Inhibitory Checkpoint Molecules Research Articles

Next generation CD44v6-specific CAR-NK cells for the treatment of solid tumors.

e14518 Background: There is a medical need to develop new and effective therapies against solid tumors. CD44v6, an adhesion molecule that is implicated in tumorigenesis and metastases, was identified as a suitable target in solid tumors such as triple negative breast cancer (TNBC) or glioblastoma. Methods: We have developed a next-generation CAR targeting CD44v6 that incorporates IL-15 superagonist and checkpoint inhibitor molecules to overcome the immunosuppressive microenvironment (TME). NK cells were used due to their potential for allogeneic usage. Results: CD44v6 CAR-NK cells demonstrated effective cytotoxicity against TNBC and glioblastoma in 3D spheroid models. The IL-15 superagonist was specifically released upon recognition of CD44v6 on TNBC and contributed to the cytotoxic attack. Competitive inhibition of PD1 neutralized inhibition by PD1 ligands. We have validated iPSC-derived CD44v6 CAR-NK cells. Conclusions: Next generation CD44v6 CAR-NK cells are resistant to TME immunosuppression and offer a new therapeutic option for the treatment of solid tumors.

Abstract LB070: In vitro modeling of antigen-driven exhaustion in human CAR T cells

Abstract A major hurdle for chimeric antigen receptor (CAR) T cells to function in solid tumors is chronic antigen exposure leading to terminal lymphocyte exhaustion and impaired anti-tumor activity. Therefore, it is necessary to establish improved in vitro models of antigen-driven CAR T cell exhaustion to evaluate new CAR design candidates prior to validation with costly in vivo studies. Using the human tumor antigen mesothelin (MSLN) and MSLN-targeting CAR T cells as a model, we developed two in vitro assays that drive an exhausted phenotype in human CAR T cells and can be used to predict anti-tumor responses in vivo: 1) a chronic stimulation assay with plate-bound recombinant antigen (MSLN), ICAM-1, and CD58 and 2) a high antigen-expressing 3D tumor spheroid co-culture assay. In the chronic antigen stimulation assay, repetitive exposure to plate-bound MSLN (alongside co-stimulation signals provided by recombinant ICAM-1 and CD58) reprogrammed the transcriptomic profile of MSLN CAR T cells leading to increased surface expression of inhibitory checkpoint molecules and reduced cytokine production. Moreover, chronically stimulated MSLN CAR T cells demonstrated a drastically impaired ability to kill MSLN+ tumor cells compared to their unstimulated or acutely stimulated counterparts. A similar exhausted phenotype was observed in the high-antigen tumor spheroid model: when MSLN CAR T cells were co-cultured with MSLN-high human NCI-H226 spheroids for 7 days in a 96-well plate, expression of inhibitory checkpoint molecules markedly increased on the surface of CAR T cells. This increased expression of exhaustion markers coincided with impairment of NCI-H226 spheroid clearance. By contrast, other tumor spheroid models expressing low to intermediate levels of MSLN (NCI-H1650, NCI-H2052, NCI-H292, and SKOV-3 tumor cells) did not substantially induce expression of inhibitory checkpoint molecules and the spheroids were successfully cleared by MSLN CAR T cells. Importantly, the ability (or inability) of a CAR construct to clear NCI-H226 high-antigen tumor spheroids in vitro accurately predicted CAR T cell performance in in vivo solid tumor studies: the CAR construct that showed effective killing in the in vitro spheroid model was the only condition to provide durable regression of NCI-H1650 tumors in a NSG xenograft mouse model. Additionally, the rank order of CAR efficacy for a series of MSLN CAR binders obtained using the tumor spheroid model mirrored in vivo efficacy. As the CAR T cell field develops novel strategies to combat antigen-driven exhaustion for use in solid tumors, these new antigen-dependent in vitro models of exhaustion can enable more cost-efficient and effective evaluation of those strategies than using in vivo models alone. Citation Format: Maria Steele, Tina Tan, Jason Yokoyama, Allan Wang, Ty Crowl, Jared Hammer, Jerry Chen, Kevin Haworth, Howell Moffett, Aaron Foster, Marc Lajoie, Scott Boyken, Rupesh Amin. In vitro modeling of antigen-driven exhaustion in human CAR T 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 LB070.

Abstract LB076: Targeting an aging-related molecule overcomes resistance to anti-PD1 treatment of cancer

Abstract Purpose: Despite receiving great attention as a promising treatment, therapeutic efficacies induced by immune checkpoint inhibitors are low in clinical settings, and thus there are high expectations for the development of innovative diagnostic and therapeutic methods. We previously demonstrated that blocking FSTL1 overcomes anti-PD1 resistance in aged mice. In this study, we comprehensively analyzed and compared gene expressions in splenic T cells harvested from young and old mice receiving anti-FSTL1 or anti-PD1 therapy after tumor implantation using the GeneChip microarray in order to identify a key gene in the anti-PD1 resistance. In addition, we evaluated anti-tumor efficacy induced by blocking the identified molecule using aged tumor models and tumor metastasis models (3LL or Colon26), which are resistant to anti-PD1 therapy. Furthermore, to validate the clinical relevancy, we analyzed immune cells isolated from malignant tumor ascites and peripheral blood cells of gastric cancer patients by flow cytometry. Results: Using the GeneChip microarray data of splenic T cells, we identified α-synuclein (SNCA), which is a neuropathological molecule involved in Parkinson's disease and other synucleinopathies, as a gene that was significantly reduced by anti-FSTL1 therapy, but not anti-PD1 therapy, in splenic T cells of aged tumor models. SNCA+ cell subsets significantly increased not only in CD3+ T cells, but also CD11b+ myeloid cells and CD45- mesenchymal stromal/stem cells, within spleen, peripheral blood, and tumor tissues of aged mice and mouse tumor models, but rarely in those of young and naive mice. Increase of the subsets were also observed in malignant tumor ascites and peripheral blood of gastric cancer patients, but not those of healthy donors. Blocking SNCA with the specific mAb significantly induced potent CD8+ T cells with greater cytotoxicity in the in vitro CTL induction setting, and also significantly induced robust anti-tumor immunity in the in vivo therapeutic setting with anti-PD1-resistant tumor models. Combination of anti-SNCA therapy successfully elicited anti-PD1 therapeutic efficacy, providing significantly better prognosis in many tumor models, including severe tumor ascites models. Conclusions: These results suggest that SNCA is a type of immune inhibitory checkpoint molecules and an immunological determinant of anti-PD1 resistance, although the further study is needed to clarify the molecular functions in immune cells. Targeting this molecule may represent a promising strategy to overcome the anti-PD1 resistance and contribute to improved clinical outcomes in cancer treatment. Citation Format: Chie Kudo-Saito, Hiroshi Imazeki, Hirokazu Shoji, Narikazu Boku. Targeting an aging-related molecule overcomes resistance to anti-PD1 treatment of cancer [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 LB076.

Therapeutic potential of interleukin-21 in cancer.

Interleukin-21 (IL-21) is an immunostimulatory cytokine which belongs to the common gamma-chain family of cytokines. It plays an import role in the development, differentiation, proliferation, and activation of immune cells, in particular T and natural killer (NK) cells. Since its discovery in 2000, IL-21 has been shown to regulate both adaptive and immune responses associates with key role in antiviral and antitumor responses. Recent advances indicate IL-21 as a promising target for cancer treatment and encouraging results were obtained in preclinical studies which investigated the potency of IL-21 alone or in combination with other therapies, including monoclonal antibodies, checkpoint inhibitory molecules, oncolytic virotherapy, and adoptive cell transfer. Furthermore, IL-21 showed antitumor effects in the treatment of patients with advanced cancer, with minimal side effects in several clinical trials. In the present review, we will outline the recent progress in IL-21 research, highlighting the potential of IL-21 based therapy as single agent or in combination with other drugs to enhance cancer treatment efficiency.

The role of tissue-resident memory T cells as mediators for response and toxicity in immunotherapy-treated melanoma-two sides of the same coin?

Tissue-resident memory T cells (TRM cells) have become an interesting subject of study for antitumor immunity in melanoma and other solid tumors. In the initial phases of antitumor immunity, they maintain an immune equilibrium and protect against challenges with tumor cells and the formation of primary melanomas. In metastatic settings, they are a prime target cell population for immune checkpoint inhibition (ICI) because they highly express inhibitory checkpoint molecules such as PD-1, CTLA-4, or LAG-3. Once melanoma patients are treated with ICI, TRM cells residing in the tumor are reactivated and expand. Tumor killing is achieved by secreting effector molecules such as IFN-γ. However, off-target effects are also observed. Immune-related adverse events, such as those affecting barrier organs like the skin, can be mediated by ICI-induced TRM cells. Therefore, a detailed understanding of this memory T-cell type is obligatory to better guide and improve immunotherapy regimens.

CAR-mediated targeting of NK cells overcomes tumor immune escape caused by ICAM-1 downregulation

BackgroundThe antitumor activity of natural killer (NK) cells can be enhanced by specific targeting with therapeutic antibodies that trigger antibody-dependent cell-mediated cytotoxicity (ADCC) or by genetic engineering to express chimeric...

Abstract B021: Clonal heterogeneity in human pancreatic adenocarcinoma and its contribution to therapeutic resistance

Abstract PDAC is a highly aggressive cancer with poor prognosis, largely due to its late diagnosis, rapid progression, and lack of responsiveness to standard therapies. The advent of large-scale genomics, single-cell RNA sequencing (scRNA-seq) and spatial transcriptomics (ST) has illuminated potential genomic targets, the intricate role of the PDAC tumor microenvironment (TME), and gene activity orchestrating cellular arrangements within cancer tissues. In recent work by our group, we characterized the composition of human PDAC at the single-cell level, revealing significant intra-tumoral heterogeneity in transcriptional subtypes and TME composition. We also found that chemotherapy profoundly alters the PDAC TME and may lead to further resistance to immunotherapy by reduced inhibitory checkpoint molecule expression and interactions involving CD8+ T cells. We have since determined that specific clones in human PDAC tumors have unique influences on the biological and clinical features of PDAC. In this study, we tested the hypothesis that the distinct genomic profiles of individual clones within PDAC tumors influence their transcriptional state and their interaction with the TME, ultimately governing disease progression and response to chemotherapy. scRNA-seq analysis was performed on 61 freshly collected human primary PDAC samples from 58 patients, either prior to or following chemotherapy treatment, including 3 matched pre- and post- treatment pairs from the same patient. CNV analysis of PDAC samples revealed significant inter- and intra- tumoral clonal heterogeneity. Using the results of differential gene expression and gene set enrichment analysis, we identified a set of common transcriptional states associated with specific CNV profiles. Further, we found that specific PDAC clones often correlated with classical and basal subtypes. Using a combination of ligand-receptor-interaction (LRI) analysis and spatial transcriptomics, we observed that distinct clones within tumors exhibited heterogeneous interactions with different cell types in the TME, and that the composition of individual clones within tumors and their LRI were significantly and differentially altered in response to chemotherapy. Our findings indicate the presence of distinct clonal neighborhoods in PDAC, and allow interrogation of clonal and cell communication network changes that occur in response to chemotherapy. This data provide new insight into potential mechanisms of treatment resistance and may help identify potential strategies to intercept or more effectively treat metastasis and improve patient outcomes. Citation Format: Despoina Kalfakakou, Emily A. Kawaler, Dylan Tamayo, Lidong Wang, Rosmel Hernandez, Amanda Ackermann, Xiaohong Jing, Cristina Hajdu, Cynthia Loomis, Adrianna Heguy, Amanda W. Lund, Theodore H. Welling, Igor Dolgalev, Aristotelis Tsirigos, Diane M. Simeone. Clonal heterogeneity in human pancreatic adenocarcinoma and its contribution to therapeutic resistance [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B021.

Blocking the CTLA-4 and PD-1 pathways during pulmonary paracoccidioidomycosis improves immunity, reduces disease severity, and increases the survival of infected mice.

Immune checkpoint pathways, i.e., coinhibitory pathways expressed as feedback following immune activation, are crucial for controlling an excessive immune response. Cytotoxic T lymphocyte antigen-4 (CTLA-4) and programmed cell death protein-1 (PD-1) are the central classical checkpoint inhibitory (CPI) molecules used for the control of neoplasms and some infectious diseases, including some fungal infections. As the immunosuppression of severe paracoccidioidomycosis (PCM), a chronic granulomatous fungal disease, was shown to be associated with the expression of coinhibitory molecules, we hypothesized that the inhibition of CTLA-4 and PD-1 could have a beneficial effect on pulmonary PCM. To this end, C57BL/6 mice were infected with Paracoccidioides brasiliensis yeasts and treated with monoclonal antibodies (mAbs) α-CTLA-4, α-PD-1, control IgG, or PBS. We verified that blockade of CTLA-4 and PD-1 reduced the fungal load in the lungs and fungal dissemination to the liver and spleen and decreased the size of pulmonary lesions, resulting in increased survival of mice. Compared with PBS-treated infected mice, significantly increased levels of many pro- and anti-inflammatory cytokines were observed in the lungs of α-CTLA-4-treated mice, but a drastic reduction in the liver was observed following PD-1 blockade. In the lungs of α-CPI and IgG-treated mice, there were no changes in the frequency of inflammatory leukocytes, but a significant reduction in the total number of these cells was observed. Compared with PBS-treated controls, α-CPI- and IgG-treated mice exhibited reduced pulmonary infiltration of several myeloid cell subpopulations and decreased expression of costimulatory molecules. In addition, a decreased number of CD4+ and CD8+ T cells but sustained numbers of Th1, Th2, and Th17 T cells were detected. An expressive reduction in several Treg subpopulations and their maturation and suppressive molecules, in addition to reduced numbers of Treg, TCD4+, and TCD8+ cells expressing costimulatory and coinhibitory molecules of immunity, were also detected. The novel cellular and humoral profiles established in the lungs of α-CTLA-4 and α-PD-1-treated mice but not in control IgG-treated mice were more efficient at controlling fungal growth and dissemination without causing increased tissue pathology due to excessive inflammation. This is the first study demonstrating the efficacy of CPI blockade in the treatment of pulmonary PCM, and further studies combining the use of immunotherapy with antifungal drugs are encouraged.

Comprehensive Immunophenotyping by Polychromatic Cytometry.

The human immune system comprises a diverse array of cells involved in innate and adaptive immunity, and these immune cells coordinate immune responses against pathogens through intricate interactions. Multicolor flow cytometry is a powerful technique for qualitatively and quantitatively measuring the characteristics of immune cells, offering advantages, such as high-dimensional analysis, elucidation of cellular heterogeneity, understanding of pathogenesis, development of therapeutic strategies, and platform flexibility. Here, we demonstrate a new immunophenotyping panel that allows simultaneous evaluation of the characteristics of T and B cells. This panel enables tracking of changes in the immune status due to aging, environmental factors, pathogen infections, and vaccine administration. Additionally, it includes co-stimulatory molecules for assessing the activation state of immune cells and inhibitory checkpoint molecules for evaluating exhaustion status, thereby providing valuable insights into the features of human immune responses. These analyses contribute to understanding the pathophysiology of diseases and developing therapeutic strategies while offering crucial information for assessing the correlation of symptoms with infections and evaluating the efficacy of vaccines.

Fine Tuning Bispecific Activity in CLL: Harmonizing a CD19/20-T Cell Bispecific with a CD28 or 4-1BBL Costimulatory Bispecific

Introduction: T cell bispecific antibodies (TCBs), such as blinatumomab, mosunetuzumab or glofitamab, redirect T cells toward cancer cells and rely on endogenous T cell activation and proliferation (Boissel et al., Nature 2023). Physiological T cell activation not only requires signal 1 provided by TCR engagement but also a signal 2 delivered by positive costimulatory molecules. Like a first-generation CAR-T, TCBs only provide signal 1 through CD3 engagement. Hence, TCB-mediated T-cell activation depends on the expression profile of inhibitory and costimulatory checkpoint molecules expressed by tumor cells (Marcinek et al., Cancer Immunology 2023). As tumor cells often lack expression of positive co-stimulatory ligands like CD80, CD86 and 4-1BBL, we hypothesized that the combination of target-antigen specific TCBs with a co-stimulatory agonistic bispecific can increase TCB efficacy by promoting T cell activity and persistence. Methods: We first evaluated the expression level of CD80, CD86 and 4-1BBL on CD19 positive, primary CLL (pCLL) cells by multi parameter flow cytometry (MPFC, n = 6). Next, we co-cultured healthy donor T cells with pCLL cells at a ratio of 1:1 and 1:5 for 6 days in the presence of a CD19- or CD20-TCB in conjunction with a co-stimulatory CD19 directed bispecific providing either CD28 or 4-1BB co-stimulation. T-cell proliferation and cytotoxicity against pCLL cells were analysed by MPFC at day 3 and day 6. Cytokine secretion (IFNγ, IL-2, TNFα) was assessed by cytokine bead array. To further dissect the impact of positive co-stimulation in counteracting evolving TCB-mediated T cell exhaustion, an in vitro model system was used. For this, healthy donor T cells were co-cultured with the lymphoma cell line OCI-Ly1 and continuously stimulated with a CD20 directed TCB over 28 days. The exhausted T cells from different time points were co-cultured with pCLL cells and stimulated with CD19 directed costimulatory bispecifics (CD19-CD28 or CD19-4-1BBL) for the assessment of T-cell fitness. Results: The phenotype profile of pCLL cells showed only dim expression of various positive costimulatory molecules (CD80: 0.5 %, CD86: 14.4 %, 4-1BBL: 9.7 %). Looking at the impact of positive costimulatory bispecifics in short term cultures with healthy donor T cells and pCLL cells, a significant increase in CD19 and CD20-TCB-mediated cytotoxicity was observed through the addition of CD19-CD28 and CD19-4-1BBL bispecific (CD19-TCB vs CD19-TCB + CD19-CD28: 29.9 % vs 56.1 %, p = 0.0014, CD20-TCB vs CD20-TCB + CD19-CD28: 30.2 % vs 64.8 %, p = 0.0112, CD20-TCB vs CD20-TCB + CD19-4-1BBL: 30.2 % to 50.4 %). In line with these findings, T-cell proliferation increased significantly through the addition of the costimulatory bispecifics (CD19-TCB vs CD19-TCB + CD19-CD28: 12.1 % vs 50.0 %, p = 0.0009; CD20-TCB vs CD20-TCB + CD19-CD28: 24.7 % vs 51.8 %, day 3, E:T = 1:1; CD19-TCB vs CD19-TCB + 4-1BBL: 30.5 % vs 63.2 %, p = 0.0048, day 6, E:T = 1:5). Interestingly, the secretion of IFNy, IL-2 and TNFα was mainly increased through the addition of the CD19-CD28 bispecific (IFNy: 141.6 vs 3151.9 pg/mL, IL-2: 21.5 vs 2311.1 pg/mL, TNFα: 60.8 vs 1945.0 pg/mL, n = 4). Importantly, exhausted T cells exposed to CD20-TCB for 28 days in our long-term culture were characterized as dysfunctional showing i.e. an increased expression of exhaustion markers (LAG3 +PD1 +Tim3 + 37.4 % to 70.4 %, day 3 vs day 28) and a reduced cytokine secretion ability (IFNγ: 7949.9 to 693.5 pg/mL, day 3 vs day 24). T cell fitness could be reinvigorated in follow up short term assays through the addition of both CD19 directed costimulatory bispecifics. Specifically, exhausted T cells stimulated with CD20-TCB and CD19-CD28 or CD19-4-1BBL showed a significant increase in TCB-mediated cytotoxicity against primary pCLL cells (CD20-TCB vs CD19-CD28: 15.5 % to 37.7 %, p = 0.0109, CD20-TCB vs CD19-4-1BBL: 15.5 % to 32.9 %, n = 3). Conclusion: In conclusion, our data demonstrates the impact of positive co-stimulation on TCB-mediated T cell function. Costimulatory bispecifics provide co-stimulation to T cells in cases of missing expression of costimulatory molecules on tumor cells as well as improving function of exhausted T cells. Clinical trials will be needed to assess the impact of combinatorial strategies to improve T-cell efficacy and to overcome resistance.

Mezigdomide Treatment in Relapsed-Refractory Myeloma Patients Shifts Bone Marrow NK and T Cell Populations from Exhaustion to Activation

Background The effect of immunomodulatory drugs (IMiDs) and novel cereblon E3 ligase modulators (CELMoDs) on the tumour microenviroment (TME) of multiple myeloma (MM) patients remains poorly understood. A recent multi-center phase 1/2 trial (CC-92480-MM-001) of CELMoD Mezigdomide (MEZI) demonstrated clinical response in triple-class refractory, heavily pre-treated MM patients (Richardson et al, EHA abstracts 2020). Here, we report results from high dimensional mass cytometry immune profiling of baseline and treatment samples from relapsed-refractory MM (RRMM) patients to understand the effect of MEZI on the bone marrow (BM) TME. In addition, we analyze baseline samples to evaluate BM immune phenotypes that may correlate with response to MEZI. Methods We identified 45 RRMM patients with paired bone marrow aspirate samples pre- (screening) and post- (cycle 2 onwards) MEZI treatment, and 32 additional patients with isolated screening samples. Of the 45 patients, 39 received a dose of 0.8-1.0mg daily for 14 or 21 days of a 28-day cycle, 6 received 0.2-0.6mg on varying schedules as part of the trial design. CD138-negative BM mononuclear cells were isolated from these patients and profiled at single-cell resolution using a previously described 39-marker TME CyTOF-panel (van Oekelen, ASH 2021). Both manual hierarchical gating and unsupervised computational workflows were used for CyTOF data analysis. Around 5 million total cells were analyzed and over 100 immune populations. Samples with <2500 total events were excluded for quality-control. Population frequencies used for comparison are reported as median percentage of the parent population. Statistical comparisons between groups were assessed using the Wilcoxon test. Results Upon MEZI treatment, both adaptive and innate BM immune compartments demonstrated a significant increase in activated populations and marked reduction in exhausted/senescent populations (Figure 1A). Total NK and NKT populations expanded on MEZI (p<0.01 for both). Both CD4 and CD8 T cell populations shifted towards an effector phenotype with significant increases in effector memory (CCR7-CD45RA-) populations (p<0.001 for both) and concurrent reductions of and naïve (p<0.001), central memory (p<0.01 for CD4, NS for CD8) and TEMRA populations (p<0.001). HLADR-expressing CD4, CD8 and NKT populations significantly increased on MEZI (p<0.001 for all) as did ICOS-expressing CD4 (p<0.01), NK (p<0.001) and NKT cells (p<0.05). Senescent NK, NKT, CD4 and CD8 populations expressing KLRG1 decreased on MEZI treatment (p<0.01 for all) and CD57 positive CD8 and NKT cell populations also reduced (p<0.05). CD8, NK and NKT cells expressing the inhibitory checkpoint molecule TIGIT, showed a near 3-fold reduction of on treatment (p<0.001 for all). Although PD1-positive populations did not change on MEZI, PD1/TIGIT double-positive CD4 and CD8 populations showed significant reduction (p<0.05 for both). A dose-dependent reduction of TIGIT-expressing CD8 and NKT populations was seen at higher doses of MEZI (p<0.05 for both), suggesting a direct effect of MEZI on the BM TME. We further correlated depth-of-response with baseline immune composition in 77 patients (Figure 1B). Patients with progression of disease (n=9) on MEZI, when compared to all other patients (n=68), were found to have higher frequencies of PD1 (p<0.05) and TIGIT (p<0.001) positive, as well as dual PD1 and TIGIT positive (p<0.001), CD8 and NKT populations. Number of prior lines (median = 6) and type of MM therapy prior to MEZI did not significantly affect baseline PD1 or TIGIT positive populations. Conclusion MEZI treatment leads to significant activation of adaptive and innate BM immune populations with concurrent reduction of exhausted and senescent populations. These findings are concordant with prior observations of IMiD-mediated immune-stimulation. Patients progressing through MEZI had higher levels of immune checkpoint expression at baseline, which may be helpful for patient selection for this novel CELMoD. These results also suggest that MEZI may be useful in combination with other agents for increasing immune activation and reducing immune exhaustion. Further analyses comparing the effect of different CELMoDs on the BM TME are ongoing.

C-Met+ Cytotoxic T Lymphocytes Exhibit Enhanced Cytotoxicity in Mice and Humans In Vitro Tumor Models.

CD8+ cytotoxic T lymphocytes (CTLs) play a crucial role in anti-tumor immunity. In a previous study, we identified a subset of murine effector CTLs expressing the hepatocyte growth factor (HGF) receptor, c-Met (c-Met+ CTLs), that are endowed with enhanced cytolytic capacity. HGF directly inhibited the cytolytic function of c-Met+ CTLs, both in 2D in vitro assays and in vivo, leading to reduced T cell responses against metastatic melanoma. To further investigate the role of c-Met+ CTLs in a three-dimensional (3D) setting, we studied their function within B16 melanoma spheroids and examined the impact of cell-cell contact on the modulation of inhibitory checkpoint molecules' expression, such as KLRG1, PD-1, and CTLA-4. Additionally, we evaluated the cytolytic capacity of human CTL clones expressing c-Met (c-Met+) and compared it to c-Met- CTL clones. Our results indicated that, similar to their murine counterparts, c-Met+ human CTL clones exhibited increased cytolytic activity compared to c-Met- CTL clones, and this enhanced function was negatively regulated by the presence of HGF. Taken together, our findings highlight the potential of targeting the HGF/c-Met pathway to modulate CTL-mediated anti-tumor immunity. This research holds promise for developing strategies to enhance the effectiveness of CTL-based immunotherapies against cancer.

SLC22A3 that encodes organic cation transporter-3 is associated with prognosis and immunogenicity of human lung squamous cell carcinoma.

SLC22A3, the gene which encodes organic cation transporter (OCT)-3, has been linked to the prognosis of several types of cancer. However, its role in lung squamous cell carcinoma (LSCC) has not been addressed elsewhere. We analyzed gene expression, DNA methylation, and clinicopathological data from The Cancer Genome Atlas - Lung Squamous Cell Carcinoma (TCGA-LUSC) (n=501), a publicly available database exclusively consisting of LSCC patients. Using a 5 FPKM (fragments per kilobase of exon per million mapped fragments) cut-off, we divided LSCC patients into two groups: patients with tumors possessing high and low SLC22A3 expression (SLC22A3-high and SLC22A3-low, respectively). Prognostic significance was determined through Cox analyses and Kaplan-Meier curves for overall survival (OS) and disease-free survival (DFS). Differential methylation position (DMP), differentially gene expression, and pathway analyses were performed. Validation was carried out in GSE74777 (n=107), GSE37745 (n=66), GSE162520 (n=45) and GSE161537 (n=17). SLC22A3-high LSCC patients had lower OS and DFS rates than SLC22A3-low LSCC patients. The different expression levels of SLC22A3 in LSCC were correlated with the methylation status of the SLC22A3 gene. Pathway analysis indicated that SLC22A3 expression levels were positively correlated with immune-related pathways such as inflammatory response and abundance of infiltrating immune cells in the tumor microenvironment (TME). Notably, in the SLC22A3-high group, many genes encoding immunological checkpoint inhibitory molecules were upregulated. In addition, SLC22A3 expression positively correlated with the Hot Oral Tumor (HOT) score, indicating high tumor immunogenicity. These findings suggest that high expression of SLC22A3 is associated with poor prognosis and high immunogenicity in LSCC tumors.

Inhibitory checkpoint molecule mRNA expression in canine soft tissue sarcoma.

Canine soft tissue sarcomas (STS) are common neoplasms and considered immune deserts. Tumour infiltrating lymphocytes are sparse in STS and, when present, tend to organize around blood vessels or at the periphery of the neoplasm. This pattern is associated with an immunosuppressive tumour microenvironment linked to overexpression of molecules of the PD-axis. PD-1, PD-L1 and PD-L2 expression correlates with malignancy and poor prognosis in other neoplasms in humans and dogs, but little is known about their role in canine STS, their relationship to tumour grade, and how different therapies affect expression. The objective of this study was to evaluate the expression of checkpoint molecules across STS tumour grades and after tumour ablation treatment. Gene expression analysis was performed by reverse-transcriptase real-time quantitative PCR in soft tissue sarcomas that underwent histotripsy and from histologic specimens of STS from the Virginia Tech Animal Laboratory Services archives. The expression of PD-1, PD-L1 and PD-L2 was detected in untreated STS tissue representing grades 1, 2, and 3. Numerically decreased expression of all markers was observed in tissue sampled from the treatment interface relative to untreated areas of the tumour. The relatively lower expression of these checkpoint molecules at the periphery of the treated area may be related to liquefactive necrosis induced by the histotripsy treatment, and would potentially allow TILs to infiltrate the tumour. Relative increases of these checkpoint molecules in tumours of a higher grade and alongside immune cell infiltration are consistent with previous reports that associate their expression with malignancy.

Ex vivo treatment with poly (I:C) alleviates the exhausted phenotype of tumor-infiltrating TCD8+ cells of gastric cancer patients.

Gastric cancer is associated with the phenotypic and functional exhaustion of TCD8+ cells. On the other hand, Toll-like receptor (TLR) agonists are known to reinforce immune responses when used as adjuvants in cancer immunotherapies. Since the compromised signaling of pro-inflammatory pathways is usually associated with T cell exhaustion, the aim of the present study was to evaluate the impact of polyinosinic-polycytidylic acid (poly (I:C))-mediated TLR3 activation in restoring the normal phenotype and function of tumor-infiltrating TCD8+ cells. Peripheral blood and tumor-infiltrating TCD8+ cells of 35 gastric cancer patients were in vitro treated with increasing concentrations of poly (I:C) and the expressions of programmed death-1 (PD-1) and lymphocyte-activation gene 3 (LAG3) on these cells were examined. The peripheral TCD8+ cells of gastric cancer patients showed higher expressions of PD-1 and LAG3 along with lower proliferation compared to TCD8+ cells of the age-matched healthy control individuals. The in vitro treatment of TCD8+ cells with 100 μg/mL concentration of poly (I:C) alleviated the expression of PD-1 and LAG3 inhibitory checkpoint molecules on both peripheral and tumor-infiltrating TCD8+ cells. The mentioned dose of poly (I:C) improved the proliferation of TCD8+ cells in response to a polyclonal activator. Besides, the releases of Interferon gamma (IFN-γ) and Tumor necrosis factor alpha (TNF-α) were increased in the poly (I:C)-treated TCD8+ cells. Poly (I:C) demonstrated a potential to reduce the phenotypic and functional exhaustion of the peripheral and tumor-infiltrating TCD8+ cells and caused them to undergo more proliferation and cytokine release.

BSBM-14 MOLECULAR PROFILING OF CHECKPOINT LIGANDS AND KEY NK-ACTIVATION AND SUPPRESSIVE LIGANDS BY METASTATIC MELANOMA CELLS

Abstract Melanoma is an aggressive skin cancer with high propensity for brain metastasis. For advanced melanoma, immune checkpoint blockade (ICB) drugs, like pembrolizumab, have shown remarkable promise for progression-free survival. The ICB drugs have primarily targeted melanoma suppression of cytotoxic T-lymphocytes (CTLs). Earlier studies showed positive responses in up to 40% of cases, which has improved with discovery of new inhibitory ligands and combination therapy. However, not all patients respond, likely because of the capacity of cancer cells to regulate inhibitory and activating ligands, circumventing both immunomodulatory axes currently targeted by immunotherapies. In this study, we aim to characterize additional axes, prioritizing NK cells of the innate immune system. NK cells can mediate cytotoxic killing similar to CTLs but possess the advantages of germline-encoded receptors that recognize a broad range of ligands, thus, requiring relatively little priming and circumventing antigen escape. We have acquired up to ten patient-derived New Zealand Melanoma lines in collaboration with Auckland Cancer Society Research Centre, of which three are from brain metastases. We used NanoString technology to assess melanoma gene expression of immunoregulatory ligands governing NK activation, inhibition or both (via dual-functional ligands dictated by NK-receptors). The results showed expression of several inhibitory ligands but very few activating ligands. The expression trends were largely similar but varied in a select few ligands. Flow cytometry was used to characterize cell-surface expression of the immunoregulatory proteins. These were compared with the classical inhibitory checkpoint molecules evidencing that melanoma cells expressed more NK inhibitory molecules. We hypothesize that melanoma cells both express inhibitory ligands and suppress activation ligands, to hinder NK cell activity. Here we aim to identify novel immune axes leading towards expansion of ICB therapies. This will afford clinicians a personalized approach and access to combination immunotherapies, especially in combating refractory metastatic melanoma, which have poor prognoses.

Programming Injectable DNA Hydrogels Yields Tumor Microenvironment-Activatable and Immune-Instructive Depots for Augmented Chemo-Immunotherapy.

Injectable hydrogels have attracted increasing attention for promoting systemic antitumor immune response through the co-delivery of chemotherapeutics and immunomodulators. However, the biosafety and bioactivity of conventional hydrogel depots are often impaired by insufficient possibilities for post-gelling injection and means for biofunction integration. Here, an unprecedented injectable stimuli-responsive immunomodulatory depot through programming a super-soft DNAhydrogel adjuvant is reported. This hydrogel system encoded with adenosine triphosphate aptamers can be intratumorally injected in a gel formulation and then undergoes significant molecular conformation change to stimulate the distinct release kinetics of co-encapsulated therapeutics. In this scenario, doxorubicinis first released to induce immunogenic cell death that intimately works together with the polymerized cytosine-phosphate-guanine oligodeoxynucleotide (CpG ODN) in gel scaffold for effectively recruiting and activating dendritic cells. The polymerized CpG ODN not only enhances tumor immunogenicity but minimizes free CpG-induced splenomegaly. Furthermore, the subsequently released anti-programmed cell death protein ligand 1 (aPDL1) blocks the corresponding immune inhibitory checkpoint molecule on tumor cells to sensitize antitumor T-cell immunity. This work thus contributes to the first proof-of-concept demonstration of a programmable super-soft DNA hydrogel system that perfectly matches the synergistic therapeutic modalities based on chemotherapeutic toxicity, in situ vaccination, and immune checkpoint blockade.

Aging modifies endometrial dendritic cell function and unconventional double negative T cells in the human genital mucosa

BackgroundImmune function in the genital mucosa balances reproduction with protection against pathogens. As women age, genital infections, and gynecological cancer risk increase, however, the mechanisms that regulate cell-mediated immune protection in the female genital tract and how they change with aging remain poorly understood. Unconventional double negative (DN) T cells (TCRαβ + CD4-CD8-) are thought to play important roles in reproduction in mice but have yet to be characterized in the human female genital tract. Using genital tissues from women (27–77 years old), here we investigated the impact of aging on the induction, distribution, and function of DN T cells throughout the female genital tract.ResultsWe discovered a novel site-specific regulation of dendritic cells (DCs) and unconventional DN T cells in the genital tract that changes with age. Human genital DCs, particularly CD1a + DCs, induced proliferation of DN T cells in a TFGβ dependent manner. Importantly, induction of DN T cell proliferation, as well as specific changes in cytokine production, was enhanced in DCs from older women, indicating subset-specific regulation of DC function with increasing age. In human genital tissues, DN T cells represented a discrete T cell subset with distinct phenotypical and transcriptional profiles compared to CD4 + and CD8 + T cells. Single-cell RNA and oligo-tag antibody sequencing studies revealed that DN T cells represented a heterogeneous population with unique homeostatic, regulatory, cytotoxic, and antiviral functions. DN T cells showed relative to CD4 + and CD8 + T cells, enhanced expression of inhibitory checkpoint molecules and genes related to immune regulatory as well as innate-like anti-viral pathways. Flow cytometry analysis demonstrated that DN T cells express tissue residency markers and intracellular content of cytotoxic molecules. Interestingly, we demonstrate age-dependent and site-dependent redistribution and functional changes of genital DN T cells, with increased cytotoxic potential of endometrial DN T cells, but decreased cytotoxicity in the ectocervix as women age, with implications for reproductive failure and enhanced susceptibility to infections respectively.ConclusionsOur deep characterization of DN T cell induction and function in the female genital tract provides novel mechanistic avenues to improve reproductive outcomes, protection against infections and gynecological cancers as women age.

Transgenic murine models for the study of drug hypersensitivity reactions linked to HLA-I molecules.

Immune-mediated drug hypersensitivity reactions (DHRs) can be life-threatening and an impediment to drug development. Mechanism of disease studies are difficult to perform in humans. Here we review human leukocyte antigens class I (HLA-I) transgenic murine models and highlight how these systems have helped to elucidate drug-specific and host immune factors that initiate, propagate and control severe drug toxicities to skin and liver. HLA transgenic mice have been developed and used to study immune-mediated drug reactions in vitro and in vivo. CD8+ T cells from HLA-B∗57:01-expressing mice respond strongly to abacavir (ABC) in vitro but have self-limited responses to drug exposure in vivo. Immune tolerance can be overcome by depleting regulatory T cells (Treg) allowing antigen-presenting dendritic cells to express CD80/86 costimulatory molecules and signal through CD28 on the CD8+ T cell. Depletion of Treg also removes competition for interleukin 2 (IL-2) to allow T cell expansion and differentiation. Fine tuning of responses depends on inhibitory checkpoint molecules such as PD-1. Improved mouse models express only HLA in the absence of PD-1. These models show enhanced liver injury to flucloxacillin (FLX) which depends on drug priming, CD4+ T cell depletion, and lack of PD-1 expression. Drug-specific HLA-restricted cytotoxic CD8+ T cells can infiltrate the liver but are suppressed by Kupffer and liver sinusoidal endothelial cells. HLA-I transgenic mouse models are now available to study ABC, FLX and carbamazepine-induced adverse reactions. In vivo studies range from characterizing drug-antigen presentation, T cell activation, immune-regulatory molecules and cell-cell interaction pathways that are specifically involved in causing or controlling unwanted DHRs.

Eribulin mesylate exerts antitumor effects via CD103

ABSTRACT Eribulin mesylate (ERB) is a synthetic analog of halichondrin B, inhibiting tumor cell growth by disrupting microtubule function. Recently, anticancer drugs have been shown to not only act directly on tumor cells but also to exert antitumor effects by modifying the tumor environment. Although ERB has also been speculated to modify the tumor microenvironment including the immune response to tumors, the precise mechanism remains unclear. In our study, ERB suppressed the tumor growth of MC38 colon cancer in wildtype mice, whereas ERB failed to inhibit the tumor growth in Rag1-deficient mice which lack both B and T cells. Moreover, depletion of either CD4+ or CD8+ T cells abrogated the antitumor effect of ERB, indicating that both CD4+ and CD8+ T cells play an important role in ERB-induced antitumor effects. Furthermore, ERB treatment increased the number of tumor infiltrating lymphocytes (TILs) as well as the expression of activation markers (CD38 and CD69), immune checkpoint molecules (LAG3, TIGIT and Tim3) and cytotoxic molecules (granzyme B and perforin) in TILs. ERB upregulated E-cadherin expression in MC38. CD103 is a ligand of E-cadherin and induces T-cell activation. ERB increased the proportion of CD103+ cells in both CD4+ and CD8+ TILs. The ERB-induced antitumor effect with the increased TIL number and the increased expression of activation markers, inhibitory checkpoint molecules and cytotoxic molecules in TILs was abrogated in CD103-deficient mice. Collectively, these results suggest that ERB exerts antitumor effects by upregulation of E-cadherin expression in tumor cells and subsequent activation of CD103+ TILs.