively, our findings establish a firm molecular link of multiaggressiveness among SCP3, NANOG, cyclin D1, and CDK4/6 and identify CDK4/6 inhibitors as actionable drugs for controlling SCP3high immune-refractory cancer.Significance: These findings reveal cyclin D1-CDK4/6 inhibition as an effective strategy for controlling SCP3high immune-refractroy cancer. Cancer Res; 78(10); 2638-53. ©2018 AACR.

Abstract PR06: The effect of low-dose fractionated versus high-dose hypofractionated radiotherapy on antitumor immunity

Abstract Fractionated, low-dose (1.8-2 Gy) ionizing radiation (IR) is an integral component of head and neck cancer management. The mechanism of tumor control has traditionally been attributed to direct cytolytic effects on tumor cells. Substantial evidence supports enhanced antitumor immune responses when IR is given in large, single or hypofractionated doses. The effect of low-dose fractionated IR on antitumor immunity is less clear. We hypothesized that high-dose (8 Gy) IR would induce antigen release and priming of antigen-specific T-lymphocytes and sensitize tumor cells to killing by effector T-lymphocytes (CTLs) to a greater degree than low-dose (2 Gy) IR. To test this hypothesis, we stably transduced murine oral cancer 1 (MOC1) cells with full-length ovalbumin as a model antigen (MOC1ova). Upon exposure of MOC1ova cells to 8 Gy IR in vitro, splenocytes endocytosed and cross-presented SIINFEKL on H2-Kb and induced proliferation of CFSE-labeled, SIINFEKL-specific OT-1 T-lymphocytes to a greater degree than when exposed to MOC1ova cells irradiated to 0 or 2 Gy. Antigen cross-presentation was dependent upon type I interferon. In in vivo correlative experiments, MOC1ova tumors irradiated to 8 Gy induced expansion of adoptively transferred OT-1 T-lymphocytes located within the spleen, draining lymph node, and tumor to a greater degree than tumors exposed to 0 or 2 Gy. To assess if higher doses of IR enhance CTL killing of target cells, a real-time impedance-based cytotoxicity assay was utilized. Exposure of target cells to 8 Gy IR dramatically enhanced OT-1 CTL killing of MOC1ova cells to a greater degree than cells exposed to 0 or 2 Gy in a process independent of antigen-presentation. We next hypothesized that 10 days of 2Gy/day (low-dose fractionated) IR would be immunosuppressive in vivo compared to 2 fractions of 8 Gy (high-dose, hypofractionated) IR. We performed a time course analysis of effector and immunosuppressive cell peripheral accumulation and tumor infiltration as well as antigen-specific draining lymph node T-lymphocyte responses comparing these two IR regimens. Cumulatively, results demonstrated significantly greater correlative and functional T-lymphocyte immune responses with high-dose hypofractionated IR compared to daily low-dose IR. Analysis of myeloid derived suppressor cell and regulatory CD4+ T-lymphocyte changes displayed mixed alterations between regimens. Taken together, these results suggest that higher-dose, hypofractionated IR results in enhanced immune activation and less immunosuppression compared to daily fractionated low-dose IR. These results have significant implications in the rational design of combination therapies utilizing IR and immunotherapy. This abstract is also being presented as Poster 32. Citation Format: Megan V. Morisada, Ellen C. Moore, Jay Friedman, Clint T. Allen. The effect of low-dose fractionated versus high-dose hypofractionated radiotherapy on antitumor immunity [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; April 23-25, 2017; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(23_Suppl):Abstract nr PR06.