Upregulation Of Inhibitory Receptors Research Articles

CD8+ T Cells Mediate Lethal Lung Pathology in the Absence of PD-L1 and Type I Interferon Signalling following LCMV Infection.

CD8+ T cells are critical to the adaptive immune response against viral pathogens. However, overwhelming antigen exposure can result in their exhaustion, characterised by reduced effector function, failure to clear virus, and the upregulation of inhibitory receptors, including programmed cell death 1 (PD-1). However, exhausted T cell responses can be "re-invigorated" by inhibiting PD-1 or the primary ligand of PD-1: PD-L1. Further, the absence of the type I interferon receptor IFNAR1 also results in T cell exhaustion and virus persistence in lymphocytic choriomeningitis virus Armstrong (LCMV-Arm)-infected mice. In this study, utilizing single- and double-knockout mice, we aimed to determine whether ablation of PD-1 could restore T cell functionality in the absence of IFNAR1 signalling in LCMV-Arm-infected mice. Surprisingly, this did not re-invigorate the T cell response and instead, it converted chronic LCMV-Arm infection into a lethal disease characterized by severe lung inflammation with an infiltration of neutrophils and T cells. Depletion of CD8+ T cells, but not neutrophils, rescued mice from lethal disease, demonstrating that IFNAR1 is required to prevent T cell exhaustion and virus persistence in LCMV-Arm infection, and in the absence of IFNAR1, PD-L1 is required for survival. This reveals an important interplay between IFNAR1 and PD-L1 with implications for therapeutics targeting these pathways.

Expression profiling of cerebrospinal fluid identifies dysregulated antiviral mechanisms in multiple sclerosis.

Despite the overwhelming evidence that multiple sclerosis is an autoimmune disease, relatively little is known about the precise nature of the immune dysregulation underlying the development of the disease. Reasoning that the CSF from patients might be enriched for cells relevant in pathogenesis, we have completed a high-resolution single-cell analysis of 96 732 CSF cells collected from 33 patients with multiple sclerosis (n = 48 675) and 48 patients with other neurological diseases (n = 48 057). Completing comprehensive cell type annotation, we identified a rare population of CD8+ T cells, characterized by the upregulation of inhibitory receptors, increased in patients with multiple sclerosis. Applying a Multi-Omics Factor Analysis to these single-cell data further revealed that activity in pathways responsible for controlling inflammatory and type 1 interferon responses are altered in multiple sclerosis in both T cells and myeloid cells. We also undertook a systematic search for expression quantitative trait loci in the CSF cells. Of particular interest were two expression quantitative trait loci in CD8+ T cells that were fine mapped to multiple sclerosis susceptibility variants in the viral control genes ZC3HAV1 (rs10271373) and IFITM2 (rs1059091). Further analysis suggests that these associations likely reflect genetic effects on RNA splicing and cell-type specific gene expression respectively. Collectively, our study suggests that alterations in viral control mechanisms might be important in the development of multiple sclerosis.

T-Cell Exhaustion in HIV-1/Hepatitis C Virus Coinfection Is Reduced After Successful Treatment of Chronic Hepatitis C.

T-cell responses during chronic viral infections become exhausted, which is reflected by upregulation of inhibitory receptors (iRs) and increased interleukin 10 (IL-10). We assessed 2 iRs-PD-1 (programmed cell death protein 1) and Tim-3 (T-cell immunoglobulin and mucin domain-containing protein 3)-and IL-10 mRNAs in peripheral blood mononuclear cells (PBMCs) and their soluble analogs (sPD-1, sTim-3, and IL-10) in plasma in chronic HIV-1/hepatitis C virus (HCV) coinfection and explored the effect of HCV treatment on these markers. We also aimed to establish whether iR expression may be determined by the HCV CD8+ T-cell immunodominant epitope sequence. Plasma and PBMCs from 31 persons with chronic HIV-1/HCV coinfection from the Swiss HIV Cohort Study were collected before and after HCV treatment. As controls, 45 persons who were HIV-1 negative with chronic HCV infection were recruited. Exhaustion markers were assessed by enzyme-linked immunosorbent assay in plasma and by quantitative reverse transcription polymerase chain reaction in PBMCs. Analysis of an HCV epitope sequence was conducted by next-generation sequencing: HLA-A*02-restricted NS31073-1081 and NS31406-1415 and HLA-A*01-restricted NS31436-1444. The study revealed higher plasma sPD-1 (P = .0235) and IL-10 (P = .002) levels and higher IL-10 mRNA in PBMCs (P = .0149) in HIV-1/HCV coinfection. A decrease in plasma sPD-1 (P = .0006), sTim-3 (P = .0136), and IL-10 (P = .0003) and Tim-3 mRNA in PBMCs (P = .0210) was observed following successful HCV treatment. Infection with the HLA-A*01-restricted NS31436-1444 ATDALMTGY prototype variant was related to higher sTim-3 levels than infection with the ATDALMTGF escape variant (P = .0326). The results underscore the synergistic effect of coinfection on expression of exhaustion markers, their reduction following successful HCV treatment and imply that iR levels may operate on an epitope-specific manner.

Modeling the Development of Cellular Exhaustion and Tumor-Immune Stalemate.

Cellular exhaustion in various immune cells develops in response to prolonged stimulation and overactivation during chronic infections and in cancer. Marked by an upregulation of inhibitory receptors and diminished effector functions, exhausted immune cells are unable to fully eradicate the antigen responsible for the overexposure. In cancer settings, this results in a relatively small but constant tumor burden known as a localized tumor-immune stalemate. In recent years, studies have elucidated key aspects of the development and progression of cellular exhaustion and have re-addressed previous misconceptions. Biological publications have also provided insight into the functional capabilities of exhausted cells. Complementing these findings, the model presented here serves as a mathematical framework for the establishment of cellular exhaustion and the development of the localized stalemate against a solid tumor. Analysis of this model indicates that this stalemate is stable and can handle small perturbations. Additionally, model analysis also provides insight into potential targets of future immunotherapy efforts.

Acute Myeloid Leukemia Causes T Cell Exhaustion and Depletion in a Humanized Graft-versus-Leukemia Model.

Allogeneic hematopoietic stem cell transplantation (alloSCT) is, in many clinical settings, the only curative treatment for acute myeloid leukemia (AML). The clinical benefit of alloSCT greatly relies on the graft-versus-leukemia (GVL) effect. However, AML relapse remains the top cause of posttransplant death; this highlights the urgent need to enhance GVL. Studies of human GVL have been hindered by the lack of optimal clinically relevant models. In this article, we report, the successful establishment of a novel (to our knowledge) humanized GVL model system by transplanting clinically paired donor PBMCs and patient AML into MHC class I/II knockout NSG mice. We observed significantly reduced leukemia growth in humanized mice compared with mice that received AML alone, demonstrating a functional GVL effect. Using this model system, we studied human GVL responses against human AML cells invivo and discovered that AML induced T cell depletion, likely because of increased T cell apoptosis. In addition, AML caused T cell exhaustion manifested by upregulation of inhibitory receptors, increased expression of exhaustion-related transcription factors, and decreased T cell function. Importantly, combined blockade of human T cell-inhibitory pathways effectively reduced leukemia burden and reinvigorated CD8 T cell function in this model system. These data, generated in a highly clinically relevant humanized GVL model, not only demonstrate AML-induced inhibition of alloreactive T cells but also identify promising therapeutic strategies targeting T cell depletion and exhaustion for overcoming GVL failure and treating AML relapse after alloSCT.

Tumor-infiltrating gamma delta T-cells reveal exhausted subsets with remarkable heterogeneity in colorectal cancer.

The γδT-cells recognize infected or transformed cells. However, unlike αβT-cells, γδT-cells are innate-like immune cells, with no major histocompatibility complex restriction requirements. γδT-cells are the main population of intestinal intraepithelial lymphocytes (IELs) and are associated with the antitumor immune response, particularly in colorectal cancer (CRC). Although CD8+ T-cells exhibit dysfunction and even exhaustion in the tumor microenvironment (TME), which contributes to tumor immune escape, whether the same applies to tumor-infiltrating (TI)-γδT-cells is not completely understood. Here, we sought to investigate the expression pattern of inhibitory receptors and functional state of TI-γδT-cells, and reveal the features of exhausted TI-γδT-cells in the CRC TME. We demonstrated that TI-γδT-cells exhibited exhaustion phenotypes and displayed more severe functional exhaustion than TI-CD8+ T-cells or NK-cells in the TME of CRC. In addition, scRNA-seq analysis of TI-γδT-cells revealed three exhausted subsets with remarkable heterogeneity. The presence of three heterogeneous exhausted γδT-cell (Tex) populations, including Texprog , Textran and Texterm were further confirmed by flow cytometry, on the basis of PD-1 and TIM-3 expression. Finally, we revealed that c-Maf not only contributed to γδT-cell exhaustion via upregulation of inhibitory receptors, but also involved in the exhaustion of CD8+ T and NK-cells. c-Maf may also be an important contributor to γδT-cell exhaustion in CRC patients. These findings indicated that TI-γδT-cells exhibit phenotypic and functional exhaustion in the CRC TME. The revealed features of exhausted TI-γδT-cells may provide help for understanding the mechanisms and the association of γδT-cell exhaustion with tumor development and pathogenesis.

Discovery of a molecular clock that controls CD8+ T cell function and exhaustion

Abstract During cancer and chronic viral infections, the persistence of antigen progressively causes CD8+ T cells to differentiate into a dysfunctional PD1+ “exhausted” state, with reduced production of inflammatory cytokines relative to effector cells that form during acute infections. Antigen and costimulation signals activate kinase cascades to induce distinct T cell transcription programs, but how T cells distinguish acute and chronic signals to program the exhausted or effector transcriptional states remains poorly understood. We found that members of the protein kinase C (PKC) family function together as a “molecular clock,” sensing acute or chronic agonism to drive distinct transcriptional programs. Continuous stimulation of PKC induces many features of T cell exhaustion, including a loss of production of the cytokines IFNγ and TNF, upregulation of inhibitory receptors and TOX, and altered expression of the proteins in the AP-1 family. Mechanistically, CD8+ T cells express several different PKC proteins, and chronic agonism of PKC leads to degradation of multiple family members and selective maintenance of only one PKC protein, PKC-η. This “PKC switch” alters downstream signaling to support the transcriptional reprogramming of T cells into a terminally exhausted state. In summary, continuous signaling through PKCs causes changes in the output from these kinases initially at the protein level, driving transcriptional changes downstream of PKC targets in the AP-1 transcription factor family and thus allowing further widespread transcriptional and functional changes that characterize T cell exhaustion. Supported by a Damon Runyon Cancer Research Fellowship (DRG-2358-19) and an NIH training grant, T32-CA009370.

Imbalanced receptors, gene signatures, and function of NK subsets predicts poor clinical outcomes in AML

Abstract NK cells mediate anti-leukemic immunosurveillance in AML. We hypothesized that impaired NK cells predict poor outcomes in AML. We analyzed the expression of receptors on CD56 NK cell subsets and their corresponding ligands on the CD45dim blast by flow cytometry in 100 AML and 17 healthy bone marrows. Risk stratification, complete remission, relapse, measurable residual disease, overall survival, relapse-free survival, NK cell ligand expression, NK function, and degranulation were evaluated. Additionally, we mapped the transcriptional landscape of AML-NK cells using single-cell RNA-seq. The expression of activating receptors by CD56dim NK was decreased in adverse risk, relapse, and MRD+ patients. Inhibitory receptors were increased in adverse risk, relapse, and MRD+. NK cell activating ligands were decreased in patients with higher expression of the corresponding receptor while inhibitory ligands were increased. Patients with a lower frequency of CD56dim NK showed less degranulation and killing rates. Transcriptionally, we observed an enrichment of inflammatory-associated pathways and an upregulation of inhibitory receptors in AML, especially in patients with the worst prognosis. Our data indicate that NK exhaustion, with a high frequency of inhibitory receptors, leads to impaired cytotoxicity and can predict poor outcomes in AML. Also, a selective pressure in the expression of NK cell ligands on the AML blast together with imbalanced receptors may vary in both NK and leukemic cells. Closing these gaps in the knowledge of NK-mediated immune evasion in leukemia is of significant interest for targeting the leukemic microenvironment by NK cell-mediated immunotherapy. "division funds" - 3117175

Comprehensive single-cell transcriptomic and proteomic analysis reveals NK cell exhaustion and unique tumor cell evolutionary trajectory in non-keratinizing nasopharyngeal carcinoma

BackgroundNonkeratinizing nasopharyngeal carcinoma (NK-NPC) has a strong association with Epstein-Barr virus (EBV) infection. The role of NK cells and the tumor cell evolutionary trajectory in NK-NPC remain unclear. In this study, we aim to investigate the function of NK cell and the evolutionary trajectory of tumor cells in NK-NPC by single-cell transcriptomic analysis, proteomics and immunohistochemistry.MethodsNK-NPC (n = 3) and normal nasopharyngeal mucosa cases (n = 3) were collected for proteomic analysis. Single-cell transcriptomic data of NK-NPC (n = 10) and nasopharyngeal lymphatic hyperplasia (NLH, n = 3) were obtained from Gene Expression Omnibus (GSE162025 and GSE150825). Quality control, dimension reduction and clustering were based on Seurat software (v4.0.2) process and batch effects were removed by harmony (v0.1.1) software. Normal cells of nasopharyngeal mucosa and tumor cells of NK-NPC were identified using copykat software (v1.0.8). Cell-cell interactions were explored using CellChat software (v1.4.0). Tumor cell evolutionary trajectory analysis was performed using SCORPIUS software (v1.0.8). Protein and gene function enrichment analyses were performed using clusterProfiler software (v4.2.2).ResultsA total of 161 differentially expressed proteins were obtained between NK-NPC (n = 3) and normal nasopharyngeal mucosa (n = 3) by proteomics (log2 fold change > 0.5 and P value < 0.05). Most of proteins associated with the nature killer cell mediated cytotoxicity pathway were downregulated in the NK-NPC group. In single cell transcriptomics, we identified three NK cell subsets (NK1-3), among which NK cell exhaustion was identified in the NK3 subset with high ZNF683 expression (a signature of tissue-resident NK cell) in NK-NPC. We demonstrated the presence of this ZNF683 + NK cell subset in NK-NPC but not in NLH. We also performed immunohistochemical experiments with TIGIT and LAG3 to confirm NK cell exhaustion in NK-NPC. Moreover, the trajectory analysis revealed that the evolutionary trajectory of NK-NPC tumor cells was associated with the status of EBV infection (active or latent). The analysis of cell-cell interactions uncovered a complex network of cellular interactions in NK-NPC.ConclusionsThis study revealed that the NK cell exhaustion might be induced by upregulation of inhibitory receptors on the surface of NK cells in NK-NPC. Treatments for the reversal of NK cell exhaustion may be a promising strategy for NK-NPC. Meanwhile, we identified a unique evolutionary trajectory of tumor cells with active status of EBV-infection in NK-NPC for the first time. Our study may provide new immunotherapeutic targets and new sight of evolutionary trajectory involving tumor genesis, development and metastasis in NK-NPC.

Abstract 5877: Intratumoral lymphocyte networks harbor TCF1+ PD1+ progenitor CD8 T cells in lung cancer

Abstract Cancer immunotherapies that activate cytotoxic T cell responses against tumors have been remarkably effective in some patients, but the determinants of response remain unclear. To investigate how spatial organization of T cells might influence immunotherapy response, we developed a tissue-based cyclic immunofluorescence (t-CyCIF) platform to characterize preclinical genetically engineered mouse models (GEMMs) of cancer. We optimized a 21-antibody panel delineating major myeloid and lymphoid cell subsets, that included key markers for characterizing T cell phenotypes and function. The panel was used to characterize the Kras/p53-mutant GEMM of lung adenocarcinoma, treated with anti-PD-1/anti-CTLA-4 immune checkpoint blockade (ICB) therapy or a neoantigen-targeted long-peptide vaccine. Spatial profiling by t-CyCIF identified intratumoral lymphocyte networks (“lymphonets”) as a key feature associated with a productive immune response against lung tumors. Lymphonets were predominantly composed of B cells and conventional CD4 T cells, but harbored tumor-reactive CD8 T cell populations. TCF1+ PD1+ progenitor CD8 T cells, a subset previously correlated with patient response to ICB, were almost exclusively localized to lymphonets inside tumors. Lymphonets of similar composition harboring TCF1+ PD1+ cells were also found in early-stage human lung adenocarcinomas. In response to ICB and vaccine therapies in mice, cytotoxic CD8 T cells (Granzyme B+/Perforin+) colocalized with TCF1+ PD1+ cells in lymphonets, likely having differentiated from this progenitor pool. Notably, vaccination further induced a highly proliferative (Ki67hi) and cytotoxic (GranzymeBhiPerforinhi) population of CD8 T cells localized outside of tumors that we posit enter tumors without passing through the intratumoral progenitor cell state. Upon becoming dysfunctional, marked by loss of Granzyme B/Perforin expression and upregulation of inhibitory receptors, CD8 T cells were excluded from lymphonets and localized predominantly to the tumor margin. Altogether, these results shed light on the spatial dynamics associated with a productive response to immunotherapies and open avenues for exploration into the function of lymphonets in supporting cytotoxic CD8 T cell responses in tumors. Citation Format: Megan L. Burger, Giorgio Gaglia, Claire C. Ritch, Danae Rammos, Yang Dai, Grace E. Crossland, Sara Z. Tavana, Simon Warchol, Alex M. Jaeger, Santiago Naranjo, Shannon Coy, Ajit J. Nirmal, Robert Krueger, Jia-Ren Lin, Hanspeter Pfister, Peter K. Sorger, Tyler Jacks, Sandro Santagata. Intratumoral lymphocyte networks harbor TCF1+ PD1+ progenitor CD8 T cells in lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5877.

Combination of two novel blocking antibodies, anti-PD-1 antibody ezabenlimab (BI 754091) and anti-LAG-3 antibody BI 754111, leads to increased immune cell responses

ABSTRACT Upregulation of inhibitory receptors, such as lymphocyte activation gene-3 (LAG-3), may limit the antitumor activity of therapeutic antibodies targeting the programmed cell death protein-1 (PD-1) pathway. We describe the binding properties of ezabenlimab, an anti-human PD-1 antibody, and BI 754111, an anti-human LAG-3 antibody, and assess their activity alone and in combination. Ezabenlimab bound with high affinity to human PD-1 (KD = 6 nM) and blocked the interaction of PD-1 with PD-L1 and PD-L2. Ezabenlimab dose-dependently increased interferon-γ secretion in human T cells expressing PD-1 in co-culture with PD-L1-expressing dendritic cells. Administration of ezabenlimab to human PD-1 knock-in mice dose-dependently inhibited growth of MC38 tumors. To reduce immunogenicity, ezabenlimab was reformatted from a human IgG4 to a chimeric variant with a mouse IgG1 backbone (BI 905725) for further in vivo studies. Combining BI 905725 with anti-mouse LAG-3 antibodies improved antitumor activity versus BI 905725 monotherapy in the MC38 tumor model. We generated BI 754111, which bound with high affinity to human LAG-3 and prevented LAG-3 interaction with its ligand, major histocompatibility complex class II. In an in vitro model of antigen-experienced memory T cells expressing PD-1 and LAG-3, interferon-γ secretion increased by an average 1.8-fold versus isotype control (p = 0.027) with BI 754111 monotherapy, 6.9-fold (p < 0.0001) with ezabenlimab monotherapy and 13.2-fold (p < 0.0001) with BI 754111 plus ezabenlimab. Overall, ezabenlimab and BI 754111 bound to their respective targets with high affinity and prevented ligand binding. Combining ezabenlimab with BI 754111 enhanced in vitro activity versus monotherapy, supporting clinical investigation of this combination (NCT03156114; NCT03433898).

Comparable transforming growth factor beta-mediated immune suppression in ex vivo-expanded natural killer cells from cord blood and peripheral blood: implications for adoptive immunotherapy

T cell-based therapies like genetically modified immune cells expressing chimeric antigen receptors have shown robust anti-cancer activity in vivo, especially in patients with blood cancers. However, extending this approach to an “off-the-shelf” setting can be challenging, as allogeneic T cells carry a significant risk of graft-versus-host disease (GVHD). By contrast, allogeneic natural killer (NK) cells recognize malignant cells without the need for prior antigen exposure and have been used safely in multiple cancer settings without the risk of GVHD. However, similar to T cells, NK cell function is negatively impacted by tumor-induced transforming growth factor beta (TGF-β) secretion, which is a ubiquitous and potent immunosuppressive mechanism employed by most malignancies. Allogeneic NK cells for adoptive immunotherapy can be sourced from peripheral blood (PB) or cord blood (CB), and the authors’ group and others have previously shown that ex vivo expansion and gene engineering can overcome CB-derived NK cells’ functional immaturity and poor cytolytic activity, including in the presence of exogenous TGF-β. However, a direct comparison of the effects of TGF-β-mediated immune suppression on ex vivo-expanded CB- versus PB-derived NK cell therapy products has not previously been performed. Here the authors show that PB- and CB-derived NK cells have distinctive gene signatures that can be overcome by ex vivo expansion. Additionally, exposure to exogenous TGF-β results in an upregulation of inhibitory receptors on NK cells, a novel immunosuppressive mechanism not previously described. Finally, the authors provide functional and genetic evidence that both PB- and CB-derived NK cells are equivalently susceptible to TGF-β-mediated immune suppression. The authors believe these results provide important mechanistic insights to consider when using ex vivo-expanded, TGF-β-resistant PB- or CB-derived NK cells as novel immunotherapy agents for cancer.

Perturbed function of natural killer cells by inflammatory cytokines in acute (AML) and chronic (CML) myeloid leukemias

Abstract Natural killer (NK) cells have the capacity to eliminate malignant cells by releasing cytotoxic granules. Indeed, mature NK cell counts are associated with favorable prognosis in AML and prolonged TKI-free remission in CML. However, NK cell maturation and anti-leukemic activity are suppressed during the disease, and the environmental drivers of this impairment remain unclear. Given an established role of inflammatory cytokines (particularly IL-1β, IL-6, and TNFα) in the progression of myeloid malignancies, we define their impact on NK cell function in chimeric mouse models of leukemia – Flt3-ITD/TET2, AML/ETO9a, Meis/Hox (AML), and BCR-ABL1 (CML), where NK cells do not carry the oncogene. Reflecting clinical observations, NK cell frequencies and numbers are reduced, implying oncogene-independent mechanisms of NK dysfunction in different leukemic subtypes. Moreover, both AML- and CML-exposed NK cells display immature phenotypes, diminished expression of activating receptors, and impaired ex vivo target-specific degranulation. The single cell RNA-seq analysis of CML-exposed NK cells confirmed decreased expression of NK maturation and cytotoxicity markers and upregulation of inhibitory receptors and genes associated with inflammatory cytokine response. Furthermore, IL-6-STAT3 and IL-1β/TNFα transcriptional signatures are enriched during the disease, which is likely to be triggered by leukemic cytokines. Thus, we next found that leukemic serum dampens NK cell degranulation ex vivo, compared to serum from control mice. These data suggest that soluble leukemic factors contribute to NK cell dysfunction and represent an optimal target for NK-boosting immunotherapies across different types of myeloid malignancies.

Beyond PD-L1-Identification of Further Potential Therapeutic Targets in Oral Cancer.

Simple SummaryTumor immunotherapy is rapidly evolving and approved for the treatment of advanced OSCC cases. In addition, the currently observed shift in the use of checkpoint inhibitors from palliative to neoadjuvant treatment may improve survival. However, not all patients respond to currently applied immune checkpoint inhibitors. Therefore, further immune targets for therapeutic approaches are urgently needed. However, there are limited data on immune checkpoint expression in OSCC. This study aimed to perform a comparative analysis of a large number of immune modulators in OSCC compared with healthy controls by NanoString mRNA analysis in order to identify possible targets for therapeutic applications. We were able to ascertain several cellular markers, checkpoints and their correlation, as well as their association with histomorphological parameters. Hence, the study contributes to the understanding of immune escape in OSCC and reveals potential targets for immunotherapy of oral cancer.Background: The involvement of immune cell infiltration and immune regulation in the progression of oral squamous cell carcinoma (OSCC) is shown. Anti-PD-1 therapy is approved for the treatment of advanced OSCC cases, but not all patients respond to immune checkpoint inhibitors. Hence, further targets for therapeutic approaches are needed. The number of identified cellular receptors with immune checkpoint function is constantly increasing. This study aimed to perform a comparative analysis of a large number of immune checkpoints in OSCC in order to identify possible targets for therapeutic application. Materials and Methods: A NanoString mRNA analysis was performed to assess the expression levels of 21 immune regulatory checkpoint molecules in OSCC tissue (n = 98) and healthy oral mucosa (NOM; n = 41). The expression rates were compared between the two groups, and their association with prognostic parameters was determined. Additionally, relevant correlations between the expression levels of different checkpoints were examined. Results: In OSCC tissue, significantly increased expression of CD115, CD163, CD68, CD86, CD96, GITRL, CD28 and PD-L1 was detected. Additionally, a marginally significant increase in CD8 expression was observed. BTLA and PD-1 levels were substantially increased, but the differential expression was not statistically significant. The expression of CD137L was significantly downregulated in OSCC compared to NOM. Correlations between immune checkpoint expression levels were demonstrated, and some occurred specifically in OSCC tissue. Conclusions: The upregulation of inhibitory receptors and ligands and the downregulation of activators could contribute to reduced effector T-cell function and could induce local immunosuppression in OSCC. Increased expression of activating actors of the immune system could be explained by the increased infiltration of myeloid cells and T-cells in OSCC tissue. The analysis contributes to the understanding of immune escape in OSCC and reveals potential targets for oral cancer immunotherapy.

ZAP70, too little, too much can lead to autoimmunity.

Establishing both central and peripheral tolerance requires the appropriate TCR signaling strength to discriminate self‐ from agonist‐peptide bound to self MHC molecules. ZAP70, a cytoplasmic tyrosine kinase, directly interacts with the TCR complex and plays a central and requisite role in TCR signaling in both thymocytes and peripheral T cells. By studying ZAP70 hypomorphic mutations in mice and humans with a spectrum of hypoactive or hyperactive activities, we have gained insights into mechanisms of central and peripheral tolerance. Interestingly, both hypoactive and hyperactive ZAP70 can lead to the development of autoimmune diseases, albeit through distinct mechanisms. Immature thymocytes and mature T cells rely on normal ZAP70 function to complete their development in the thymus and to modulate T cell responses in the periphery. Hypoactive ZAP70 function compromises key developmental checkpoints required to establish central tolerance, allowing thymocytes with potentially self‐reactive TCRs a greater chance to escape negative selection. Such ‘forbidden clones’ may escape into the periphery and may pose a greater risk for autoimmune disease development since they may not engage negative regulatory mechanisms as effectively. Hyperactive ZAP70 enhances thymic negative selection but some thymocytes will, nonetheless, escape negative selection and have greater sensitivity to weak and self‐ligands. Such cells must be controlled by mechanisms involved in anergy, expansion of Tregs, and upregulation of inhibitory receptors or signaling molecules. However, such potentially autoreactive cells may still be able to escape control by peripheral negative regulatory constraints. Consistent with findings in Zap70 mutants, the signaling defects in at least one ZAP70 substrate, LAT, can also lead to autoimmune disease. By dissecting the similarities and differences among mouse models of patient disease or mutations in ZAP70 that affect TCR signaling strength, we have gained insights into how perturbed ZAP70 function can lead to autoimmunity. Because of our work and that of others on ZAP70, it is likely that perturbations in other molecules affecting TCR signaling strength will be identified that also overcome tolerance mechanisms and cause autoimmunity. Delineating these molecular pathways could lead to the development of much needed new therapeutic targets in these complex diseases.

IL-27–producing B-1a cells suppress neuroinflammation and CNS autoimmune diseases

Regulatory B cells (Breg cells) that secrete IL-10 or IL-35 (i35-Breg) play key roles in regulating immunity in tumor microenvironment or during autoimmune and infectious diseases. Thus, loss of Breg function is implicated in development of autoimmune diseases while aberrant elevation of Breg prevents sterilizing immunity, exacerbates infectious diseases, and promotes cancer metastasis. Breg cells identified thus far are largely antigen-specific and derive mainly from B2-lymphocyte lineage. Here, we describe an innate-like IL-27-producing natural regulatory B-1a cell (i27-Breg) in peritoneal cavity and human umbilical cord blood. i27-Bregs accumulate in CNS and lymphoid tissues during neuroinflammation and confers protection against CNS autoimmune disease. i27-Breg immunotherapy ameliorated encephalomyelitis and uveitis through up-regulation of inhibitory receptors (Lag3, PD-1), suppression of Th17/Th1 responses, and propagating inhibitory signals that convert conventional B cells to regulatory lymphocytes that secrete IL-10 and/or IL-35 in eye, brain, or spinal cord. Furthermore, i27-Breg proliferates in vivo and sustains IL-27 secretion in CNS and lymphoid tissues, a therapeutic advantage over administering biologics (IL-10, IL-35) that are rapidly cleared in vivo. Mutant mice lacking irf4 in B cells exhibit exaggerated increase of i27-Bregs with few i35-Bregs, while mice with loss of irf8 in B cells have abundance of i35-Bregs but defective in generating i27-Bregs, identifying IRF8/BATF and IRF4/BATF axis in skewing B cell differentiation toward i27-Breg and i35-Breg developmental programs, respectively. Consistent with its developmental origin, disease suppression by innate i27-Bregs is neither antigen-specific nor disease-specific, suggesting that i27-Breg would be effective immunotherapy for a wide spectrum of autoimmune diseases.

NK Cells in a Tug-of-War With Cancer: The Roles of Transcription Factors and Cytoskeleton.

Natural killer (NK) cells are innate immune cells which play a key role in shaping the immune response against cancer. Initially hailed for their potential to recognise and eliminate tumour cells, their application has been greatly hindered by the immunosuppressive tumour microenvironment (TME) which suppresses NK functions (e.g., cytotoxicity). This dysfunctional state that is accompanied by phenotypic changes such as upregulation of inhibitory receptors and downregulation of activating receptors, forms the basis of what many researchers have referred to as ‘exhausted’ NK cells. However, there is no consensus on whether these phenotypes are sufficient to define an exhausted state of the NK cell. While recent advances in checkpoint inhibition appear to show promise in early-stage pre-clinical studies, much remains to be fully explored and understood in the context of the TME. The TME is where the NK cells are subjected to interaction with various cell types and soluble factors, which could exert an inhibitory effect on NK cytotoxicity. In this review, we provide an overview of the general markers of NK cell exhaustion viz, the surface activating and inhibitory receptors. We also highlight the potential role of T-box transcription factors in characterising such a dysfunctional state and discuss the often-overlooked mechanism of cell cytoskeletal dynamics in regulating NK cell function. These aspects may further contribute to NK exhaustion or NK revival in cancer and may open new avenues to explore cancer treatment strategies.

Neoadjuvant endobronchial delivery of gene mediated cytotoxic immunotherapy (GMCI) for non-small cell lung cancer (NSCLC): Safety and immunologic activity.

9050 Background: GMCI is a tumor-specific immuno-oncology approach implemented through local delivery of aglatimagene besadenovec(AdV-tk) followed by anti-herpetic prodrug. This leads to immunogenic tumor cell death, antigen presenting cell activation, and T cell stimulation resulting in CD8+ T cell dependent protection, as demonstrated in preclinical models and clinical trials in other tumor types. This is the first study to assess endobronchial delivery of AdV-tk for NSCLC. Methods: This Phase I dose escalation trial enrolled patients with suspected NSCLC who were candidates for surgery. A single AdV-tk injection was performed by endobronchial ultrasound (n = 11) or mediastinoscopy (n = 1) during the diagnostic staging procedure 3 weeks prior to surgery. Three dose levels were evaluated: 2.5x 1011, 5x1011, and 1x1012 vector particles (vp) in a 3+3 design. Valacyclovir was administered for 14 days, starting the day after AdV-tk injection. To assess the local and systemic effects of GMCI, immune biomarkers were evaluated in blood and tumor samples before and after GMCI. Results: From 2017-2019, 12 patients (9 men, 3 women, median age 65 [range 55-80]) received GMCI followed by surgery. Average tumor size was 5.1 cm (largest diameter) and final pathologic stage was I (n = 4), II (n = 3), and III (n = 5). Treatment-related adverse events were CTC grade 1 fever (n = 1), flu-like symptoms (n = 1) and nausea/vomiting/diarrhea (n = 1). The only &gt; grade 2 lab abnormality was transient grade 3 lymphopenia (n = 2). A measurable reduction in tumor size was observed in one patient. The average amount of tumor necrosis was 29.4%. Significant infiltration of CD8+T cells (5.2-fold compared to baseline, p = 0.001) was found in tumor 19-22 days after AdV-tk injection. Within the CD8+tumor infiltrating lymphocytes, there was increased expression of CD38 (2.5-fold, p = 0.002), Ki67 (4.8-fold, p = 0.02), PD1 (1.9-fold, p = 0.002), CD39 (2.9-fold, p = 0.04) and CTLA-4 (4.8-fold, p &lt; 0.001), without significant detected differences in Tim3 or TIGIT. Simultaneously, peripheral blood CD8+ cells displayed significant increases in CD38 (3.4-fold, p = 0.006), HLA-DR (4.2-fold, p = 0.002), and Ki67 (5.8-fold, p = 0.017). Conclusions: Intratumoral injection of AdV-tk into lung tumors was safe and feasible. Further, AdV-tk effectively induced peripheral blood and intra-tumoral CD8 T cell activation. Consequent upregulation of inhibitory receptors suggests a potential benefit for combination therapies. Clinical trial information: NCT03131037.

Assessing CD8+ cytotoxic T-cell dysfunction in obesity and its implications in anti-PD-1 blockade therapy

Abstract In chronic conditions, notably cancers, CD8+ cytotoxic T cells gradually lose their effector functions due to constant exposure to antigens and consequent upregulation of inhibitory receptors like PD-1. In this regard, PD-1 blockade therapy was shown to bolster cytotoxic T-cell response and improve disease prognosis in some cases. PD-1 mediated T cell dysfunction is also involved in obesity-induced tumorigenesis. In this study, we used a molecular cytometry approach to better understand the disturbances caused by obesity in immune cells. A detailed single-cell analysis of various cell compartments revealed a unique population of CD8+ cytotoxic T cells in the epididymal fat of mice consuming a high fat diet for 16 weeks. These cells showed high expression of proteins and/or mRNA transcripts for various inhibitory receptors, including PD-1. Based on these results, we created a 28-color panel for high-throughput cell analysis at multiple time points over the 16-week period, using a BD FACSymphony™. The frequency of CD8+PD-1+ T cells in the fat correlated with the numbers of inflammatory cells, suggesting that the dysfunctional phenotype resulted from a growth in inflammation over time. The functional state of the CD8+PD-1+ cells was also examined by challenging these cells with or without PD-1 blockade. Taken together the data delineated a refined application for a broad analysis of immune cells in the fat and provided insights into the effects of obesity-induced T-cell dysfunction in mice. For Research Use Only. Not for use in diagnostic or therapeutic procedures. Class 1 laser product. BD, the BD Logo, FACSymphony is trademark of Becton, Dickinson and Company. © 2019 BD and its subsidiaries. All rights reserved.

Dendritic Cells Control Regulatory T Cell Function Required for Maintenance of Intestinal Tissue Homeostasis.

Dendritic cells (DCs) together with regulatory T cells (Tregs) are essential mediators of immune homeostasis. Disruption of function or frequency of either cell type can lead to fatal autoimmunity. We previously described that mice constitutively lacking DCs (∆DC) develop autoimmunity characterized by reduced body weight, autoantibodies, and pronounced intestinal inflammation. In this study, we show that lack of DCs leads to an altered gene expression profile in peripheral but not thymic Tregs with increased expression of inhibitory receptors. The suppressive function of Tregs from ΔDC mice was impaired in T cell cocultures. In a model of transfer colitis, Tregs from ∆DC mice were only functional in the presence of DCs in recipient mice. Lack of MHC class II on DCs also resulted in upregulation of inhibitory receptors on Tregs, reduced body weight, and elevated serum IgA levels. Further analysis of the IgA response revealed an expansion of IgA+ germinal center B cells and plasma cells in mesenteric lymph nodes and more IgA-coated commensal bacteria in feces of ∆DC mice. Thus, we show a critical role for DCs to establish intestinal homeostasis by regulating Treg function for prevention of spontaneous inflammation.