Intratumoral Infiltration Of CD8 Research Articles

Synergistic Effects of Nanoscale CaO2 Combined with PD-1 Inhibitors in the Treatment of Hepatocellular Carcinoma: A Promising Combination.

To explore the effect of calcium peroxide nanoparticles (CaO2 NPs) combined with programmed cell death protein 1 (PD-1) inhibitors in the treatment of liver cancer and its related mechanism. Hepa1-6 cells were cultured to construct the Hepa1-6 mouse liver cancer model. In vivo mechanism study, a unilateral tumor model was established. Eighteen tumor-bearing mice were randomly divided into the control group (intra-tumoral injection of PBS solution) and the experimental group (intra-tumoral injection of CaO2 NPs). A hypoxic probe, pH probe, and micro-CT were used to evaluate the effect of CaO2 NPs on improving hypoxia, neutralizing acidity, and inducing calcium overload within the tumor. To study the effect of CaO2 NPs combined with PD-1 inhibitors on proximal and distal tumors, the bilateral tumor model was established. Forty tumor-bearing mice were randomly divided into the control group (intra-tumoral/intra-peritoneal injection of PBS solution), CaO2 NPs group (intra-tumoral injection of CaO2 NPs), PD-1 group (intra-peritoneal injection of PD-1 inhibitor), and the combination group (intra-tumoral injection of CaO2 NPs and intra-peritoneal injection of PD-1 inhibitors). The administered side was recorded as the proximal tumor. Tumor volume and body weight were measured every 2 days after treatment. On day 8, serum and tumor samples were collected. The immune factors in serum (Interferon-γ (IFN-γ), Tumour necrosis factor-α (TNF-α), Interleukin-2 (IL-2), and Interleukin-10 (IL-10)) and tumor tissue (IFN-γ and TNF-α) were detected by ELISA. H&E staining was used to detect tumor necrosis. Immunohistochemical staining was used to detect the amount of CD4+ and CD8+ T cells within the tumor. By analyzing the tumor volume, pathological indexes, and immune-related indexes, the effects of CaO2 NPs combined with PD-1 inhibitors on proximal and distal tumors were evaluated, and they mediated immunomodulatory effects (including local and systemic immunity), and their effects on tumor burden were studied. In addition, a unilateral tumor model was established to study the effect of CaO2 NPs combined with PD-1 inhibitors on survival time. The results of in vivo mechanism study showed that CaO2 NPs can improve hypoxia, neutralize acidity, and induce calcium overload within tumors. The results of the study on the effect of CaO2 NPs combined with PD-1 inhibitor on proximal and distal tumors showed that, compared with the other three groups, the bilateral tumor burden of the combination group was significantly reduced, the intra-tumoral infiltration of CD8+ and CD4+ T cells were significantly increased, the secretion of anti-tumor immune factors in tumor and serum was increased, and the secretion of pro-tumor immune factors was decreased. Mice in the combination group showed the longest survival compared with the other groups. CaO2 NPs can improve hypoxia, neutralize acidity, and induce calcium overload within tumors, so as to reduce tumor burden and realize an immunosuppressive tumor transformation to a hot tumor, and play a synergistic role with PD-1 inhibitors in anti-liver cancer.

Co-delivering irinotecan and imiquimod by pH-responsive micelle amplifies anti-tumor immunity against colorectal cancer

Irinotecan (IRT), a classic clinical chemotherapeutic agent for treating colorectal cancer, has been found to induce immunogenic cell death (ICD) while exerting cytotoxicity in tumor cells. This effect is likely to be amplified in combination with immune modulators. Unfortunately, free drugs without targeting capacity would receive poor outcomes and strong side effects. To address these issues, in this work, an acid-sensitive micelle based on an amphiphilic poly(β-amino ester) derivative was constructed to co-deliver IRT and the immune adjuvant imiquimod (IMQ), termed PII. PII kept stable under normal physiological conditions. After internalization by tumor cells, PII dissociated in acidic lysosomes and released IRT and IMQ rapidly. In the CT26 tumor mouse model, PII increased the intra-tumoral SN38 (the active metabolite of IRT) and IMQ concentrations by up to 9.39 and 3.44 times compared with the free drug solution. The tumor inhibition rate of PII achieved 87.29%. This might profit from that IRT induced ICD, which promoted dendritic cells (DCs) maturation and intra-tumoral infiltration of CD8+ T cells. In addition, IMQ enhanced the antigen presenting ability of DCs and stimulated tumor associated macrophages to secrete tumor-killing cytokines. PII provided an effective strategy to combat colorectal cancer by synergy of chemotherapy and immunoregulation.

PLA2G2A+ cancer-associated fibroblasts mediate pancreatic cancer immune escape via impeding antitumor immune response of CD8+ cytotoxic T cells

Our previous research defined a novel metabolic cancer associated fibroblasts subset (meCAFs) enriched in loose-type pancreatic ductal adenocarcinoma (PDAC) and related to CD8+ T cells accumulation. Consistently, the abundance of meCAFs was associated with poor prognosis but better immunotherapy responses in PDAC patients. However, the metabolic characteristic of meCAFs and its cross-talk with CD8+ T cells remain to be elucidated. In this study, we identified PLA2G2A as a marker of meCAFs. In particular, the abundance of PLA2G2A+ meCAFs was positively related to the accumulation of total CD8+ T cells and negatively correlated with clinical outcomes of PDAC patients and infiltration of intratumoral CD8+ T cells. We demonstrated that PLA2G2A+ meCAFs substantially attenuated the antitumor ability of tumor infiltrating CD8+ T cells and facilitated tumor immune escape in PDAC. Mechanistically, PLA2G2A regulated the function of CD8+ T cells as a pivotal soluble mediator via MAPK/Erk and NF-κB signaling pathways. In conclusion, our study identified the unrecognized role of PLA2G2A+ meCAFs in promoting tumor immune escape by impeding the antitumor immune function of CD8+ T cells, and strongly suggested PLA2G2A as a promising biomarker and therapeutic target for immunotherapy in PDAC.

Modulating the tumor microenvironment improves antitumor effect of anti-PD-L1 mAb in breast cancer.

Introduction: Immune checkpoint inhibitors (ICIs) have provided noteworthy benefits in multiple cancer patients. However, the efficacy of monotherapy of ICIs was very limited. In this study, we endeavored to explore whether losartan can modulate the solid tumor microenvironment (TME) and improve the therapeutic efficacy of anti-PD-L1 mAb in 4T1 mouse breast tumor model and the underlying mechanism. Methods: The tumor-bearing mice were treated with control agents, losartan, anti-PD-L1 mAb or the dual agents. The blood and tumor tissues were respectively used for ELISA and immunohistochemical analysis. CD8-depletion and lung metastatic experiments were performed. Results: Compared to control group, losartan inhibited the expression of alpha-smooth muscle actin (α-SMA), deposition of collagen I in the tumor tissues. The concentration of transforming growth factor-β1 (TGF-β1) in the serum was low in the losartan treated group. Although losartan alone was ineffective, the combination of losartan and anti-PD-L1 mAb elicited dramatic antitumor effect. Immunohistochemical analysis revealed that there were more intra-tumoral infiltration of CD8+ T cells and increased granzyme B production in the combination therapy group. In addition, the size of spleen was smaller in the combination therapy group, compared to monotherapy. The CD8-depleting Abs abrogated the antitumor efficacy of losartan and anti-PD-L1 mAb in vivo. The combination of losartan and anti-PD-L1 mAb significantly inhibited 4T1 tumor cells lung metastatic in vivo. Conclusion: Our results indicated that losartan can modulate the tumor microenvironment, and improve the efficacy of anti-PD-L1 mAb.

Aptamer-conjugated nano-liposome for immunogenic chemotherapy with reversal of immunosuppression.

Cancer cells have various immune evasion mechanisms that resist the immune cells by reprogramming the tumor microenvironment (TME), such as programmed death-ligand 1 (PD-L1) and indoleamine 2,3-dioxygenase-1 (IDO1) overexpression. One of the approaches to restore antitumor immune response by T-cells is through induction of immunogenic cell death (ICD). Thus, drug carrier containing IDO1 siRNA and ICD inducer would be effective anticancer regimen to modulate the immunosuppressive TME by reversing the IDO1-mediated immunosuppression in a synergistic combination with ICD induction. However, numerous nanocarrier platforms for co-delivery of multiple drugs mostly depend on the enhanced permeation and retention (EPR), which is insufficient to achieve selectivity in tumor sites harboring various types of cells. We designed a targeted drug delivery system using nano-sized liposomes functionalized with anti-CD44 and anti-PD-L1 DNA aptamers, which target breast cancer cells and inhibit PD-1/PD-L1 interaction between cancer cells and T-cells. To reverse immunosuppressive TME and reactivate immune response, cancer-targeting nano-liposomes were prepared to contain immunogenic cell death inducer (Doxorubicin, DOX) and IDO1 siRNA, namely Aptm[DOX/IDO1]. The Aptm[DOX/IDO1] specifically delivered the loaded DOX and IDO1 siRNA into target breast cancer cells through aptamer-mediated endocytosis. Cancer-targeted DOX/IDO1 siRNA delivery enhanced ICD and suppressed IDO1 expression with significantly high toxicity in cancer cells. We demonstrated that Aptm[DOX/IDO1] could achieve synergistic antitumor effects by facilitating ICD response and simultaneous reversal of the immunosuppressive TME with IDO1 knockdown in the subcutaneous breast cancer model mice, thus reducing tumor size. These antitumor effects were exerted with intratumoral infiltration of CD8+ cytotoxic T lymphocyte as well as attenuation of regulatory T-cell recruitment in the tumor sites. We further proved that our Aptm[DOX/IDO1] strategy significantly reduced tumor metastasis in tumor-xenograft mice through a synergistic combination of cancer cell-targeted ICD induction and reversal of the IDO1-mediated immunosuppressive TME. Our nanocarrier platform based on cationic liposomes containing DOX and IDO1 siRNA, which are conjugated with two DNA aptamers targeting the cancer cell surface, accomplished synergistic chemoimmunotherapy through tumor-specific immune modulation into immune-favorable TME in vivo.

Tumor-infiltrating OX40+ lymphocytes is an independent positive prognostic factor for patients with pancreatic ductal adenocarcinoma.

OX40 signaling pathway occupies a vital place in anti-tumor immunity; however, the role of tumor-infiltrating OX40+ lymphocytes in pancreatic ductal adenocarcinoma (PDAC) remains to be identified. A total of 325 sequential PDAC patients who received curative tumor resection between January 2014 and December 2016 were enrolled. Tissues of these patients were immunohistochemically assessed for tumor infiltration of CD4+ T cells, CD8+ cytotoxic T cells (CTLs), and OX40+ lymphocytes. The frequency of OX40+ tumor-infiltrating lymphocytes (TILs) was then analyzed to various clinicopathological features, densities of tumor infiltration of CD4+ T cells and CTLs, and survival analysis was conducted using Kaplan-Meier (KM) curves. The risk scores of associated markers were calculated by the Cox proportional-hazards model. Our results showed that higher OX40+ lymphocytes infiltration was significantly correlated with superior median overall survival (OS) (25.8 vs 13.4months, P < 0.001). Additionally, using univariate and multivariate Cox proportional hazards analyses, this study revealed that together with tumor differentiation, tumor size, serum CA199 levels, serum CA125 levels, and the infiltration of intratumoral CD8+ T cells. The abundance of OX40+ lymphocytes within the tumor was continued to be an independent predictor for OS (P = 0.023, HR = 0.713, 95% CI: 0.532-0.954). This study demonstrated that intratumoral infiltration by a high number of OX40+ lymphocytes is a novel biomarker for favorable prognosis in resected PDAC patients, which implies that OX40-agonist-based immunotherapy might be a potential target in PDAC patients.

Abstract LB087: Characterization of JNJ-70218902, a TMEFF2 x CD3 bispecific antibody, in prostate cancer models

Abstract TMEFF2, a transmembrane protein with epidermal growth factor-like and 2 follistatin-like domains, is primarily restricted in expression to healthy brain and prostate and is enriched in prostate cancer. JNJ-70218902 (JNJ-902) is a bispecific antibody that engages TMEFF2-expressing tumor cells and CD3 on T cells causing exposure dependent pro-inflammatory responses and target tumor cell lysis. Here we report the preclinical characterization of JNJ-902 using in vitro and in vivo prostate cancer models. Expression of TMEFF2 transcript and protein in brain and prostate was confirmed in healthy human tissue, as was its elevated expression in advanced prostate cancer tumors, including in 41/45 (91%) metastatic castration-resistant prostate cancer (mCRPC) samples. JNJ-902 bound to TMEFF2-positive LNCaP-AR cells (androgen-sensitive human prostate cancer cell line) in a concentration-dependent manner (EC50 = 9.6 nM); no binding to TMEFF2-negative DU145 prostate cancer cells was observed. The binding affinity (KD) of JNJ-902 for CD3 on primary human T cells was determined to be ~151 nM. T cell-mediated killing, as measured by caspase-3 activity, was induced upon incubation of JNJ-902 with healthy human donor T cells (n = 5) and LNCaP-AR cells at a 3:1 effector to target ratio (EC50 = 1.4 nM). No cytotoxicity was detected using DU145 cells or with control antibodies. Corresponding T cell activation was confirmed by concentration-dependent increases in cell surface CD8+CD25+ expression and proinflammatory cytokine production (eg, GM-CSF, IFN-γ, IL-10, TNF-α). Antitumor activity of JNJ-902 at increasing concentrations up to 5 mg/kg was observed in T cell humanized NSG mice bearing LNCaP xenografts (tumor growth inhibition [∆%TGI] of 75-122%) and in LuCaP 86.2 patient-derived xenografts (∆%TGI of 72-88%) as compared with control-treated mice. JNJ-902-dependent intratumoral infiltration of CD8+ T cells was detected in both models with increases in frequency of CD8+granzymeB+ effector cells. The data summarized here are the first report on the pharmacological activity of a novel TMEFF2 × CD3 bispecific antibody, supporting further investigation of TMEFF2 in mCRPC given its expression in advanced prostate cancer. Targeting TMEFF2 with the TMEFF2 × CD3 bispecific antibody JNJ-902 elicited robust T cell-mediated responses in both prostate cancer cells and xenograft models. Based on these data, a phase 1 dose escalation trial of JNJ-902 in patients with mCRPC is currently ongoing (NCT04397276). Citation Format: Subhasree Basu, Mike Russell, Katrin Sproesser, Bethany Mattson, Cassandra L. Lowenstein, Sathya Venkataramani, Maura Diamond, Robin E. Ernst, Stephen Jarantown, Jackson Wong, Gerald Chu, Kathryn Packman, Shaozhou Ken Tian, Scott R. Brodeur, Margaret Yu, Brent A. Rupnow. Characterization of JNJ-70218902, a TMEFF2 x CD3 bispecific antibody, in prostate cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB087.

Abstract 6250: Investigating the immunomodulatory mechanisms of Smyd3 depletion in HPV-negative head and neck squamous cell carcinoma

Abstract Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer type in the world, with Human Papilloma virus (HPV)-negative patients having a ~50% recurrence rate and poor median overall survival of ~13 months in the recurrent/metastatic setting. Pembrolizumab, a programmed-death-1 (PD-1) checkpoint inhibitor is active but with a response rate as low as 19%. Our preliminary work has identified SET and MYND Domain containing 3 (SMYD3) as a chromatin modifier that is significantly overexpressed in HPV-negative HNSCC tumor tissues compared to normal epithelium and is associated with low CD8+ T-cell infiltration in HPV-negative HNSCC. Furthermore, we have shown that knockdown of human SMYD3 upregulates the expression of multiple type I IFN response and antigen presentation machinery (APM) genes. This study aims to investigate whether Smyd3 depletion potentiates immune-mediated antitumor efficacy in anti-PD-1 resistant syngeneic mouse models of HPV-negative HNSCC (MOC1), as well as relevant mechanisms. SiRNA-mediated knockdown of Smyd3 in mouse HPV-negative MOC1 cells followed by RNA-seq revealed upregulation of multiple type I IFN response and APM genes in vitro. Multicolor flow cytometry of MOC1 tumors treated with control anti-sense oligonucleotides (ASOs) or Smyd3 ASOs showed that Smyd3 depletion in a syngeneic, heterotopic mouse model (C57BL/6) of flank MOC1 tumors induced intratumoral infiltration of CD8+ T-cells, as well as upregulation of H2-Kb (MHC class I) and mouse Pd-l1 in MOC1 cells. Combined treatment of Smyd3 ASOs with anti-PD-1 induced complete regressions of flank MOC1 tumors in 4/8 treated mice and significant tumor growth restriction in 2/8 mice, while 2/8 tumors “escaped” the treatment effect. Single-cell RNA seq of MOC1 tumors treated with control or Smyd3 ASOs is ongoing to evaluate the effects of Smyd3 depletion on MOC1 cancer cells and immune cell subsets of the tumor microenvironment and to identify potential mechanisms of resistance of Smyd3 ASO + anti-PD-1 combination treatment. Genome-wide mapping of Smyd3 and the repressive mark H4K20me3, which is known to be written by Smyd3, in MOC1 cells using CUT&amp;RUN assays is also ongoing and aims to decipher whether Smyd3 directly binds to and regulates the expression of immune-related genes through H4K20me3. Ex-vivo cytotoxicity assays aim to evaluate whether CRISPR Smyd3 KO in MOC1 cells sensitizes cancer cells to TIL-mediated cytotoxicity. Smyd3 depletion induces an inflamed tumor microenvironment and may potentiate the immune-mediated antitumor efficacy of anti-PD-1 in syngeneic mouse models of HPV-negative HNSCC. This work may lay the biological rationale to combine SMYD3 inhibition with T-cell based immunotherapeutic approaches in HPV-negative HNSCC. Citation Format: Daniel E. Tsai, Yvette Robbins, Angel Huynh, Andrew Sinkoe, Cem Sievers, Madhavi Murali, Xiaolin Luo, Clint T. Allen, Vassiliki Saloura. Investigating the immunomodulatory mechanisms of Smyd3 depletion in HPV-negative head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6250.

Abstract 74: Engineering tertiary lymphoid structures for glioblastoma: A novel gene combination promotes therapeutic TLS formation in an immune-competent mouse model of GBM

Abstract The absence of conventional lymphatic access to the brain parenchyma restricts the capacity of the peripheral immune system to recognize and elicit durable responses against glioma antigens. In some peripheral solid tumor types and autoimmune diseases such as multiple sclerosis, the spontaneous development of tertiary lymphoid structures (TLS) with varying degrees of organization have been observed in human patients and mice experiencing chronic inflammation. The presence of TLS in the context of cancer is generally associated with improved prognosis, particularly when these TLS are characterized by intratumoral infiltration of CD8+ T-cells and B-cells. We sought to engineer the development of TLS in the brain tumor setting, utilizing our SB28 glioblastoma (GBM) model which is sparsely infiltrated by lymphocytes and characterized by a significant myeloid-derived suppressor cell population. To determine the most critical chemokines and cytokines required to elicit TLS formation in the murine GBM context, we stably transduced SB28 with a combination of TLS-stimulating factors that we identified and injected these cells into the brain parenchyma of syngeneic C57BL/6J mice. A combination of the chemoattractant and lymphoid follicle-stimulating cytokines interleukin (IL)-7, LIGHT (TNFSF14), CCL21, and IL-17 promoted infiltration of CD8+CD3+ T-cells into the tumor and nearby parenchyma, but it also promoted enhanced tumor proliferation. A modified gene combination including IL-7, LIGHT, and CCL21 promoted CD8+ T-cell infiltration and clustering by immunofluorescence analysis. This combination also significantly decreased tumor burden as measured by bioluminescent imaging, resulting in complete tumor rejection in a subset of mice. Finally, modifying this combination to include CXCL13 promoted the infiltration and clustering of B-cells along with the observed CD8+ T-cell infiltration. This combination elicited the formation of vascular structures that stained for PNAd (MECA-79) and LYVE-1, markers for high endothelial venules (HEV) and lymphatic endothelium, respectively. Taken together, these analyses suggest that these factors are sufficient to stimulate the formation of TLS in the GBM setting. Future studies will evaluate a replication competent viral vector based approach for gene delivery and whether TLS formation can promote tumor specific T-cell memory and persistence in GBM. Ultimately, we aim to promote therapeutic TLS in a manner that is highly translational to brain tumor patients and complementary to existing T-cell therapies. Citation Format: Kira Morgan Downey, Bindu Hegde, Zinal Chheda, Jason Zhang, Hideho Okada. Engineering tertiary lymphoid structures for glioblastoma: A novel gene combination promotes therapeutic TLS formation in an immune-competent mouse model of GBM [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 74.

Apatinib combined with PD-L1 blockade synergistically enhances antitumor immune responses and promotes HEV formation in gastric cancer.

Apatinib, an antiangiogenic drug, has shown beneficial effects only in a fraction of advanced gastric cancer (GC) patients. Given the recent success of immunotherapies, combination of apatinib with immune checkpoint inhibitor may provide sustained and potent antitumor responses. Immunocompetent mice with subcutaneous MFC tumors grown were given a combination of apatinib and anti-PD-L1 antibody therapy. GC tissues from patients undergoing curative resection in China were collected, and the density of HEVs, MSI status and tumor-infiltrated lymphocytes were analyzed by immunohistochemical staining. Combined apatinib and PD-L1 blockade therapy synergistically delayed tumor growth and increased survival in MFC-bearing immunocompetent mice. The combination therapy promoted antitumor immunity by increasing the ratio of CD8+ cytotoxic T cells to Foxp3+ Treg cells, the accumulation of CD20+ B cells and the Th1/Th2 cytokine ratio (IFN-γ/IL-10). The combination therapy induced the formation of HEVs through activation of LTβR signaling, thus promoting CD8+ cytotoxic T cell and CD20+ B cell infiltration in tumors. In clinical GC samples, the density of HEVs positively correlated with the intratumoral infiltration of CD8+ cytotoxic T cells and CD20+ B cells. MSI-high GC showed a higher density of HEVs, CD8+ cytotoxic T cells and CD20+ B cells than MSS/MSI-low GC. GC patients with high densities of HEVs, CD8+ cytotoxic T cells and CD20+ B cells had an improved prognosis with superior overall survival. Combining apatinib with PD-L1 blockade treatment synergistically enhances antitumor immune responses and promotes HEV formation in GC.

Senescent Tumor Cells Build a Cytokine Shield in Colorectal Cancer.

Cellular senescence can either support or inhibit cancer progression. Here, it is shown that intratumoral infiltration of CD8+ T cells is negatively associated with the proportion of senescent tumor cells in colorectal cancer (CRC). Gene expression analysis reveals increased expression of C‐X‐C motif chemokine ligand 12 (CXCL12) and colony stimulating factor 1 (CSF1) in senescent tumor cells. Senescent tumor cells inhibit CD8+ T cell infiltration by secreting a high concentration of CXCL12, which induces a loss of CXCR4 in T cells that result in impaired directional migration. CSF1 from senescent tumor cells enhance monocyte differentiation into M2 macrophages, which inhibit CD8+ T cell activation. Neutralization of CXCL12/CSF1 increases the effect of anti‐PD1 antibody in allograft tumors. Furthermore, inhibition of CXCL12 from senescent tumor cells enhances T cell infiltration and results in reducing the number and size of tumors in azoxymethane (AOM)/dextran sulfate sodium (DSS)‐induced CRC. These findings suggest senescent tumor cells generate a cytokine barrier protecting nonsenescent tumor cells from immune attack and provide a new target for overcoming the immunotherapy resistance of CRC.

IMMU-28. INDUCTION OF TERTIARY LYMPHOID STRUCTURE-LIKE T-CELL CLUSTERS BY DELIVERY OF A NOVEL GENE COMBINATION INTO AN IMMUNOCOMPETENT MOUSE MODEL OF GLIOBLASTOMA

Abstract The lack of conventional lymphatic drainage to and from the brain parenchyma restricts the capacity of the peripheral immune system to recognize and respond to glioma antigens. In some peripheral solid tumor types and central nervous system autoimmunity, the spontaneous development of tertiary lymphoid structures (TLS) with varying degrees of organization have been observed in human patients and mice following chronic inflammation. In the cancer setting, presence of TLS are generally associated with improved prognosis, especially when they are characterized by intratumoral infiltration of CD8+ T-cells. We aimed to induce the development of TLS in vivo, utilizing our SB28 glioblastoma model which is sparsely infiltrated by lymphocytes. As a proof-of-concept study, we stably transduced SB28 with a combination of several TLS-stimulating factors that we’ve identified and injected these cells into the brain parenchyma of syngeneic C57BL/6J mice. A combination of the chemoattractant and lymphoid follicle-stimulating cytokines LIGHT, CCL21, IL-7, and IL-17 produced substantial infiltration of CD8+CD3+ T-cells into the tumor and nearby parenchyma. However, this combination was also associated with accelerated tumor growth. A modified gene combination including LIGHT, CCL21, and IL-7 promoted CD8+CD3+ T-cell infiltration by flow cytometry, T-cell clustering by immunofluorescence analysis, and inhibited tumor burden compared with the control as measured by bioluminescent imaging. There was also evidence of increased lymphatic vasculature around the margins of T-cell clustering as demonstrated by LYVE-1 staining. Together, these analyses highlight a role for these factors in stimulating the recruitment and clustering of T-cell to the glioblastoma microenvironment in a TLS-like phenomenon. Future studies will evaluate whether the recruitment of other lymphocytes and stromal cells to these TLS-like clusters can promote T-cell memory and persistence. Ultimately, we aim to provide these factors utilizing a gene delivery method that will prove translatable to the clinic and complementary to existing T-cell therapies.

Abstract B4: High-dose ascorbic acid synergizes with anti-PD1 in a lymphoma mouse model

Abstract Major efforts are under way to identify agents that can potentiate effects of immune checkpoint inhibition. Ascorbic acid (AA) has been previously shown to cause DNA demethylation and corresponding increase in hydroxymethylation in multiple malignancies by enhancing the activity of the Ten-Eleven Translocation (TET) enzymes (Shenoy et al., BCJ 2017; Shenoy et al., JCI 2019; Cimmino et al., Cancer Cell 2017; Agathocleous et al., Nature 2017). Furthermore, AA has re-emerged as a promising anticancer agent based on recent knowledge of pharmacokinetics, discovery of unexpected mechanisms of action, and early phase trials with IV AA (Shenoy et al., Cancer Cell 2018). Here, we report that AA treatment caused genome-wide demethylation and enhanced expression of endogenous retroviral elements in lymphoma cells. AA also increased 5-hydroxymethylcytosine (5hmC) levels of CD8+ T cells and enhanced their cytotoxic activity in a lymphoma coculture system. High-dose AA treatment synergized with anti-PD1 therapy in a syngeneic lymphoma mouse model, resulting in marked inhibition of tumor growth compared with either agent alone. Analysis of the intratumoral epigenome revealed increased 5hmC with AA treatment, consistent with in vitro findings. Analysis of the tumor immune microenvironment revealed that AA strikingly increased intratumoral infiltration of CD8+ T cells and macrophages, suggesting enhanced tumor immune recognition. The combination treatment markedly enhanced intratumoral infiltration of macrophages and CD8+ T lymphocytes, granzyme B production by cytotoxic cells (cytotoxic T cells and natural killer cells), and IL-12 production by antigen-presenting cells compared with single-agent anti-PD1. These data strongly indicate that AA potentiates anti-PD1 checkpoint inhibition through synergistic mechanisms. The study provides a compelling rationale for testing combinations of AA and anti-PD1 agents in lymphoma patients as well as in preclinical models of other malignancies. Citation Format: Rebecca Luchtel, Tushar Bhagat, Kith Pradhan, William Jacobs, Mark Levine, Amit Verma, Niraj Shenoy. High-dose ascorbic acid synergizes with anti-PD1 in a lymphoma mouse model [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 B4.

Abstract B051: Antitumor activity of dual PD-1 and LAG-3 blockade reversed by adoptive transfer of tumor-draining lymph node cells

Abstract Immune checkpoint inhibitors (ICIs) emerged as promising therapies for various cancer types. Yet, therapeutics that target single immune checkpoint protein (ICP) have met with obstacles including low response rates and acquired tumor resistance. To overcome these obstacles, combination therapies that simultaneously target multiple ICPs are being actively pursued in the field. In this study, we utilized the B16F10 tumors, a syngeneic mouse melanoma model that showed limited responses to numerous ICIs against single ICP, and assessed the efficacy of dual blockade of PD-1 and LAG-3 (an ICP expressed on the surface of T and NK cells) through i.p. injection of neutralizing antibodies [anti-PD-1 (clone: RMP1-14) and anti-LAG-3 (clone: C9B7W), 10 mg/kg each, twice weekly]. Moreover, to interrogate the effects of altered immune landscape in the tumor microenvironment (TME) on tumor responses to ICIs, we performed a single-time adoptive transfer of tumor-draining lymph node (TDLN) cells harvested from B16F10 tumor-bearing donor mice (5 M TDLN cells per recipient mouse, i.v.), 2 days before the first antibody treatments, and assessed its impact on tumor responses in recipient mice. TDLN cells were either used immediately after isolation or cultured in vitro with anti-CD3/IL-2 for 5 days that resulted in rapid proliferation and overpopulation (&amp;gt;98%) of T cells. Our results showed that combination treatment with anti-PD-1 and anti-LAG-3, compared to isotype controls, significantly inhibited B16F10 tumor growth (&amp;gt;60%) in mice that did not received donor TDLN cells. FACS analyses of blood and tumor samples indicated that anti-PD-1/anti-LAG-3 significantly enhanced the percentages of CD8+ T cells and CD49b+ NK cells as well as the ratio between CD8+ T cells and CD4+CD25+CD127-/low regulatory T cells, in B16F10 tumors but not in the peripheral blood. Interestingly, adoptive transfer of freshly-isolated TDLN cells completely abrogated the antitumor activity, whereas infusion with anti-CD3/IL-2-treated TDLN cells only partially alleviated the antitumor activity of anti-PD-1/anti-LAG-3. FACS analyses indicated that, in mice receiving donor TDLN cells, anti-PD-1/anti-LAG-3 was not able to stimulate intratumoral infiltration of CD8+ T cells or NK cells or increase the ratio between CD8+ T cells and regulatory T cells. In conclusion, combined treatment of anti-PD-1 and anti-LAG-3 significantly inhibits B16F10 tumor growth associated with activation of immune responses in the TME, which is at least partially reversed by adoptive transfer of TDLN cells. Our results support the usage of the B16F10 mouse melanoma model for testing the efficacy of combination therapies or bi-specific antibodies against both PD-1 and LAG-3. The reason behind tumor resistance induced by adoptive transfer of TDLN cells remains unclear. It is important to identify this mechanism of resistance, as the knowledge can benefit patient selection or provide a rationale for future combination therapies that concomitantly target PD-1/LAG-3 and this resistance machinery. Citation Format: Wei Liang, Lilibet Valdovinos, Kaitlyn Yapjoco, Kaede Hinata, Geeta Sharma, Thomas B Broudy. Antitumor activity of dual PD-1 and LAG-3 blockade reversed by adoptive transfer of tumor-draining lymph node cells [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B051. doi:10.1158/1535-7163.TARG-19-B051

Immune microenvironment modulation unmasks therapeutic benefit of radiotherapy and checkpoint inhibition

BackgroundImmune checkpoint inhibitors (ICIs) for solid tumors, including those targeting programmed cell death 1 (PD-1) and cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), have shown impressive clinical efficacy, however, most patients do not achieve durable responses. One major therapeutic obstacle is the immunosuppressive tumor immune microenvironment (TIME). Thus, we hypothesized that a strategy combining tumor-directed radiation with TIME immunomodulation could improve ICI response rates in established solid tumors.MethodsUsing a syngeneic mouse model of human papillomavirus (HPV)-associated head and neck cancer, mEER, we developed a maximally effective regimen combining PD-1 and CTLA-4 inhibition, tumor-directed radiation, and two existing immunomodulatory drugs: cyclophosphamide (CTX) and a small-molecule inducible nitric oxide synthase (iNOS) inhibitor, L-n6-(1-iminoethyl)-lysine (L-NIL). We compared the effects of the various combinations of this regimen on tumor growth, overall survival, establishment of immunologic memory, and immunologic changes with flow cytometry and quantitative multiplex immunofluorescence.ResultsWe found PD-1 and CTLA-4 blockade, and radiotherapy alone or in combination, incapable of clearing established tumors or reversing the unfavorable balance of effector to suppressor cells in the TIME. However, modulation of the TIME with cyclophosphamide (CTX) and L-NIL in combination with dual checkpoint inhibition and radiation led to rejection of over 70% of established mEER tumors and doubled median survival in the B16 melanoma model. Anti-tumor activity was CD8+ T cell-dependent and led to development of immunologic memory against tumor-associated HPV antigens. Immune profiling revealed that CTX/L-NIL induced remodeling of myeloid cell populations in the TIME and tumor-draining lymph node and drove subsequent activation and intratumoral infiltration of CD8+ effector T cells.ConclusionsOverall, this study demonstrates that modulation of the immunosuppressive TIME is required to unlock the benefits of ICIs and radiotherapy to induce immunologic rejection of treatment-refractory established solid tumors.

Induction of antigen specific CD8+ T cell infiltration by a novel neoadjuvant vaccine containing HSP70 and GPC3 peptides plus soluble LAG-3 and Poly-IC:LC: Interim results of a Phase I study.

e14306 Background: Even with curative resection, the recurrence rate of HCC is still high, and no effective adjuvant therapy is currently available. Our previous Phase I study with novel therapeutic peptides and immune adjuvants demonstrated the safety, antigen specific CTL induction in PBMC and a sign of efficacy (ASCO 2017 Abstract # 3086); thus, we started Phase I study of the same therapy as a perioperative immunotherapy setting in patients with resectable HCC (UMIN000029991). Methods: Two mg each of HLA-A*24:02, 02:01, or 02:06 restricted HSP70- and GPC3-derived peptides, in combination with hLAG-3Ig (1.0 mg) + Poly-IC:LC (1.4 mg) were injected intradermally at four sites of the inguinal and axillary regions every week for 6 weeks before surgery. Patients subsequently received 10 injections of adjuvant immunotherapy over 4 months. Surgical specimens and PBMCs were analyzed by mass cytometry (CyTOF), using 66 antibodies to monitor T cell exhaustion, T cell activation, Effector Treg induction, etc. Tumor specimens were also subjected to immunohistochemical staining of CD3, CD8, PD1, HSP70, and GPC3. The reason for early reporting is the interesting findings at the foci of HCC, and the interim analyses was approved by the Data and Safety Monitoring Committee. Results: Of the 11 screened patients, 5 completed the treatments and were analyzed. We found massive CD8+ T lymphocyte infiltration in the intratumor foci of HCC, which is usually accompanied by peritumoral lymphocytic infiltration. Moreover, the density of lymphocytes was markedly higher in areas of HSP70 or GPC3 antigen expression. One case out of five recurred 5 month after surgery and it showed low CD8+ and PD1+ cell infiltration and high effector Treg (CD4+/CD25+/CD45RA-/FoxP3 +) infiltration. This trend was not observed in PBMC, suggesting the importance of TIL analysis. The high PD1 expression was accompanied by massive intratumoral infiltration of CD8+ lymphocytes. Conclusions: The novel therapeutic peptide and immune adjuvant combination induced sustained immune cell infiltration into tumor microenvironments, especially those presenting target tumor-associated antigens. Our novel immunotherapy may convert cold tumors into hot tumors containing PD1+ lymphocytes. Thus, the combination of this novel strategy with PD (L) 1 antibody is warranted. Clinical trial information: 000029991.

Cytoplasmic Expression of P16 is Related to Low Infiltration of CD8+ T cell and Poor Prognosis in Digestive Neuroendocrine Carcinoma

Background and Aims: The aim of this study is to examine the predictive and prognostic value of cytoplasmic P16Ink4A (cP16) and poor infiltration of CD8+ T cell in primary digestive neuroendocrine carcinoma (dNEC). Methods: We retrospectively reviewed 86 cases of NEC and mixed NEC (all cases thereafter refer to NEC), which had primary sites in digestive system. Kaplan-Meier survival curves and log-rank test were used for the survival analysis. Bioinformatics analysis was performed to identify the P16-related pathways. Results: The expression frequency of P16 in dNEC and adjacent tissues was 81.4% and 19.3% respectively (p<0.0001). There was no difference in overall survival (OS) between dNEC patients with P16 positive or negative expression. However, there was a statistically lower OS in dNEC patients with cP16 expression than those without cP16 expression (p=0.050). In addition, cP16 expression was correlated with poor infiltration of intratumoral CD8+ T cells. Furthermore,we found that dNEC patients with two features of cP16 expression in tumor and poor infiltration of CD8+ T cells in intratumoral tissue showed more poor survival (p=0.0133) than patients with single feature alone respectively. Bioinformatics analysis suggested that EGR1 and HLA class I might be involved in the inhibitory effect of cP16 on intratumoral infiltrated T cells. Conclusions: Therefore, the cP16 and poor intratumoral infiltration of CD8+ T cells may serve as poor prognostic biomarkers in dNEC. CP16 expression is associated with decreased intratumoral infiltration of CD8+ T cells probably related to deregulation of EGR1/ HLA class I pathway.

Intraepithelial Attack Rather than Intratumorally Infiltration of CD8+T Lymphocytes is a Favorable Prognostic Indicator in Pancreatic Ductal Adenocarcinoma.

Background: Tumor-infiltrating lymphocytes (TILs) are one of the major participants in the tumor microenvironment of pancreatic ductal adenocarcinoma (PDAC). However, the mechanism of interaction between TILs and tumors is complex and remains unclear.Objective: To evaluate the state of immunoreactions in PDAC tissues, and explore the prognostic value of these markers in a large sample, to provide a new theoretical basis for PDAC immunotherapy.Method: Immunohistochemical staining of CD4+ and CD8+T cells was performed in a tissue microarray (TMA) of 143 cases of PDAC. Two major variables for the spatial distributions of CD4+T and CD8+T cells in PDAC tissues, intraepithelial attack and intratumoral infiltration, were used to evaluate the state of immunoreactions, and the interrelationships with the clinicopathological variables were analyzed.Results: Our data showed that both the intraepithelial CD4+T and CD8+T attack were less frequent than the intratumoral infiltration. CD8+T intraepithelial attack and intratumoral infiltration were more intense than CD4+T. CD8+T intraepithelial attack was an independent favorable prognostic factor for overall survival, correlating negatively with vascular invasion and positively with CD4+T and CD8+T high intratumoral infiltration. CD8+T high intratumoral infiltration without CD8+T intraepithelial attack was a poor prognostic factor. CD8+T high intratumoral infiltration was accompanied by T stage progression. Conclusively, in PDAC progression, imbalances of T cells occurred in CD4+ and CD8+ immunoreactions. The CD8+T intraepithelial attack was an independent favorable prognostic indicator, however the intraepithelial attack of CD4+T and the both intratumoral infiltration of CD8+T and CD4+T played an ambiguous role.Conclusion: Our data suggested that it is a potential approach to increasing the number of intraepithelial attacking CD8+T cells for tumor immunotherapy, and exploring a new mechanism for immunosuppression in a tumor microenvironment with high T cell infiltration without attack.

生物标志物在阻断PD-1/PD-L1通路治疗肿瘤中的预测性作用

Identifying biomarkers predicting clinical response to treatment of PD-1/PD-L1 pathway blockade can guide patient selection and therapeutic individualization. Some researches have shown that patients with cancer will acquire better clinical effects by blocking PD-1/PD-L1 pathway whose characteristics include higher pretreatment expression of PD-L1 by tumor cells or tumor infiltrating immune cells, the massive infiltration of intratumoral CD8+ T cells, the high mutation frequency of tumor cell gene. These biomarkers are expected to become indicators which can select patients. Key words: Neoplasms; Therapy; Biological markers; PD-1/PD-L1 pathway

Enhanced local and systemic anti-melanoma CD8+ T cell responses after memory T cell-based adoptive immunotherapy in mice.

Adoptive cell transfer (ACT) melanoma immunotherapy typically employs acutely activated effector CD8+ T cells for their ability to rapidly recognize and clear antigen. We have previously observed that effector CD8+ T cells are highly susceptible to melanoma-induced suppression, whereas memory CD8+ T cells are not. Although memory T cells have been presumed to be potentially advantageous for ACT, the kinetics of local and systemic T cell responses after effector and memory ACT have not been compared. B16F10 melanoma cells stably transfected to express very low levels of the lymphocytic choriomeningitis virus (LCMV) peptide GP33 (B16GP33) were inoculated into syngeneic C57BL/6 mice. Equal numbers of bona fide naïve, effector, or memory phenotype GP33-specific CD8+ T cells were adoptively transferred into mice 1 day after B16GP33 inoculation. The efficacy of ACT immunotherapy was kinetically assessed using serial tumor measurements and flow cytometric analyses of local and systemic CD8+ T cell responses. Control of B16GP33 tumor growth, persistence of adoptively transferred CD8+ cells, intratumoral infiltration of CD8+ T cells, and systemic CD8+ T cell responsiveness to GP33 were strongest after ACT of memory CD8+ T cells. Following surgical tumor resection and melanoma tumor challenge, only mice receiving memory T cell-based ACT immunotherapy exhibited durable tumor-specific immunity. These findings demonstrate how the use of non-expanded memory CD8+ T cells may enhance ACT immunotherapeutic efficacy.