Immunogenic Phenotype Research Articles

Epigenetic modification of CSDE1 locus dictates immune recognition of nascent tumorigenic cells.

Weak immunogenicity of tumor cells is a root cause for the ultimate failure of immunosurveillance and immunotherapy. Although tumor evolution can be shaped by immunoediting toward a less immunogenic phenotype, mechanisms governing the initial immunogenicity of primordial tumor cells or original cancer stem cells remain obscure. Here, using a single tumor-repopulating cell (TRC) to form tumors in immunodeficient or immunocompetent mice, we demonstrated that immunogenic heterogeneity is an inherent trait of tumorigenic cells defined by the activation status of signal transducer and activator of transcription 1 (STAT1) protein in the absence of immune pressure. Subsequent investigation identified that the RNA binding protein cold shock domain-containing protein E1 (CSDE1) can promote STAT1 dephosphorylation by stabilizing T cell protein tyrosine phosphatase (TCPTP). A methyltransferase SET and MYN domain-containing 3 (SMYD3) was further identified to mediate H3K4 trimethylation of CSDE1 locus, which was under the regulation of mechanotransduction by cell-matrix and cell-cell contacts. Thus, owing to the differential epigenetic modification and subsequent differential expression of CSDE1, nascent tumorigenic cells may exhibit either a high or low immunogenicity. This identified SMYD3-CSDE1 pathway represents a potential prognostic marker for cancer immunotherapy effectiveness that requires further investigation.

CAR-T Cells with Phytohemagglutinin (PHA) Provide Anti-Cancer Capacity with Better Proliferation, Rejuvenated Effector Memory, and Reduced Exhausted T Cell Frequencies.

The development of genetic modification techniques has led to a new era in cancer treatments that have been limited to conventional treatments such as chemotherapy. intensive efforts are being performed to develop cancer-targeted therapies to avoid the elimination of non-cancerous cells. One of the most promising approaches is genetically modified CAR-T cell therapy. The high central memory T cell (Tcm) and stem cell-like memory T cell (Tscm) ratios in the CAR-T cell population increase the effectiveness of immunotherapy. Therefore, it is important to increase the populations of CAR-expressing Tcm and Tscm cells to ensure that CAR-T cells remain long-term and have cytotoxic (anti-tumor) efficacy. In this study, we aimed to improve CAR-T cell therapy's time-dependent efficacy and stability, increasing the survival time and reducing the probability of cancer cell growth. To increase the sub-population of Tcm and Tscm in CAR-T cells, we investigated the production of a long-term stable and efficient cytotoxic CAR-T cell by modifications in the cell activation-dependent production using Phytohemagglutinin (PHA). PHA, a lectin that binds to the membranes of T cells and increases metabolic activity and cell division, is studied to increase the Tcm and Tscm population. Although it is known that PHA significantly increases Tcm cells, B-lymphocyte antigen CD19-specific CAR-T cell expansion, its anti-cancer and memory capacity has not yet been tested compared with aCD3/aCD28-amplified CAR-T cells. Two different types of CARs (aCD19 scFv CD8-(CD28 or 4-1BB)-CD3z-EGFRt)-expressing T cells were generated and their immunogenic phenotype, exhausted phenotype, Tcm-Tscm populations, and cytotoxic activities were determined in this study. The proportion of T cell memory phenotype in the CAR-T cell populations generated by PHA was observed to be higher than that of aCD3/aCD28-amplified CAR-T cells with similar and higher proliferation capacity. Here, we show that PHA provides long-term and efficient CAR-T cell production, suggesting a potential alternative to aCD3/aCD28-amplified CAR-T cells.

HROP68: A rare case of medullary pancreatic cancer-characterization and chemosensitivity of the first patient-derived cell line.

Medullary pancreatic carcinoma (MPC) is a rare subtype of pancreatic ductal adenocarcinoma. MPCs represent less than 1% of all pancreatic cancers, and, with only 26 cases in the literature, knowledge regarding drug response and treatment outcome is very limited. We present the case of a 64-year-old male patient with MPC who was treated by left pancreatic resection and adjuvant chemotherapy. Due to local recurrence, the patient underwent intended curative reoperation. From both surgical specimens, patient-derived xenografts (PDXs) and, from the recurrence, a patient-derived cell line (PDCL) were established. We subsequently performed an in-depth characterization of this cell line including phenotypic characterization, surface protein expression, growth, and migratory performance as well as mutational analysis using whole-exome sequencing (WES). Additionally, in vitro drug sensitivity toward the standard-of-care chemotherapeutic regimen and selected targeted therapies was evaluated. The pathological and molecular properties of this rare MPC case observed in the patient's tumors are preserved in the corresponding PDX and the PDCL of HROP68Tu2. Despite displaying an "immunogenic phenotype" with marked T-cell infiltration and a high-level expression of HLA II and Programmed death-ligand 1 (PD-L1), molecular analysis revealed microsatellite stability but a multitude of mutations affecting KRAS, TP53, KAT6B, FOXG1, RUNX1, and GRIK2 among others. Furthermore, HROP68Tu2 cells were susceptible toward 5-FU, irinotecan, oxaliplatin, gemcitabine, paclitaxel, and erlotinib as single agents, but only a moderate synergistic response was seen to the drugs of the FOLFIRINOX regimen. Even worse, the drugs of the two combinations gemcitabine plus paclitaxel and gemcitabine plus erlotinib showed antagonistic effects. Moreover, lapatinib, PRIMA-Met1, and olaparib selected as targeted therapeutics according to the mutational profiles and protein expression inhibited HROP68Tu2 cells' growth. This study illustrates the establishment of the first preclinical MPC models as well as the first in-depth characterization of an MPC PDCL. Since the scientific and clinical knowledge of this rare pancreatic cancer type is very limited, the presented models contribute to a better understanding of MPC and might be a valuable tool for the development of future treatment options.

Blockade of CCR1 induces a phenotypic shift in macrophages and triggers a favorable antilymphoma activity

AbstractTumor-associated macrophages (TAMs) within the tumor microenvironment (TME) play an important role in tumor growth and progression. TAMs have been involved in producing immunosuppressive TME via various factors; however, the underlying mechanisms remain unclear in B-cell lymphoma, including mantle cell lymphoma (MCL). We identified that chemokine receptor-1 (CCR1) is highly expressed on monocytes (Mo) and macrophages (MΦ), and CCR1 pharmacological inhibition or CCR1 siRNA abolished lymphoma-mediated Mo/MΦ migration in a chemotaxis assay. The deficiency of host CCR1 (CCR1 KO) was associated with decreased infiltration of peritoneal-MΦ compared with WT-CCR1. Functional studies indicated that the genetic depletion of CCR1 or treatment inhibited protumor MΦ (M2-like) phenotype by decreasing CD206 and IL-10 expression. Moreover, CCR1 depletion reprogrammed MΦ toward an MHCII+/TNFα+ immunogenic phenotype. Mechanistically, protumor MΦ driven–IL-10 provides a positive feedback loop to tumor-CCL3 by regulating the CCL3 promoter via STAT1 signaling. Therapeutic in vivo targeting of CCR1 with CCR1 antagonist BX-471 significantly reduced FC-muMCL1 mouse tumors in the syngeneic MCL model by the depletion of M2-TAMs and increased infiltration of cytotoxic CD8+ T cells. Our study established that CCR1 exerts a pivotal role in macrophage programming, thus shaping protumor TME and lymphoma progression. CCR1 inhibition through CCR1 antagonists may be a promising therapeutic strategy to reprogram macrophages in lymphoma-TME and achieve better clinical outcomes in patients.

Genomic analysis of Mycobacterium brumae sustains its nonpathogenic and immunogenic phenotype.

Mycobacterium brumae is a rapid-growing, non-pathogenic Mycobacterium species, originally isolated from environmental and human samples in Barcelona, Spain. Mycobacterium brumae is not pathogenic and it's in vitro phenotype and immunogenic properties have been well characterized. However, the knowledge of its underlying genetic composition is still incomplete. In this study, we first describe the 4 Mb genome of the M. brumae type strain ATCC 51384T assembling PacBio reads, and second, we assess the low intraspecies variability by comparing the type strain with Illumina reads from three additional strains. Mycobacterium brumae genome is composed of a circular chromosome with a high GC content of 69.2% and containing 3,791 CDSs, 97 pseudogenes, one prophage and no CRISPR loci. Mycobacterium brumae has shown no pathogenic potential in in vivo experiments, and our genomic analysis confirms its phylogenetic position with other non-pathogenic and rapid growing mycobacteria. Accordingly, we determined the absence of virulence-related genes, such as ESX-1 locus and most PE/PPE genes, among others. Although the immunogenic potential of M. brumae was proved to be as high as Mycobacterium bovis BCG, the only mycobacteria licensed to treat cancer, the genomic content of M. tuberculosis T cell and B cell antigens in M. brumae genome is considerably lower than those antigens present in M. bovis BCG genome. Overall, this work provides relevant genomic data on one of the species of the mycobacterial genus with high therapeutic potential.

Normofractionated irradiation and not temozolomide modulates the immunogenic and oncogenic phenotype of human glioblastoma cell lines

PurposeGlioblastoma multiforme (GBM) is the most aggressive primary brain tumor, with an overall poor prognosis after diagnosis. Conventional treatment includes resection, chemotherapy with temozolomide (TMZ), and concomitant radiotherapy (RT). The recent success of immunotherapy approaches in other tumor entities, particularly with immune checkpoint inhibitors, could not be clinically transferred to GBM treatment so far. Therefore, preclinical analyses of the expression of both immune-suppressive and immune-stimulatory checkpoint molecules following treatment of human glioblastoma cells with RT and/or temozolomide is needed to design feasible radio(chemo)immunotherapy trials for GBM in the future.MethodsFive human glioblastoma cell lines (H4, HROG-06, U118, U138, U251) were analyzed regarding their clonogenic survival and cell death forms after chemotherapy (CT) with TMZ and/or normofractionated RT (5 × 2 Gy) via multicolor flow cytometry. Further, the tumor cell surface expression of immune-activating (OX40L, CD137L, CD70, and ICOSL) and immune-suppressive (PD-L1, PD-L2, HVEM) checkpoint molecules and of an oncogenic molecule (EGFR) were measured via multicolor flow cytometry after CT and RT alone or after RCT.ResultsNormofractionated RT and not TMZ was the trigger of induction of predominantly necrosis in the glioblastoma cells. Notably, clonogenicity did not correlate with cell death induction by RT. The basal expression level of immune-suppressive PD-L1, PD-L2, and HVEM varied in the analyzed glioblastoma cells. RT, but not TMZ, resulted in a significant upregulation of PD-L1 and PD-L2 in all tumor cells investigated. Also, the expression of HVEM was increased after RT in most of the GBM cell lines. In contrast, normofractionated RT individually modulated expression of the stimulating immune checkpoint molecules CD70, CD137L, OX40L, and ICOSL1. The oncogenic factor EGFR was significantly increased by irradiation in all examined cell lines, albeit to a different extent. None of the investigated molecules were downregulated after the treatments.ConclusionNormofractionated radiotherapy modulates the immunogenic as well as the oncogenic phenotype of glioblastoma cells, partly individually. Therefore, not only PD-L1 and PD-L2, but also other immunogenic molecules expressed on the surface of glioblastoma cells could serve as targets for immune checkpoint blockade in combination with RT in the future.

Altered distribution and function of NK-cell subsets lead to impaired tumor surveillance in JAK2V617F myeloproliferative neoplasms.

In cancer, tumor cells and their neoplastic microenvironment can sculpt the immunogenic phenotype of a developing tumor. In this context, natural killer (NK) cells are subtypes of lymphocytes of the innate immune system recognized for their potential to eliminate neoplastic cells, not only through direct cytolytic activity but also by favoring the development of an adaptive antitumor immune response. Even though the protective effect against leukemia due to NK-cell alloreactivity mediated by the absence of the KIR-ligand has already been shown, and some data on the role of NK cells in myeloproliferative neoplasms (MPN) has been explored, their mechanisms of immune escape have not been fully investigated. It is still unclear whether NK cells can affect the biology of BCR-ABL1-negative MPN and which mechanisms are involved in the control of leukemic stem cell expansion. Aiming to investigate the potential contribution of NK cells to the pathogenesis of MPN, we characterized the frequency, receptor expression, maturation profile, and function of NK cells from a conditional Jak2V617F murine transgenic model, which faithfully resembles the main clinical and laboratory characteristics of human polycythemia vera, and MPN patients. Immunophenotypic analysis was performed to characterize NK frequency, their subtypes, and receptor expression in both mutated and wild-type samples. We observed a higher frequency of total NK cells in JAK2V617F mutated MPN and a maturation arrest that resulted in low-numbered mature CD11b+ NK cells and increased immature secretory CD27+ cells in both human and murine mutated samples. In agreement, inhibitory receptors were more expressed in MPN. NK cells from Jak2V617F mice presented a lower potential for proliferation and activation than wild-type NK cells. Colonies generated by murine hematopoietic stem cells (HSC) after mutated or wild-type NK co-culture exposure demonstrated that NK cells from Jak2V617F mice were deficient in regulating differentiation and clonogenic capacity. In conclusion, our findings suggest that NK cells have an immature profile with deficient cytotoxicity that may lead to impaired tumor surveillance in MPN. These data provide a new perspective on the behavior of NK cells in the context of myeloid malignancies and can contribute to the development of new therapeutic strategies, targeting onco-inflammatory pathways that can potentially control transformed HSCs.

Mesenchymal and adrenergic cell lineage states in neuroblastoma possess distinct immunogenic phenotypes.

Apart from the anti-GD2 antibody, immunotherapy for neuroblastoma has had limited success due to immune evasion mechanisms, coupled with an incomplete understanding of predictors of response. Here, from bulk and single-cell transcriptomic analyses, we identify a subset of neuroblastomas enriched for transcripts associated with immune activation and inhibition and show that these are predominantly characterized by gene expression signatures of the mesenchymal lineage state. By contrast, tumors expressing adrenergic lineage signatures are less immunogenic. The inherent presence or induction of the mesenchymal state through transcriptional reprogramming or therapy resistance is accompanied by innate and adaptive immune gene activation through epigenetic remodeling. Mesenchymal lineage cells promote T cell infiltration by secreting inflammatory cytokines, are efficiently targeted by cytotoxic T and natural killer cells and respond to immune checkpoint blockade. Together, we demonstrate that distinct immunogenic phenotypes define the divergent lineage states of neuroblastoma and highlight the immunogenic potential of the mesenchymal lineage.

The impact of tumor immunogenicity on cancer pain phenotype using syngeneic oral cancer mouse models.

Head and neck squamous cell carcinoma (HNSCC) patients report severe function-induced pain at the site of the primary tumor. The current hypothesis is that oral cancer pain is initiated and maintained in the cancer microenvironment due to secretion of algogenic mediators from tumor cells and surrounding immune cells that sensitize the primary sensory neurons innervating the tumor. Immunogenicity, which is the ability to induce an adaptive immune response, has been widely studied using cancer cell transplantation experiments. However, oral cancer pain studies have primarily used xenograft transplant models in which human-derived tumor cells are inoculated in an athymic mouse lacking an adaptive immune response; the role of inflammation in oral cancer-induced nociception is still unknown. Using syngeneic oral cancer mouse models, we investigated the impact of tumor cell immunogenicity and growth on orofacial nociceptive behavior and oral cancer-induced sensory neuron plasticity. We found that an aggressive, weakly immunogenic mouse oral cancer cell line, MOC2, induced rapid orofacial nociceptive behavior in both male and female C57Bl/6 mice. Additionally, MOC2 tumor growth invoked a substantial injury response in the trigeminal ganglia as defined by a significant upregulation of injury response marker ATF3 in tongue-innervating trigeminal neurons. In contrast, using a highly immunogenic mouse oral cancer cell line, MOC1, we found a much slower onset of orofacial nociceptive behavior in female C57Bl/6 mice only as well as sex-specific differences in the tumor-associated immune landscape and gene regulation in tongue innervating sensory neurons. Together, these data suggest that cancer-induced nociceptive behavior and sensory neuron plasticity can greatly depend on the immunogenic phenotype of the cancer cell line and the associated immune response.

Stem Cells in the Tumor Immune Microenvironment -Part of the Cure or Part of the Disease? Ontogeny and Dichotomy of Stem and Immune Cells has Led to better Understanding.

Stem cells are at the basis of tissue homeostasis, hematopoiesis and various regenerative processes. Epigenetic changes in their somatically imprinted genes, prolonged exposure to mutagens/carcinogens or alteration of their niche can lead to the development of an enabling environment for tumor growth and progression. The involvement of stem cells in both health and disease becomes even more compelling with ontogeny as embryonic and extraembryonic stem cells which persist into adulthood in well established and specific niche may have distinct implications in tumorigenesis. Immune surveillance plays an important role in this interplay since the response of immune cells toward the oncogenic process can range from reactivity to placidity and even complicity, being orchestrated by intercellular molecular dialogues with the other key players of the tumor microenvironment. With the current understanding that every developing and adult tissue contains inherent stem and progenitor cells, in this manuscript we review the most relevant interactions carried out between the stem cells, tumor cells and immune cells in a bottom-up incursion through the tumor microenvironment beginning from the perivascular niche and going through the tumoral parenchyma and the related stroma. With the exploitation of various factors that influence the behavior of immune effectors toward stem cells and other resting cells in their niche, new therapeutic strategies to tackle the polarization of immune effectors toward a more immunogenic phenotype may arise.

Abstract 3501: Induction of tumor cell apoptosis & immunogenic cell death by CD40L-armed oncolytic adenoviruses

Abstract Low tumor immunogenicity and a cold tumor microenvironment are two main characteristics occurring in different cancer types responsible for immunotherapy resistance. Immunostimulatory gene therapy using oncolytic viruses (OVs) has been developed as a new promising cancer therapy for its capacity to induce an anti-tumor immune response and sensitize the non-responding patients to checkpoint blockade therapy. Lokon adenoviruses (LOAds) are serotype 5/35 oncolytic adenoviruses that in addition to their oncolytic capacity encode for immunostimulatory molecules. Here we have evaluated the anti-tumor effects of trimerized membrane-bound (TMZ)-CD40L-armed LOAd700 and TMZ-CD40L/4-1BBL-armed LOAd703 in a variety of solid tumors to understand the underlying tumor modulating mechanisms. A panel of eight different human solid cancer cell lines (T24, PEA2, Mel526, H727, Skov3, MiaPaca2, H29 and A549) was infected with LOAd(-), a virus without transgenes, LOAd700 or LOAd703 and assessed for their response in different assays. MTS assay was used to measure the cell viability, flow cytometry for evaluating the cell surface markers and luminescent assay for measuring apoptosis (caspase 3/7 activity). LOAd viruses efficiently killed all tested cell lines. However, the oncolytic capacity of CD40L-armed LOAd700 and LOAd703 was significantly higher than LOAd(-) in two cell lines, T24 and PEA2, which were found to express the CD40 receptor. Death receptor TRAIL-R1 was significantly upregulated in T24 infected with LOAd703 and PEA2 infected with either LOAd700 or LOAd703. TRAIL-R2 was upregulated post LOAd infection in 7/8 cell lines where the upregulation was significant in T24, PEA2 and H29. Interestingly, the immunogenic cell death marker calreticulin, a damage-associated molecular pattern (DAMP) known as an “eat-me” signal to dendritic cells, was upregulated post LOAd infection in all cell lines. The increase in calreticulin level was statistically significant in 4/8 cell lines (T24, PEA2, Mel526 and H29) post LOAd700 and/or LOAd703 infection. Furthermore, LOAd700 and LOAd703 increased the effector caspase 3/7 activity in 5/8 cell lines (T24, PEA2, Mel526, Skov3 and MiaPaCa2) indicating an augmented apoptosis level. In conclusion, CD40L-armed LOAd viruses were, in addition to their high oncolytic capacity, demonstrated to induce apoptosis. Upregulation of cell surface calreticulin as well as the death receptors TRAIL-R1 and -R2 indicated generation of a more immunogenic phenotype of cancer cells. Thus, our findings suggest that CD40L-armed LOAd viruses could be inducers of apoptosis and immunogenic cell death in a variety of cancer types with an augmented effect in cancer cells expressing the CD40 receptor. Citation Format: Sedigheh Naseri, Mariela Mejia Cordova, Jessica Wenthe, Ann-Charlotte Hellström, Tanja Lövgren, Angelica Loskog, Emma Eriksson, Gustav Ullenhag. Induction of tumor cell apoptosis & immunogenic cell death by CD40L-armed oncolytic adenoviruses [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 3501.

In Situ Micro-Nano Conversion Augmented Tumor-Localized Immunochemotherapy.

The immune checkpoint blockade (ICB) therapy based on monoclonal antibodies still suffers from a lower immune response rate and severe immune-related side effects, which greatly compromise its therapeutic benefits. Herein, ultrasound (US) microbubbles (MBs) that locally delivered the camptothecin-floxuridine (CF) drug combination and anti-PD-L1 blocking antibody (αPD-L1) to tumors were developed to improve ICB therapy. The resulting αPCF MBs exhibited good stability, allowing their use as US imaging contrast agents to trace the drug delivery in vivo. Furthermore, the combination of αPCF MBs treatment and disrupted US irradiation triggered tumor in situ conversion of αPCF MBs to αPCF NPs while promoting higher tumor cell uptake and deeper tumor penetration as confirmed by the US/fluorescence bimodal imaging. Camptothecin (CPT) and floxuridine (FUDR) were further released at a fixed 1:1 molar ratio within the tumor microenvironment (TME) to synergistically elicit an immunogenic tumor phenotype and sensitize tumors to αPD-L1-mediated ICB therapy, while the local simultaneous delivery of immunotherapeutic αPD-L1 further reversed the immunosuppressive tumor microenvironment and promoted the infiltration of cytotoxic T lymphocytes (CTLs), thus achieving a synergistic therapeutic effect of chemotherapy and immunotherapy in the CT26 tumor-bearing mice. Thus, αPCF MBs + US mediated local co-delivering of the drug combination and αPD-L1 well augmented the ICB therapy while effectively minimizing the off-target side effects, providing a safe and universal therapeutic strategy for tumor immunotherapy.

Prognostic implications of the peripheral platelet-to-lymphocyte ratio and neutrophil-to-lymphocyte ratio in predicting pathologic complete response after neoadjuvant chemotherapy in breast cancer patients.

The inflammatory response is extremely important in tumor progression, and it is very difficult to identify prognostic indicators for neoadjuvant therapy in breast cancer patients. The aim of this study was to mine the potential prognostic significance of the platelet-to-lymphocyte ratio (PLR) and neutrophil-to-lymphocyte ratio (NLR) in breast cancer patients receiving anthracycline- or taxane-based neoadjuvant chemotherapy (NACT). A total of 67 women diagnosed with breast cancer who received neoadjuvant therapy were enrolled in the study. Before starting NACT, the PLR and NLR were calculated. The optimal cutoff value was calculated using receiver operating characteristic (ROC) curve analyses, which indicated that 106.3 and 2.464 were the best cutoff values for the PLR and NLR, respectively. The optimal cutoff values for them were used to divide patients into low and high NLR groups and low and high PLR groups. Independent prognostic biomarkers and the value of PLR and NLR were assessed. The connection between the NLR/PLR and pathologic complete response (pCR), together with other clinical/pathological factors was evaluated. Logistic regression model analyses revealed that patients with a high PLR correlated remarkably with better pCR than those with a low PLR. The results indicated that by using the cutoff value of 106.3, PLR had prognostic significance. However, there was no significant difference in NLR if analyzed separately. By combining PLR and NLR, the NLRhigh and PLRhigh subgroups achieved a significantly higher rate of pCR than the NLRIow/PLRIow subgroup [odds ratio (OR) 0.153, 95% confidence interval (CI): 0.068 to 0.876, P=0.008]. Therefore, the combination of NLRhigh/PLRhigh was an independent prognostic factor different from others, such as PLR, Ki-67, and chemotherapy regimen. The PLR may serve as a potential marker of the efficacy of neoadjuvant therapy in breast cancer, enabling oncologists to intervene earlier. Peripheral blood NLR and PLR can reflect the immune status of patients. Indicating that an immunogenic phenotype is a good predictor of chemotherapy response and that combined studies can better identify immunophenotypes in patients.

193-LB: Differentiation of PTPN2 Knockout Pluripotent Stem Cells into Stem-Cell–Derived ß-Like Cells Leads to Increased Stimulation of Diabetogenic TCR T Cell Transductants

Type 1 diabetes (T1D) is an autoimmune disease marked by permanent loss of insulin-producing pancreatic β cells. Genome wide association studies have identified protein tyrosine non receptor type 2 (PTPN2) as a high-risk T1D gene. PTPN2 has been classically implicated in immune cell function, but is expressed in T cells, B cells, and in pancreatic β cells. Mice with a β cell specific knock out (KO) of ptpn2 exhibit impaired function and increased susceptibility to stress induced β cell death. We hypothesized that altered PTPN2 expression in pancreatic beta cells may contribute to T1D disease development. In order to study PTPN2 in the human context we generated CRISPR/Cas9 mediated clonal PTPN2 knock out (KO) hPSCs, abolishing the functional domain of PTPN2. PTPN2 KO and WT hPSCs were differentiated into functional stem cell derived beta-like cells (sBCs) using our published protocol without significant differences in key developmental markers or insulin content. Compared to WT sBC, PTPN2 KO sBCs at steady state and under T1D stress conditions show increased expression of HLA Class I molecules important in T cell recognition. To investigate the interaction of KO or WT sBCs with diabetogenic T cells we employed an HLA-peptide-TCR matched coculture system we recently established. Using this system, we observed increased stimulation of diabetogenic TCR T cell transductants by KO sBC compared to WT. Our data provide evidence for a potential mechanism by which PTPN2, a known T1D high-risk gene may contribute to an immunogenic phenotype of β cells. Our findings suggest that dysregulation of PTPN2 expression contributes to an autoimmune response towards human β cells thus contributing to the pathogenesis of type 1 diabetes. Disclosure J. Q. Matuschek: None. H. A. Russ: Consultant; Eli Lilly and Company, Sigilon Therapeutics, Inc. T. M. Triolo: None. A. W. Michels: Employee; ImmunoMolecular Therapeutics, Stock/Shareholder; ImmunoMolecular Therapeutics. K. Mcdaniel: None. M. S. Hansen: None. S. Williams: None. R. Castro-gutierrez: None. A. Shilleh: None. Funding NIH: K12DK094712

Immune Checkpoint Blockades in Triple-Negative Breast Cancer: Current State and Molecular Mechanisms of Resistance.

Immune checkpoint blockades (ICBs) have revolutionized cancer treatment. Recent studies have revealed a subset of triple-negative breast cancer (TNBC) to be considered as an immunogenic breast cancer subtype. Characteristics of TNBC, such as higher mutation rates and number of tumor-infiltrating immune cells, render the immunogenic phenotypes. Consequently, TNBCs have shown durable responses to ICBs such as atezolizumab and pembrolizumab in clinic. However, a significant number of TNBC patients do not benefit from these therapies, and mechanisms of resistance are poorly understood. Here, we review biomarkers that predict the responsiveness of TNBCs to ICB and recent advances in delineating molecular mechanisms of resistance to ICBs.

Combining Targeted Radionuclide Therapy and Immune Checkpoint Inhibition for Cancer Treatment.

The development of immunotherapy, in particular immune checkpoint inhibitors (ICI), has revolutionized cancer treatment in the past decades. However, its efficacy is still limited to subgroups of patients with cancer. Therefore, effective treatment combination strategies are needed. Here, radiotherapy is highly promising, as it can induce immunogenic cell death, triggering the release of pro-inflammatory cytokines, thereby creating an immunogenic phenotype and sensitizing tumors to ICI. Recently, targeted radionuclide therapy (TRT) has attained significant interest for cancer treatment. In this approach, a tumor-targeting radiopharmaceutical is used to specifically deliver a therapeutic radiation dose to all tumor cells, including distant metastatic lesions, while limiting radiation exposure to healthy tissue. However, fundamental differences between TRT and conventional radiotherapy make it impossible to directly extrapolate the biological effects from conventional radiotherapy to TRT. In this review, we present a comprehensive overview of studies investigating the immunomodulatory effects of TRT and the efficacy of combined TRT-ICI treatment. Preclinical studies have evaluated a variety of murine cancer models in which α- or β-emitting radionuclides were directed to a diverse set of targets. In addition, clinical trials are ongoing to assess safety and efficacy of combined TRT-ICI in patients with cancer. Taken together, research indicates that combining TRT and ICI might improve therapeutic response in patients with cancer. Future research has to disclose what the optimal conditions are in terms of dose and treatment schedule to maximize the efficacy of this combined approach.

The Hidden Side of Complement Regulator C4BP: Dissection and Evaluation of Its Immunomodulatory Activity.

C4b-binding protein (C4BP) is a well-known regulator of the complement system that holds additional and important activities unrelated to complement inhibition. Recently, we have described a novel immunomodulatory activity in the minor C4BP(β-) isoform directly acting over inflammatory phagocytes. Here we show that incorporation of the β-chain to the C4BP α-chain oligomer interferes with this immunomodulatory activity of C4BP. Moreover, an oligomeric form including only the complement control protein 6 (CCP6) domain of the C4BP α-chain (PRP6-HO7) is sufficient to “reprogram” monocyte-derived DCs (Mo-DCs) from a pro-inflammatory and immunogenic phenotype to an anti-inflammatory and tolerogenic state. PRP6-HO7 lacks complement regulatory activity but retains full immunomodulatory activity over inflammatory Mo-DCs induced by TLRs, characterized by downregulation of relevant surface markers such as CD83, HLA-DR, co-stimulatory molecules such as CD86, CD80 and CD40, and pro-inflammatory cytokines such as IL-12 and TNF-α. Furthermore, PRP6-HO7-treated Mo-DCs shows increased endocytosis, significantly reduced CCR7 expression and CCL21-mediated chemotaxis, and prevents T cell alloproliferation. Finally, PRP6-HO7 shows also full immunomodulatory activity over Mo-DCs isolated from lupus nephritis patients with active disease, even without further pro-inflammatory stimulation. Therefore PRP6-HO7, retaining the immunomodulatory activity of C4BP(β-) and lacking its complement regulatory activity, might represent a promising and novel alternative to treat autoimmune diseases.

Interspatial Distribution of Tumor and Immune Cells in Correlation with PD-L1 in Molecular Subtypes of Gastric Cancers.

Simple SummaryThe majority of gastric cancers are negative for the Epstein–Barr virus (EBV) and mismatch repair-proficient, or microsatellite stable. Molecular subtyping currently does not influence clinical decision making, and accurate response prediction towards immunotherapy remains a major challenge. We therefore analyzed PD-L1 expression, tumor-infiltrating lymphocytes, and their spatial relationship to tumor cells in EBV-negative mismatch repair-proficient gastric cancers compared to EBV-positive and mismatch repair-deficient tumors to identify an immunogenic phenotype that is susceptible to immunotherapy in this large group. We demonstrated a close relationship between the total number of tumor-infiltrating lymphocytes, their proximity to tumor cells, and the expression of PD-L1 across all subtypes, including the EBV-negative and mismatch repair-proficient cancers. However, we also identified a subgroup of PD-L1-negative, EBV-negative, and mismatch repair-proficient cancers with high numbers of tumor-associated CD8+ lymphocytes. This is indicative of an immunoreactive phenotype in a subgroup of gastric cancers along or independent of PD-L1 status and molecular type.(1) Background: EBV-positive and mismatch repair-deficient (MMRd) gastric cancers (GCs) show higher levels of tumor-infiltrating lymphocytes (TILs) and PD-L1 expression and thus a more profound response to immunotherapy. However, the majority of GCs are EBV-negative (EBV−) and MMR proficient (MMRp). We analyzed PD-L1 expression and TILs in EBV-MMRpGCs in comparison to EBV-positive (EBV+) and MMRdGCs to identify an immunogenic phenotype susceptible to immunotherapy. (2) Methods: A next-generation tissue microarray of 409 primary resected GCs was analyzed by Epstein-Barr encoding region (EBER) in situ hybridization for MSH1, PMS2, MSH2, MSH6, PD-L1, and CD8 immunohistochemistry. PD-L1 positivity was defined as a combined positive score (CPS) of ≥1. CD8+ TILs and their proximity to cancer cells were digitally analyzed on the HALO™ image analysis platform. (3) Results: Eleven cases were EBV+, 49 cases MMRd, and 349 cases EBV-MMRpGCs. The highest rate of PD-L1 positivity was seen in EBV+GCs, followed by MMRdGCs and EBV-MMRpGCs (81.8%, 73.5%, and 27.8%, respectively). EBV+ and MMRdGCs also demonstrated increased numbers and proximity of CD8+ TILs to tumor cells compared to EBV-MMRpGCs (p < 0.001 each). PD-L1 status positively correlated with the total numbers of CD8+ TILs and their proximity to tumor cells in all subtypes, including EBV-MMRpGCs (p < 0.001 each). A total of 28.4% of EBV-MMRpGCs showed high CD8+ TILs independent of PD-L1. (4) Conclusions: PD-L1 and CD8 immunohistochemistry, supplemented by digital image analysis, may identify EBV-MMRpGCs with high immunoreactivity indices, indicating susceptibility to immunotherapy.

Chaperone-Mediated Autophagy Ablation in Pericytes Reveals New Glioblastoma Prognostic Markers and Efficient Treatment Against Tumor Progression.

Background: The lack of knowledge of the progression mechanisms of glioblastoma (GB), the most aggressive brain tumor, contributes to the absence of successful therapeutic strategies. Our team has recently demonstrated a crucial new role for chaperone-mediated autophagy (CMA) in pericytes (PC)-acquired immunosuppressive function, which prevents anti-tumor immune responses and facilitates GB progression. The possible impact that GB-induced CMA in PC has on other functions that might be useful for future GB prognosis/treatment, has not been explored yet. Thus, we proposed to analyze the contribution of CMA to other GB-induced changes in PC biology and determine if CMA ablation in PC is a key target mechanism for GB treatment. Methods: Studies of RNA-seq and secretome analysis were done in GB-conditioned PC with and without CMA (from knockout mice for LAMP-2A) and compared to control PC. Different therapeutic strategies in a GB mouse model were compared. Results: We found several gene expression pathways enriched in LAMP2A-KO PC and affected by GB-induced CMA in PC that correlate with our previous findings. Phagosome formation, cellular senescence, focal adhesion and the effector function to promote anti-tumor immune responses were the most affected pathways, revealing a transcriptomic profiling of specific target functions useful for future therapies. In addition, several molecules associated with tumor mechanisms and related to tumor immune responses such as gelsolin, periostin, osteopontin, lumican and vitamin D, were identified in the PC secretome dependent on GB-induced CMA. The CMA ablation in PC with GB cells showed an expected immunogenic phenotype able to phagocyte GB cells and a key strategy to develop future therapeutic strategies against GB tumor progression. A novel intravenous therapy using exofucosylated CMA-deficient PC was efficient to make PC reach the tumor niche and facilitate tumor elimination. Conclusion: Our results corroborate previous findings on the impaired immunogenic function of PC with GB-induced CMA, driving to other altered PC functions and the identifications of new target markers related to the tumor immune responses and useful for GB prognosis/therapy. Our work demonstrates CMA ablation in PC as a key target mechanism to develop a successful therapy against GB progression.

Immune mechanisms behind prostate cancer in men of African ancestry: A review.

Men of African ancestry (AA) with prostate cancer suffer from worse outcomes. However, a recent analysis of patients treated with the dendritic cell vaccine sipuleucel-T for prostate cancer suggested that AA patients could have improved outcomes relative to whites. We conducted a focused literature review of Medline-indexed articles and clinical trials listed on clinicaltrials.gov. We identify several studies pointing to enrichment of inflammatory cellular infiltrates and cytokine signaling among AA patients with prostate cancer. We outline potential genomic and transcriptomic alterations that may contribute to immunogenicity. Last, we investigate differences in host immunity and vaccine responsiveness that may be enhanced in AA patients. AA patients with prostate cancer may be enriched for an immunogenic phenotype. Dedicated studies are needed to better understand the immune mechanisms that contribute to existing cancer disparities and test immune-based therapies in this population.