Cells In Tumor-draining Lymph Nodes Research Articles

Abstract 4221: Characterization of OVA-expressing immunogenic syngeneic tumor models for immune checkpoint inhibitor evaluation

Abstract BACKGROUND: Syngeneic mouse tumor models are widely used as preclinical models for immuno-oncology (I/O) research. However, their response to immune checkpoint inhibitors (ICIs) in vivo is limited, despite the increase in tumor infiltrating lymphocytes (TILs), which is potentially due to low intrinsic immunogenicity. Expression of chicken ovalbumin (OVA) engineered into syngeneic tumors has previously been shown to render the cells more immunogenic and increases the response to ICIs such as anti-PD-1. In this study we investigated the potential to increase the response to ICIs through OVA expression and characterized the model for TILs and T cell-mediated immunity compared with parent cell line. METHODS: A series of murine cells were transduced by lentiviral vector (pLVX-EF1a_IRES-PURO) with a chicken OVA coding cDNA. OVA expression was confirmed by western blot following single clone selection under puromycin. MC38-OVA, B16-F10-OVA, CT26.WT-OVA and EMT6-OVA cells were subcutaneously inoculated into immunocompetent C57BL/6 or BALB/c mice to evaluate the tumorigenicity and their in vivo response to anti-PD-1(10mg/kg, IP) treatment. In the study, blood, spleen and tumors were collected at termination for FACS analysis of the immune cell populations and the OVA-specific T cells. The effects of CD4+ and CD8+ T lymphocyte depletion was investigated using anti-CD4 and anti-CD8 antibodies in the PD-1 cured EMT6-OVA mice. Adoptive cell transfer (ACT) was evaluated by re-challenge studies in both OVA expressing and WT tumor-bearing mice with isolated T cells derived from OVA untreated mice, anti-PD-1 cured mice or OT-I mice. RESULTS: Anti-PD-1 treatment resulted in complete regression in most mice bearing CT26.WT-OVA, MC38-OVA and EMT6-OVA, whereas an improvement in the response was shown in B16-F10-OVA in vivo. T cell and NK cells expansion was observed after anti-PD-1 treatment in peripheral blood with increased T cells, Treg cells and NK cells in both tumor-draining lymph nodes and spleen. In the re-challenge study anti-PD-1 cured mice generated robust tumor specific memory T cell, which successfully inhibited OVA expressing tumor growth following ACT. CD3+ T cells from EMT6-OVA-bearing mice showed anti-tumor immunity in vivo. CD4 and CD8 depletion studies provided further insight into the T cell role in PD-1 treatment. CONCLUSIONS: The panel of OVA expressing murine cell lines displayed increased T cell-mediated immunity and enhanced anti-PD-1 responses compared to WT models for different cancer indications in vivo, providing novel immunogenic syngeneic models for preclinical immunotherapy evaluation. Citation Format: Chenpan Nie, Yi Zhang, Jia Zheng, Annie X. An, Jun Zhou, Henry Q.X. Li, Jingjing Wang. Characterization of OVA-expressing immunogenic syngeneic tumor models for immune checkpoint inhibitor evaluation [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 4221.

Innate lymphoid cells: NK and cytotoxic ILC3 subsets infiltrate metastatic breast cancer lymph nodes

ABSTRACT Innate lymphoid cells (ILCs) – which include cytotoxic Natural Killer (NK) cells and helper-type ILC – are important regulators of tissue immune homeostasis, with possible roles in tumor surveillance. We analyzed ILC and their functionality in human lymph nodes (LN). In LN, NK cells and ILC3 were the prominent subpopulations. Among the ILC3s, we identified a CD56+/ILC3 subset with a phenotype close to ILC3 but also expressing cytotoxicity genes shared with NK. In tumor-draining LNs (TD-LNs) and tumor samples from breast cancer (BC) patients, NK cells were prominent, and proportions of ILC3 subsets were low. In tumors and TD-LN, NK cells display reduced levels of NCR (Natural cytotoxicity receptors), despite high transcript levels and included a small subset CD127− CD56− NK cells with reduced function. Activated by cytokines CD56+/ILC3 cells from donor and patients LN acquired cytotoxic capacity and produced IFNg. In TD-LN, all cytokine activated ILC populations produced TNFα in response to BC cell line. Analyses of cytotoxic and helper ILC indicate a switch toward NK cells in TD-LN. The local tumor microenvironment inhibited NK cell functions through downregulation of NCR, but cytokine stimulation restored their functionality.

In Situ Tumor Vaccination with Calcium-Linked Degradable Coacervate Nanocomplex Co-Delivering Photosensitizer and TLR7/8 Agonist to Trigger Effective Anti-Tumor Immune Responses.

In situ anti-tumor vaccination is an attractive type of cancer immunotherapy which relies on the effectiveness of dendritic cells (DCs) to engulf tumor antigens, become activated, and present antigens to T cells in lymphoid tissue. Here, a multifunctional nanocomplex based on calcium crosslinked polyaspartic acid conjugated to either a toll-like receptor (TLR)7/8 agonist or a photosensitizer is reported. Intratumoral administration of the nanocomplex followed by laser irradiation induces cell killing and hence generation of a pool of tumor-associated antigens, with concomitant promotion of DCs maturation and expansion of T cells in tumor-draining lymph nodes. Suppression of tumor growth is observed both at the primary site and at the distal site, thereby hinting at successful induction of an adaptive anti-tumor response. This strategy holds promise for therapeutic application in a pre-operative and post-operative setting to leverage to mutanome of the patient's own tumor to mount immunological memory to clear residual tumor cells and metastasis.

Correlation and prognostic implications of intratumor and tumor draining lymph node Foxp3+ T regulatory cells in colorectal cancer

BackgroundThe prognostic value of intratumor T regulatory cells (Tregs) in colorectal cancer (CRC) was previously reported, but the role of these cells in tumor draining lymph nodes (TDLNs) was less addressed.MethodsA total of 150 CRC stages I-IV were retrospectively enrolled. Intratumor and TDLN Tregs were examined by immunohistochemical assay. The association of these cells was estimated by Pearson correlation. Survival analyses of subgroups were conducted by Kaplan–Meier curves, and the log-rank test and risk factors for survival were tested by the Cox proportional hazard model.ResultsHigh accumulation of Tregs in tumors was significant in patients with younger age and good histological grade, where enrichment of these cells in TDLNs was more apparent in those with node-negative disease and early TNM stage disease, both of which were more common in early T stage cases. A significant correlation of intratumoral and TDLN Tregs was detected. Patients with higher intratumoral Tregs displayed significantly better PFS and OS than those with lower Tregs. However, no such differences were found, but a similar prognostic prediction trend was found for these cells in TDLNs. Finally, intratumoral Tregs were an independent prognostic factor for both PFS (HR = 0.97, 95% CI 0.95–0.99, P < 0.01) and OS (HR = 0.98, 95% CI 0.95–1.00, P = 0.04) in the patients.ConclusionsHigher intratumor Tregs were associated with better survival in CRC. Although no such role was found for these cells in TDLNs, the positive correlation and similar prognostic prediction trend with their intratumoral counterparts may indicate a parallelized function of these cells in CRC.

Regorafenib enhances antitumor immune efficacy of anti-PD-L1 immunotherapy on oral squamous cell carcinoma

Oral squamous cells carcinoma (OSCC) is the most common oral malignancy that majorly originated from oral cavity. Though the prognostic and predictive value of targeting checkpoint molecules has been reported on OSCC, the treatment efficacy of monotherapy is remaining limited. Several studies suggested that multikinase inhibitors may show potential to facilitate anti-PD-L1-induced anti-tumor immunity. Regorafenib, an oral multikinase inhibitor has been approved by FDA for various types of cancer treatment. Here, we aim to identify whether regorafenib may boost anti-tumor immunity of anti-PD-L1 in MOC1-bearing OSCC animal model. The alteration of immune cells such as M1/M2-like macrophages (MΦ), cytotoxicity T cells, regulatory T cells (Tregs), and myeloid-derived suppressor cells (MDSCs) after combination of anti-PD-L1 and regorafenib was validated by flow cytometry and immunohistochemistry staining. The combination index analysis (CI=0.89) supported that regorafenib effectively induce anti-OSCC efficacy of anti-PD-L1. Combination of anti-PD-L1 and regorafenib may not only trigger the polarization of M1-like MΦ (CD11b+CD86+) in mice bone marrow (BM) and spleen (SP), but also induce the accumulation and function of CD8+ T cells in tumor-draining lymph node (TDLN) and tumor. In addition, immunosuppressive related cells (MDSCs and Treg) and factors were all decreased by combination therapy in BM, SP and tumor. In sum, regorafenib may improve anti-OSCC efficacy of anti-PD-L1 through systemically and locally upregulating the immunostimulation immunity and suppressing immunosuppression immunity.

Abstract P013: Germinal center hypoxia in tumor-draining lymph nodes negatively regulates humoral immune responses and affects the activation of tumor-infiltrating T cells

Abstract Efforts to understand intrinsic and acquired resistance to anti-tumor immunotherapies have largely focused on characterizing factors within the primary tumor. However, lymph nodes are specialized hubs of immune response development, and an improved understanding of the microenvironmental conditions impacting immune cell activation in tumor-draining lymph nodes (TDLNs) may yield novel approaches for enhancing responses to anti-tumor immunotherapies. Using mouse models of breast cancer, we identified hypoxic regions amongst the B cell germinal center reactions of TDLNs. Injection of lethally irradiated tumor cells was sufficient to induce GC hypoxia, and hypoxia in TDLNs associated with the frequency of antibody- secreting cells. In vitro, hypoxia impaired the proliferation of activated B cells, and inhibited class-switching to IgG1 and IgA immunoglobulin isotypes. To assess the role of the hypoxic response in tumor-reactive GCs in vivo, we deleted von Hippel- Lindau factor (VHL) in class-switched B cells and found decreased GC B cells in TDLNs, and reduced class-switched and tumor-specific antibodies in the circulation, indicating that the hypoxic response negatively-regulates tumor-induced humoral immune responses. pVHL deletion in class-switched B cells also reduced the expression of activation markers amongst tumor-infiltrating T cells and increased the presence of M2-like macrophages within the primary tumor. We detected the hypoxia marker carbonic anhydrase IX in the GCs of LNs from breast cancer patients, providing the first line of evidence that hypoxia develops within human GCs and validating our observations of hypoxia in TDLNs of pre-clinical mammary tumor models. We conclude that GC hypoxia develops in TDLNs, and that the hypoxic response negatively regulates tumor-induced humoral immune responses in pre-clinical models, with consequent impacts on the phenotype tumor-infiltrating T cells and macrophages. Citation Format: Natalie Firmino, Kevin Bennewith. Germinal center hypoxia in tumor-draining lymph nodes negatively regulates humoral immune responses and affects the activation of tumor-infiltrating T cells [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2021 Oct 5-6. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(1 Suppl):Abstract nr P013.

Abstract P011: B cell subsets that correlate with anti-PD-1 resistance in a preclinical model of HPV+ oropharyngeal cancer

Abstract A subset of head and neck squamous cell carcinomas (HNSCC) is associated with infection with oncogenic strains of human papillomavirus (HPV) and its prevalence continues to rise in the US. Cancer progression has been associated, in part, with an impaired immunity. Because of this, immunotherapies such as immune checkpoint blockade (ICB) have been FDA approved to treat different types of cancers, including HNSCC. The ICB targeting the molecule programmed death cell-1 (PD-1) is used as treatment for HPV-related HNSCC, however, only around 30% of patients respond. Most studies have focused on characterizing T cell responses, however, B cells, are also present in the tumor microenvironment of HPV-positive HNSCC. Interestingly, different B cell subsets with differential functions that may promote or prevent an effective anti-tumor response are present in tumors and tumor-draining lymph nodes. Therefore, fine regulation of anti-tumor and tumor-promoting B cell subsets may be necessary to promote an effective anti-tumor response and therefore prevent tumor progression. However, the B cells that may be associated with responsiveness to anti-PD-1 in HPV+ oropharyngeal cancer remain undefined. By using the preclinical model for HPV+ oropharyngeal cancer (named mEER) we have determined that tongue-implanted tumors are sensitive to anti-PD-1, where around 50% of the mice clear the tumors, whereas flank-implanted tumors are completely resistant to these treatments. By using this in vivo model, we were able to characterize B cell subsets in anti-PD-1 sensitive and anti-PD-1 resistant tumors. First, we compared B cell subsets in untreated mice between tongue (anti-PD-1 sensitive) and flank-implanted (anti-PD-1 resistant) tumors by flow cytometry. Interestingly, flank-implanted tumors contained more PD-1-expressing B cells, which have been identified as an immunosuppressive population in hepatocellular carcinoma. The presence of immunosuppressive B cells on flank tumors was also supported by our finding that flank implanted tumors grew at a slower rate in B cell-deficient mice compared to wild-type mice. When we compared tongue-bearing tumor mice that responded or not responded to anti-PD-1, we observed that mice that did not respond to anti-PD-1 had a higher frequency of antibody-secreting cells in tumor-draining lymph nodes compared to responders. Lastly, when we cultured naïve B cells with tumor cell supernatant from mEER cells, B cells upregulated PD-1 and were able to produce the immunosuppressive cytokine IL-10. Our data suggest that HPV+ oropharyngeal tumors modulate B cell functions and that a differential infiltration of B cell subsets into tumors and tumor-draining lymph nodes correlates with responsiveness to anti-PD-1 therapy in a preclinical model of HPV+ oropharyngeal cancer. These results will aid in our long-term goal to elucidate novel mechanisms for the modulation of anti-tumor responses by B cells and the development of novel B cell-specific therapies for HPV-related cancers and, therefore, improve clinical outcomes in these patients. Citation Format: Pamela A. Merheb, Daniel L. Castañón, Michael Rivera, Jorge Galán, Stephanie M. Dorta-Estremera. B cell subsets that correlate with anti-PD-1 resistance in a preclinical model of HPV+ oropharyngeal cancer [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2021 Oct 5-6. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(1 Suppl):Abstract nr P011.

Heterologous Prime-Boost Vaccination with a Peptide-Based Vaccine and Viral Vector Reshapes Dendritic Cell, CD4+ and CD8+ T Cell Phenotypes to Improve the Antitumor Therapeutic Effect.

Simple SummaryDeveloping new therapeutic cancer vaccines is of paramount importance to counteract tumor escape observed after conventional therapies in certain types of cancer. We have previously shown that the combination of two different vaccine platforms, targeting tumor-specific antigens, resulted in potent immune responses in preclinical models. Here, we show that the heterologous prime-boost combination with a protein vaccine and a viral vector vesicular stomatitis virus immunologically reshapes the immune-excluded TC-1 tumor model as well as the inflamed MC-38 tumor model, leading to beneficial therapeutic efficacy. Furthermore, the treatment with a multi-epitope vaccine allowed us to appreciate the various repartition among three antigen-specific cytotoxic T-cell responses combined with the viral boost. The combination leads to improved efficacy in all animals and highlights the importance of combining tumor epitopes. Our vaccine strategy could be further extended to prophylactic cancer vaccines and beyond, for infectious diseases.Heterologous prime-boost settings with a protein vaccine and the viral vector vesicular stomatitis virus, both expressing tumor-associated antigens (KISIMA-TAA and VSV-GP-TAA), have been previously shown to generate potent antitumor immunity. In the cold TC-1 model (HPV antigen) and the immune-infiltrate MC-38 model (Adpgk, Reps1 and Rpl18 neo-antigens), we further investigated pivotal immune cells that educate CD8+ T cells. Heterologous prime-boost vaccination induced a superior antitumor response characterized by the increase in number and functionality of antigen-specific CD8+ T cells, recruitment of cross-presenting dendritic cells, and polarization of CD4+ T cells towards an antitumor Th1 phenotype within the tumor and tumor-draining lymph nodes, turning the cold TC-1 tumor into a hot, inflamed tumor. In the inflamed MC-38 tumor model, treatment combination markedly prolonged the overall survival of mice. Treatment with multi-epitope vaccines also induced high frequencies of multiple antigen specificities in the periphery and in the tumor. Prime-boost treatment reduced tumor-infiltrating regulatory CD4+ T cells whilst increasing cross-presenting dendritic cells in tumor-draining lymph nodes. In conclusion, heterologous prime-boost vaccination possesses the ability to induce a potent anti-tumor response in both immune-excluded and immune-infiltrated mouse tumor models. Additionally, this study highlights the design of a multi-epitope vaccine for cancer immunotherapy.

Mycobacterium tuberculosis Rv3628 is an effective adjuvant via activation of dendritic cells for cancer immunotherapy

Tumor antigens (Ags) are weakly immunogenic and elicit inadequate immune responses, thus induction of antigen-specific immune activation via the maturation of dendritic cells (DCs) is a strategy used for cancer immunotherapy. In this study, we examined the effect of Rv3628 from Mycobacterium tuberculosis (Mtb) on activation of DCs and anti-tumor immunity in vivo. Intravenous injection of mice with Rv3628 promoted DC activation of spleen and lymph nodes. More importantly, Rv3628 also induced activation of DCs and enhanced Ag presentation in tumor-bearing mice. In mice bearing ovalbumin (OVA)-expressing tumors, combination treatment with Rv3628 and OVA peptide promoted OVA-specific T cell activation and accumulation of interferon (IFN)-γ and tumor necrosis factor (TNF)-α-producing OT-I and OT-II cells in tumor-draining lymph nodes. Moreover, three different tumor Ags in three different tumor models showed enhanced anti-tumor activity with Rv3628 as adjuvant, including inhibition of growth of OVA-expressing B16 melanoma, CT26 carcinoma, and B16 melanoma tumors, and a synergistic effect with anti-programmed cell death protein 1 (PD-1) antibody treatment. Additionally, potential application against human tumors was indicated by similar activation of human peripheral blood DCs by Rv3628. Taken together, these data demonstrate that Rv3628 could be an effective adjuvant in tumor immunotherapy via enhanced capacity of DC activation and Ag presentation.

Conventional type I dendritic cells maintain a reservoir of proliferative tumor-antigen specific TCF-1+ CD8+ T cells in tumor-draining lymph nodes

In tumors, a subset of CD8+ Tcells expressing the transcription factor TCF-1 drives the response to immune checkpoint blockade. We examined the mechanisms that maintain these cells in an autochthonous model of lung adenocarcinoma. Longitudinal sampling and single-cell sequencing of tumor-antigen specific TCF-1+ CD8+ Tcells revealed that while intratumoral TCF-1+ CD8+ Tcells acquired dysfunctional features and decreased in number as tumors progressed, TCF-1+ CD8+ Tcell frequency in the tumor draining LN (dLN) remained stable. Two discrete intratumoral TCF-1+ CD8+ Tcell subsets developed over time-a proliferative SlamF6+ subset and a non-cycling SlamF6- subset. Blocking dLN egress decreased the frequency of intratumoral SlamF6+ TCF-1+ CD8+ Tcells. Conventional type I dendritic cell (cDC1) in dLN decreased in number with tumor progression, and Flt3L+anti-CD40 treatment recovered SlamF6+ Tcell frequencies and decreased tumor burden. Thus, cDC1s in tumor dLN maintain a reservoir of TCF-1+ CD8+ Tcells and their decrease contributes to failed anti-tumor immunity.

Motility Dynamics of T Cells in Tumor-Draining Lymph Nodes: A Rational Indicator of Antitumor Response and Immune Checkpoint Blockade.

Simple SummaryImmune checkpoint blockade (ICB) therapies are attracting much attention for the clinical treatment of tumors. Combination therapies are being developed to enhance the effects of ICBs, and the importance of tumor-draining lymph nodes (TDLNs) has been reevaluated in antitumor responses via ICB therapy. The migration and motility status of T cells and their functional interaction with antigen-bearing dendritic cells are key factors for inducing adaptive immunity in LNs. Although immune checkpoint molecules are known to regulate T cell motility, their actual influence on T cell dynamics in TDLNs, particularly in ICBs, is poorly understood. In this review, we summarize the relevance of T cell dynamics and immune responses in draining LNs, and discuss the alteration of T cell motility under ICB. To develop better immunotherapies using ICBs, studying T cell dynamics in TDLNs can provide a good indicator to evaluate the efficacy of antitumor therapy.The migration status of T cells within the densely packed tissue environment of lymph nodes reflects the ongoing activation state of adaptive immune responses. Upon encountering antigen-presenting dendritic cells, actively migrating T cells that are specific to cognate antigens slow down and are eventually arrested on dendritic cells to form immunological synapses. This dynamic transition of T cell motility is a fundamental strategy for the efficient scanning of antigens, followed by obtaining the adequate activation signals. After receiving antigenic stimuli, T cells begin to proliferate, and the expression of immunoregulatory receptors (such as CTLA-4 and PD-1) is induced on their surface. Recent findings have revealed that these ‘immune checkpoint’ molecules control the activation as well as motility of T cells in various situations. Therefore, the outcome of tumor immunotherapy using checkpoint inhibitors is assumed to be closely related to the alteration of T cell motility, particularly in tumor-draining lymph nodes (TDLNs). In this review, we discuss the migration dynamics of T cells during their activation in TDLNs, and the roles of checkpoint molecules in T cell motility, to provide some insight into the effect of tumor immunotherapy via checkpoint blockade, in terms of T cell dynamics and the importance of TDLNs.

IL7-Fc Enhances the Efficacy of Adoptive T Cell Therapy under Lymphopenic Conditions in a Murine Melanoma Model.

Adoptive cell therapy (ACT) using tumor-reactive T cells is a promising form of immunotherapy to specifically target cancer. However, the survival and functional maintenance of adoptively transferred T cells remains a challenge, ultimately limiting their efficacy. Here, we evaluated the use of recombinant IL7-Fc in ACT. In a lymphopenic murine melanoma model, IL7-Fc treatment led to the enhanced inhibition of tumor growth with an increased number of adoptively transferred CD8+ T cells in tumor tissue and tumor-draining lymph nodes. Additionally, IL7-Fc further enhanced anti-tumor responses that were induced by recombinant human IL2 in the same mouse model. In contrast, in an immunocompetent murine melanoma model, IL7-Fc dampened the anti-tumor immunity. Further, IL7-Fc decreased the proliferation of adoptively transferred and immune-activated tumor-reactive CD8+ T cells in immunocompetent mice by inducing the massive expansion of endogenous T cells, thereby limiting the space for adoptively transferred T cells. Our data suggest that IL7-Fc is principally beneficial for enhancing the efficacy of tumor-reactive T-cells in lymphopenic conditions for the ACT.

Both T cell priming in lymph node and CXCR3-dependent migration are the key events for predicting the response of atezolizumab

Anti-PD-L1 antibodies benefit many cancer patients, even those with “non-inflamed tumor”. Determining which patients will benefit remains an important clinical goal. In a non-inflamed tumor mouse model, we found that PD-L1 was highly expressed on antigen-presenting cells (APCs) especially on CD103+ CD11c+ dendritic cells in tumor-draining lymph nodes (dLNs), suppressing T-cell priming by APCs. In this model, anti-PD-L1 antibodies enhanced T-cell priming and increased CXCR3+ activated T-cells in dLNs, which was followed by the trafficking of T-cells to tumors in response to CXCR3 ligands. As predictive biomarker, each APCs-related gene expression (AP score) alone or T-cells trafficking-related chemokine gene expression (T score) alone were still less than perfect among the 17 mouse models examined. However a combining score of AP score and T score (AP/T score) precisely identified anti-PD-L1-sensitive tumors. In the phase 3 trial of atezolizumab vs docetaxel in advanced NSCLC patients (OAK), the AP/T score could identify atezolizumab-treated NSCLC patients who achieved significant improvement in overall survival. This biomarker concept would be a clinically valuable for prediction of anti-PD-L1 antibody efficacy.

Abstract 515: Heterogeneity of PD-1hi CD8 T cells associates with response to PD-1 blockade in hepatocellular carcinoma

Abstract Blockade of the PD-1 pathway is a therapeutic strategy to reinvigorate T cell responses against tumors, and when combined with other biologic therapies in the first line setting this achieves significant clinical response in about 25% of hepatocellular carcinoma (HCC) patients. We hypothesize that phenotypic diversity of tumor infiltrating T cells can explain, at least partially, the disparate clinical responses to immunotherapy. Here, we analyze the molecular diversity of T cells in tumor, adjacent tissue and tumor-draining lymph node (dLN) by single-cell RNA sequencing of tissue from 23 patients with early stage HCC treated by neoadjuvant PD-1 blockade (NCT03916627; final data to be submitted to AACR 2021 as late breaking abstract). We identify distinct subsets of PD-1hi CD8 T cells with varying degrees of exhaustion and effector gene programs. Compared to parallel analysis of untreated HCC tumors, we observed that PD-1 blockade resulted in expansion of PD-1hi CD8 T cells in the tumor, regardless of clinical response. PD-1hi CD8 T cell subsets were highly clonal and enriched in the tumor compared to adjacent tissue, suggesting specificity to tumor antigens. Remarkably, we find an association between a specific transcriptomic phenotype within PD-1hi CD8 T cells and response to PD-1 blockade. Using T cell receptor (TCR) sequencing to study the differentiation patterns between T cell states, we found that expanded clonotypes present among PD-1hi CD8 T cells were also found in CD8 effector cells; these data identify characteristic clonally related T cell populations that are enriched in clinical responders. Furthermore, we find the dLNs harbor clonotypes of PD-1hi CD8 T cells expanded in the tumor. These clonally expanded CD8 T cells in dLN display features of activation and exhaustion, suggesting a continuous role of dLN in anti-tumor responses. These results will be corroborated with 8 additional patient samples in which we will further analyze the molecular characteristics of tumor-specific CD8 T cells in the TME and dLN. Furthermore, our ongoing sequencing of pre- and post-treatment lesions will enable measurement of CD8 T cell clonal expansion and molecular programs during response or resistance to PD-1 blockade. These results should help unravel the contribution of dLN T cells to response to immunotherapy as well as identify T cell molecular programs at baseline that correlate with response. Citation Format: Assaf Magen, Pauline Hamon, Etienne Humblin, Alessandra Schanoski, Joel Kim, Jessica Le Berichel, Ephraim Kenigsberg, Myron Schwartz, Thomas Marron, Alice Kamphorst, Miriam Merad. Heterogeneity of PD-1hi CD8 T cells associates with response to PD-1 blockade in hepatocellular carcinoma [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 515.

Abstract 1852: A novel PD1-CD40 bispecific antibody YH008 induces potent anti-tumor activity in vivo by PD1 dependent activation of CD40 signaling

Abstract Immunotherapies that target PD1/PDL1 pathway have marked a new era of cancer therapy. Combinatorial treatment of inhibiting PD1/PDL1 signaling and other immunomodulating agents has been widely tested and validated as a better strategy compared to single agent treatment. CD40 (TNFRSF5) is a member of the TNFR super family, a key T cell co-stimulatory signal critical for B-cell maturation and immunoglobulin class switching. Biocytogen developed a novel first in class PD1-CD40 bispecific antibody (YH008) that agonizes CD40 conditionally upon PD1 binding. In vitro study data shows YH008 can block the interaction of PD1 to PDL1 and induced dose dependent NFAT activation. YH008 was able to activate human CD40 on reporter cells in the presence of PD1-expressing jurkat cells in a dose dependent manner without relying on FcγR expressing cells. This feature is beneficial for conditionally activation of CD40+ cells in tumor microenvironment and tumor-draining lymph nodes, where tumor specific PD1+ T cells are enriched. In vivo study result shows that YH008 demonstrated robust anti-tumor efficacy against MC38 and B16F10 tumors in PD1/CD40 double humanized mice (B-hPD1/hCD40). In hCD40 mice which has no human PD1 engagement, YH008 did not show anti-tumor activity against MC38 tumor. Furthermore, YH008 did not elevate liver enzymes of MC38 bearingB-hPD1/hCD40 mice at dose as high as 26 mg/kg, demonstrating improved liver safety profile than selicrelumab. Taken together, these data demonstrated that YH008 is a promising new immunotherapeutic candidate in cancer treatment. Citation Format: Baihong Liu, Li Hui, Chonghui Liu, Zhihong Li, Fangxia Pan, Benny Yang, Qingcong Lin. A novel PD1-CD40 bispecific antibody YH008 induces potent anti-tumor activity in vivo by PD1 dependent activation of CD40 signaling [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 1852.

Lymphatic PD-L1 Expression Restricts Tumor-Specific CD8+ T-cell Responses.

Lymph node (LN)-resident lymphatic endothelial cells (LEC) mediate peripheral tolerance by self-antigen presentation on MHC-I and constitutive expression of T-cell inhibitory molecules, including PD-L1 (CD274). Tumor-associated LECs also upregulate PD-L1, but the specific role of lymphatic PD-L1 in tumor immunity is not well understood. In this study, we generated a mouse model lacking lymphatic PD-L1 expression and challenged these mice with two orthotopic tumor models, B16F10 melanoma and MC38 colorectal carcinoma. Lymphatic PD-L1 deficiency resulted in consistent expansion of tumor-specific CD8+ T cells in tumor-draining LNs in both tumor models, reduced primary tumor growth in the MC38 model, and increased efficacy of adoptive T-cell therapy in the B16F10 model. Strikingly, lymphatic PD-L1 acted primarily by inducing apoptosis in tumor-specific CD8+ central memory T cells. Overall, these findings demonstrate that LECs restrain tumor-specific immunity via PD-L1, which may explain why some patients with cancer without PD-L1 expression in the tumor microenvironment still respond to PD-L1/PD-1-targeted immunotherapy. SIGNIFICANCE: A new lymphatic-specific PD-L1 knockout mouse model reveals that lymphatic endothelial PD-L1 expression reduces tumor immunity, inducing apoptosis in tumor-specific CD8+ central memory cells in tumor-draining lymph nodes.

Diet-Induced Obesity Impairs Outcomes and Induces Multi-Factorial Deficiencies in Effector T Cell Responses Following Anti-CTLA-4 Combinatorial Immunotherapy in Renal Tumor-Bearing Mice.

Simple SummaryImmunotherapy use has become standard for many patients with advanced kidney cancer; unfortunately, <50% of patients experience durable responses. Mounting evidence suggests that modifiable factors, such as diet and obesity, impact immunotherapy outcomes. Obesity, a major U.S. health epidemic, blunts anti-tumor immunity and promotes tumor growth in multiple preclinical models. However, the full biological impact of obesity on the T cell responses needed to achieve positive immunotherapy outcomes remains unclear. Here, we studied the effects of obesity on T cell responses following combinatorial immunotherapy in a mouse model of kidney cancer. We found that obesity is associated with blunted effector T cell responses, resulting in diminished immunotherapy outcomes. This therapy produces sustained T cell responses and robust tumor control in obese-resistant mice fed the same high-fat diet. Finding ways to amplify T cell responses within renal tumors from hosts with obesity will be critical for achieving optimal immunotherapy outcomes.Associations between modifiable factors and the efficacy of cancer immunotherapies remain uncertain. We found previously that diet-induced obesity (DIO) reduces the efficacy of an immunotherapy consisting of adenovirus-encoded TRAIL plus CpG oligonucleotide (AdT/CpG) in mice with renal tumors. To eliminate confounding effects of diet and determine whether outcomes could be improved in DIO mice, we evaluated AdT/CpG combined with anti-CTLA-4 in diet-matched, obese-resistant (OB-RES) versus DIO tumor-bearing mice. Therapy-treated OB-RES mice displayed effective renal tumor control and sustained CD4+ and CD8+ T cell responses. In contrast, therapy-treated DIO mice exhibited progressive tumor outgrowth and blunted T cell responses, characterized by reduced intratumoral frequencies of IFNγ+ CD4+ and CD8+ T cells. Weak effector T cell responses in therapy-treated DIO mice were accompanied by low intratumoral concentrations of the T cell chemoattractant CCL5, heightened concentrations of pro-tumorigenic GM-CSF, and impaired proliferative capacity of CD44+CD8+ T cells in tumor-draining lymph nodes. Our findings demonstrate that in lean mice with renal tumors, combining in situ T cell priming upstream of anti-CTLA-4 enhances outcomes versus anti-CTLA-4 alone. However, host obesity is associated with heightened immunotherapy resistance, characterized by multi-factorial deficiencies in effector CD4+ and CD8+ T cell responses that extend beyond the tumor microenvironment.

The aryl hydrocarbon receptor suppresses immunity to oral squamous cell carcinoma through immune checkpoint regulation

Immune checkpoint inhibitors represent some of the most important cancer treatments developed in the last 20 y. However, existing immunotherapy approaches benefit only a minority of patients. Here, we provide evidence that the aryl hydrocarbon receptor (AhR) is a central player in the regulation of multiple immune checkpoints in oral squamous cell carcinoma (OSCC). Orthotopic transplant of mouse OSCC cells from which the AhR has been deleted (MOC1AhR-KO) results, within 1 wk, in the growth of small tumors that are then completely rejected within 2 wk, concomitant with an increase in activated T cells in tumor-draining lymph nodes (tdLNs) and T cell signaling within the tumor. By 2 wk, AhR+ control cells (MOC1Cas9), but not MOC1AhR-KO cells up-regulate exhaustion pathways in the tumor-infiltrating T cells and expression of checkpoint molecules on CD4+ T cells (PD-1, CTLA4, Lag3, and CD39) and macrophages, dendritic cells, and Ly6G+ myeloid cells (PD-L1 and CD39) in tdLNs. Notably, MOC1AhR-KO cell transplant renders mice 100% immune to later challenge with wild-type tumors. Analysis of altered signaling pathways within MOC1AhR-KO cells shows that the AhR controls baseline and IFNγ-induced Ido and PD-L1 expression, the latter of which occurs through direct transcriptional control. These observations 1) confirm the importance of malignant cell AhR in suppression of tumor immunity, 2) demonstrate the involvement of the AhR in IFNγ control of PD-L1 and IDO expression in the cancer context, and 3) suggest that the AhR is a viable target for modulation of multiple immune checkpoints.

CD8 T cell differentiation in cancer is comprised of two distinct phases

Abstract A crucial part of the immune response to tumors are CD8 T cells, with CD8 infiltration predicting disease progression in many cancers. Recent work has shown two subsets of CD8 T cells that respond to tumors, one a stem-like CD8 T cell (TCF1+) that can give rise to a more cytotoxic terminally differentiated cell (TCF1−). To understand the CD8 T cell response to tumors it is important to study how tumor-specific CD8 T cells activate and differentiate. To study this we have made a prostate cancer model which expresses the LCMV glycoprotein (GP) and acts as a tumor-specific antigen. This model allows us to transfer LCMV GP specific P14 CD8 T cells into TRAMPC1-GP bearing mice to study how tumor-specific CD8 T cells activate. These studies have shown that tumor-specific CD8 T cells are activated in tumor draining lymph nodes (TDLN), where they retain an activated undifferentiated phenotype, upregulating CD44, PD1, while maintaining TCF1. These tumor-specific CD8 T cells only differentiate (TCF1−) once they have migrated into the tumor. This model can also be seen in human prostate cancer, with CD8 T cells in TDLNs retaining an activated undifferentiated phenotype (PD1+CD45RA-TCF1+). To determine if these cells are related to the CD8s within the tumor we have shown TCR overlap between the activated CD8s T cells in human prostate TDLNs and the CD8 T cell subsets within the tumor. These data suggest a two-step differentiation process for tumor-specific CD8 T cells where they are activated in TDLNs and differentiate further only in the tumor. This model of two distinct phases of CD8 T cell differentiation adds to the basic understanding of the immune response to cancer.

CD4+ and CD8+ T cells in sentinel nodes exhibit distinct pattern of PD-1, CD69, and HLA-DR expression compared to tumor tissue in oral squamous cell carcinoma.

Anticancer immunotherapies have revolutionized cancer management, yet the effect of systemic anti‐programmed cell death protein 1 (PD‐1) treatment is predominantly studied in tumor‐infiltrating lymphocytes (TILs). Its impact on PD‐1 expressing cells in tumor‐draining lymph nodes (TDLNs) is not well understood and yet to be explored. Thus, further research aiming for better understanding of the PD‐1 pathway not only in tumor tissue but also in TDLNs is warranted. In this study, we investigated the expression of PD‐1, CD69, and HLA‐DR on CD4+ and CD8+ T cells by flow cytometry analysis of peripheral blood mononuclear cells (PBMCs), TDLNs, and tumor samples from patients with oral squamous cell carcinoma (OSCC). Our data showed that both helper and cytotoxic T lymphocytes in OSCC tissue were highly activated and expressed high level of PD‐1 (over 70% positivity). Lymphocytes in TDLNs and peripheral blood expressed significantly lower levels of PD‐1 and other activation markers compared to TILs. Moreover, we demonstrated that a significant fraction of PD‐1 negative TILs expressed high levels of human leukocyte antigen – DR isotype and CD69. In contrast, PD‐1 negative cells in TDLNs and PBMCs scarcely expressed the aforementioned activation markers. Furthermore, we proved that patients with a high percentage of CD3+ PD‐1+ cells in tumor‐draining lymph nodes had significantly lower disease‐free and overall survival rates (log‐rank test P = .0272 and P = .0276, respectively). Taken together, we proved that flow cytometry of lymph nodes in OSCC is feasible and may be used to investigate whether PD‐1 levels in TDLNs correspond with survival and potentially with response to anti‐PD‐1 therapy. Such knowledge may ultimately help guide anti‐PD‐1 treatment.