Human T-cell Activation Research Articles

Camelid-derived Tcell engagers harnessing human γδ Tcells as promising antitumor immunotherapeutic agents.

In the last decade, there has been a surge in developing immunotherapies to enhance the immune system's ability to eliminate tumor cells. Bispecific antibodies known as Tcell engagers (TCEs) present an attractive strategy in this pursuit. TCEs aim to guide cytotoxic Tcells toward tumor cells, thereby inducing a strong activation and subsequent tumor cell lysis. In this study, we investigated the activity of different TCEs on both conventional alpha-beta (αβ) Tcells and unconventional gamma delta (γδ) Tcells. TCEs were built using camelid single-domain antibodies (VHHs) targeting the tumor-associated antigen CEACAM5 (CEA), together with Tcell receptor chains or a CD3 domain. We show that Vγ9Vδ2 Tcells display stronger in vitro antitumor activity than αβ Tcells when stimulated with a CD3xCEA TCE. Furthermore, restricting the activation of fresh human peripheral Tcells to Vγ9Vδ2 Tcells limited the production of protumor factors and proinflammatory cytokines, commonly associated with toxicity in patients. Taken together, our findings provide further insights that γδ Tcell-specific TCEs hold promise as specific, effective, and potentially safe molecules to improve antitumor immunotherapies.

Inhibiting T-Cell-Mediated Rejection of the Porcine Meniscus Through Freeze-Thawing and Downregulating Porcine Xenoreactive Antigen Genes.

Immune rejection presents a significant challenge in xenogenic meniscal transplantation. Pigs are widely regarded as an advantageous tissue source for such transplants, with porcine GGTA1, CMAH, and B4GALNT2 being among the most common xenoreactive antigen (Ag) genes. While some studies have suggested that allogeneic meniscus (AM) transplants may exhibit immunoprivileged properties, our study observed slight immunological rejection has been observed following contact between human meniscal cells (HMCs) and human peripheral blood mononuclear cells (PBMCs). Given the limited systematic research on immune responses following xenograft meniscus transplantation, we established porcine meniscus transplantation (PMT) models to comprehensively assess the immunogenicity of porcine meniscus (PM) from both innate and adaptive immune perspectives. Our investigations confirmed that PMT beneath the epidermis led to innate cell infiltration into the xenografts and T-cell activation in local lymph nodes. T-cell activation upregulated the interleukin (IL)-17 signaling pathway, disrupting collagen organization and metabolic processes, thereby hindering PM regeneration. Using freeze-thaw treatment on PM alleviated T-cell activation post-transplantation by eliminating xenogenic DNA. In vitro findings demonstrated that gene editing in porcine meniscal cells (PMCs) suppressed human T-cell activation by downregulating the expression of xenoreactive Ag genes. These results suggest that GGTA1/CMAH/B4GALNT2 knockout (KO) pigs hold significant promise for advancing the field of meniscal transplantation.

Lymph Node-Targeted Vaccine Boosting of TCR T-cell Therapy Enhances Antitumor Function and Eradicates Solid Tumors.

T-cell receptor (TCR)-modified T-cell therapies have shown promise against solid tumors, but overall therapeutic benefits have been modest due in part to suboptimal T-cell persistence and activation in vivo, alongside potential tumor antigen escape. In this study, we demonstrate an approach to enhance the in vivo persistence and function of TCR T cells through combination with Amphiphile (AMP) vaccination including cognate TCR T peptides. AMP modification improves lymph node targeting of conjugated tumor immunogens and adjuvants, thereby coordinating a robust T cell-activating endogenous immune response. AMP vaccine combination with TCR T-cell therapy led to complete eradication and durable responses against established murine solid tumors refractory to TCR T-cell monotherapy. Enhanced antitumor efficacy was correlated with simultaneous in vivo invigoration of adoptively transferred TCR T cells and in situ expansion of the endogenous antitumor T-cell repertoire. Long-term protection against tumor recurrence in AMP-vaccinated mice was associated with antigen spreading to additional tumor-associated antigens not targeted by vaccination. AMP vaccination further correlated with pro-inflammatory lymph node transcriptional reprogramming and increased antigen presenting-cell maturation, resulting in TCR T-cell expansion and functional enhancement in lymph nodes and solid tumor parenchyma without lymphodepletion. In vitro evaluation of AMP peptides with matched human TCR T cells targeting NY-ESO-1, mutant KRAS, and HPV16 E7 illustrated the clinical potential of AMP vaccination to enhance human TCR T-cell proliferation, activation, and antitumor activity. Taken together, these studies provide rationale and evidence to support clinical evaluation of combining AMP vaccination with TCR T-cell therapies to augment antitumor activity.

A time-resolved meta-analysis of consensus gene expression profiles during human T-cell activation

BackgroundThe coordinated transcriptional regulation of activated T-cells is based on a complex dynamic behavior of signaling networks. Given an external stimulus, T-cell gene expression is characterized by impulse and sustained patterns over the course. Here, we analyze the temporal pattern of activation across different T-cell populations to develop consensus gene signatures for T-cell activation.ResultsHere, we identify and verify general biomarker signatures robustly evaluating T-cell activation in a time-resolved manner. We identify time-resolved gene expression profiles comprising 521 genes of up to 10 disjunct time points during activation and different polarization conditions. The gene signatures include central transcriptional regulators of T-cell activation, representing successive waves as well as sustained patterns of induction. They cover sustained repressed, intermediate, and late response expression rates across multiple T-cell populations, thus defining consensus biomarker signatures for T-cell activation. In addition, intermediate and late response activation signatures in CAR T-cell infusion products are correlated to immune effector cell-associated neurotoxicity syndrome.ConclusionThis study is the first to describe temporally resolved gene expression patterns across T-cell populations. These biomarker signatures are a valuable source, e.g., monitoring transcriptional changes during T-cell activation with a reasonable number of genes, annotating T-cell states in single-cell transcriptome studies, or assessing dysregulated functions of human T-cell immunity.

Apoptosis in mesenchymal stromal cells activates an immunosuppressive secretome predicting clinical response in Crohn’s disease

Invivo apoptosis of human mesenchymal stromal cells (MSCs) plays a critical role in delivering immunomodulation. Yet, caspase activity not only mediates the dying process but also death-independent functions that may shape the immunogenicity of apoptotic cells. Therefore, a better characterization of the immunological profile of apoptotic MSCs (ApoMSCs) could shed light on their mechanistic action and therapeutic applications. We analyzed the transcriptomes of MSCs undergoing apoptosis and identified several immunomodulatory factors and chemokines dependent on caspase activation following Fas stimulation. The ApoMSC secretome inhibited human Tcell proliferation and activation, and chemoattracted monocytes invitro. Both immunomodulatory activities were dependent on the cyclooxygenase2 (COX2)/prostaglandin E2 (PGE2) axis. To assess the clinical relevance of ApoMSC signature, we used the peripheral blood mononuclear cells (PBMCs) from a cohort of fistulizing Crohn's disease (CD) patients who had undergone MSC treatment (ADMIRE-CD). Compared with healthy donors, MSCs exposed to patients' PBMCs underwent apoptosis and released PGE2 in a caspase-dependent manner. Both PGE2 and apoptosis were significantly associated with clinical responses to MSCs. Our findings identify a new mechanism whereby caspase activation delivers ApoMSC immunosuppression. Remarkably, such molecular signatures could implicate translational tools for predicting patients' clinical responses to MSC therapy in CD.

Clinical Staphylococcus aureus inhibits human T-cell activity through interaction with the PD-1 receptor.

Therapies that target and aid the host immune defense to repel cancer cells or invading pathogens are rapidly emerging. Antibiotic resistance is among the largest threats to human health globally. Staphylococcus aureus (S. aureus) is the most common bacterial infection, and it poses a challenge to the healthcare system due to its significant ability to develop resistance toward current available therapies. In long-term infections, S. aureus further adapt to avoid clearance by the host immune defense. In this study, we discover a new interaction that allows S. aureus to avoid elimination by the immune system, which likely supports its persistence in the host. Moreover, we find that blocking the specific receptor (PD-1) using antibodies significantly relieves the S. aureus-imposed inhibition. Our findings suggest that therapeutically targeting PD-1 is a possible future strategy for treating certain antibiotic-resistant staphylococcal infections.

Large extracellular vesicles derived from human regulatory macrophages (L-EVMreg) attenuate CD3/CD28-induced T-cell activation in vitro

Macrophages belong to the innate immune system, and we have recently shown that in vitro differentiated human regulatory macrophages (Mreg) release large extracellular vesicles (L-EVMreg) with an average size of 7.5 μm which regulate wound healing and angiogenesis in vitro. The aim of this study was to investigate whether L-EVMreg also affect the CD3/CD28-mediated activation of T-cells. Mreg were differentiated using blood monocytes and L-EVMreg were isolated from culture supernatants by differential centrifugation. Activation of human T-cells was induced by CD3/CD28-coated beads in the absence or presence of Mreg or different concentrations of L-EVMreg. Inhibition of T-cell activation was quantified by flow cytometry and antibodies directed against the T-cell marker granzyme B. Phosphatidylserine (PS) exposure on the surface of Mreg and L-EVMreg was analyzed by fluorescence microscopy. Incubation of human lymphocytes with CD3/CD28 beads resulted in an increase of cell size, cell granularity, and number of granzyme B–positive cells (P < 0.05) which is indicative of T-cell activation. The presence of Mreg (0.5 × 106 Mreg/ml) led to a reduction of T-cell activation (number of granzyme B–positive cells; P < 0.001), and a similar but less pronounced effect was also observed when incubating activated T-cells with L-EVMreg (P < 0.05 for 3.2 × 106 L-EVMreg/ml). A differential analysis of the effects of Mreg and L-EVMreg on CD4+ and CD8+ T-cells showed an inhibition of CD4+ T-cells by Mreg (P < 0.01) and L-EVMreg (P < 0.05 for 1.6 × 106 L-EVMreg/ml; P < 0.01 for 3.2 × 106 L-EVMreg/ml). A moderate inhibition of CD8+ T-cells was observed by Mreg (P < 0.05) and by L-EVMreg (P < 0.01 for 1.6 × 106 L-EVMreg/ml and 3.2 × 106 L-EVMreg/ml). PS was restricted to confined regions of the Mreg surface, while L-EVMreg showed strong signals for PS in the exoplasmic leaflet. L-EVMreg attenuate CD3/CD28-mediated activation of CD4+ and CD8+ T-cells. L-EVMreg may have clinical relevance, particularly in the treatment of diseases associated with increased T-cell activity.Key messagesMreg release large extracellular vesicles (L-EVMreg) with an average size of 7.5 µmL-EVMreg exhibit phosphatidylserine positivityL-EVMreg suppress CD4+ and CD8+ T-cellsL-EVMreg hold clinical potential in T-cell-related diseases

Effect of Systemic Administration of CD4+ T cells and Local Administration of T-cell Stimulants on T-cell Activity in Psoriatic Skin Xenografts on NOG Mice.

Immunodeficient mice engrafted with psoriatic human skin are widely used for the preclinical evaluation of new drug candidates. However, the T-cell activity, including the IL23/IL17 pathway, declines in the graft over time after engraftment, which likely affects the study data. Here, we investigated whether the T-cell activity could be sustained in xenografted psoriatic skin by local stimulation of T cells or systemic injection of autologous CD4 + T cells. We surgically transplanted human psoriatic skin from 5 untreated patients onto female NOG mice. Six days after surgery, mice received an intraperitoneal injection of autologous human CD4+ T cells, a subcutaneous injection under the grafts of a T-cell stimulation cocktail consisting of recombinant human IL2, human IL23, antihuman CD3, and antihuman CD28, or saline. Mice were euthanized 21 d after surgery and spleens and graft biopsies were collected for analysis. Human T cells were present in the grafts, and 60% of the grafts maintained the psoriatic phenotype. However, neither local T-cell stimulation nor systemic injection of autologous CD4+ T cells affected the protein levels of human IL17A, IL22, IFN γ, and TNF α in the grafts. In conclusion, NOG mice seem to accept psoriatic skin grafts, but the 2 approaches studied here did not affect human T-cell activity in the grafts. Therefore, NOG mice do not appear in this regard to be superior to other immunodeficient mice used for psoriasis xenografts.

Cell therapy biomanufacturing: integrating biomaterial and flow-based membrane technologies for production of engineered T-cells.

Adoptive T-cell therapies (ATCTs) are increasingly important for the treatment of cancer, where patient immune cells are engineered to target and eradicate diseased cells. The biomanufacturing of ATCTs involves a series of time-intensive, lab-scale steps, including isolation, activation, genetic modification, and expansion of a patient's T-cells prior to achieving a final product. Innovative modular technologies are needed to produce cell therapies at improved scale and enhanced efficacy. In this work, well-defined, bioinspired soft materials were integrated within flow-based membrane devices for improving the activation and transduction of T cells. Hydrogel coated membranes (HCM) functionalized with cell-activating antibodies were produced as a tunable biomaterial for the activation of primary human T-cells. T-cell activation utilizing HCMs led to highly proliferative T-cells that expressed a memory phenotype. Further, transduction efficiency was improved by several fold over static conditions by using a tangential flow filtration (TFF) flow-cell, commonly used in the production of protein therapeutics, to transduce T-cells under flow. The combination of HCMs and TFF technology led to increased cell activation, proliferation, and transduction compared to current industrial biomanufacturing processes. The combined power of biomaterials with scalable flow-through transduction techniques provides future opportunities for improving the biomanufacturing of ATCTs.

Sustaining the T-cell activity in xenografted psoriasis skin.

Xenografting of psoriasis skin onto immune deficient mice has been widely used to obtain proof-of-principle of new drug candidates. However, the lack of human T-cell activity in the grafts limits the use of the model. Here, we show that xenografting of lesional skin from psoriasis patients onto human IL-2 NOG mice results in increased numbers of human CD3+ cells in the grafts, axillary lymph nodes and blood from human IL-2 NOG mice compared to C.B-17 scid and NOG mice. In addition, disease relevant human cytokine levels were higher in graft lysates and serum from human IL-2 NOG mice. However, the epidermis was lacking and no efficacy of ustekinumab, a human anti-P40 antibody targeting both IL-12 and IL-23, was shown. Thus, despite the sustained T-cell activity, the model needs further investigations and validation to capture more aspects of psoriasis.

The Medawar Prize Acceptance Speech 2022.

The Medawar Prize Acceptance Speech 2022.

Evaluation of Common Gamma Chain Cytokine Signaling Blockade with REGN7257, an Interleukin 2 Receptor Gamma (IL2RG) Monoclonal Antibody, on Immune Cell Populations in Monkey and Human

The common γ chain cytokine receptor (γc; IL2RG) family of cytokines includes interleukin 2 (IL2), IL4, IL7, IL9, IL15, and IL21. This set of cytokines exhibits broad pleiotropic actions on both the innate and adaptive immune system with each cytokine sharing the IL2RG chain as part of its signaling receptor complex. Mutations in the IL2RG gene result in X-linked severe combined immunodeficiency (XSCID) in humans, whereby patients present with dramatically diminished numbers of T-cells and NK-cells, and dysfunctional B-cells. Given the crucial role for γc cytokines in the development and function of lymphocytes, modulating their activities may offer therapeutic potential in a range of immune-mediated diseases. To understand the effects of γc cytokine blockade on immune cells including T-cell subsets, we utilized REGN7257, a fully human IL2RG monoclonal antibody that inhibits γc cytokine-induced signaling, and tested its ability to suppress immune cell populations and their functions in cynomolgus monkey and human. Mixed lymphocyte reaction assays were performed to look at the impact of γc cytokine signaling blockade on activation/proliferation of human T-cells. To further analyze the contribution of γc cytokines to T-cell differentiation and function, we performed in vitro transcriptomic studies on human peripheral blood mononuclear cells stimulated with anti-CD3/CD28 beads. In addition, we assessed the effects of γc cytokine signaling blockade on both human T-cell and NK-cell effector functions in target cell killing assays. Finally, we evaluated the effects of γc cytokine signaling blockade with REGN7257 on immune cell populations and their activation/proliferation status in cynomolgus monkeys by flow cytometry. γc cytokine signaling blockade with REGN7257 demonstrated potent inhibition of allogeneic responses in mixed human lymphocyte reaction assays, by preventing T-cell activation and proliferation. Two-tailed gene set enrichment analysis identified 4 distinct patterns of significantly enriched gene sets that were impacted by γc cytokine signaling upon CD3/CD28-induced activation of human T-cells: gene sets involved in inflammatory responses, differentiation and proliferation, activation and immune responses, as well as those related to adhesion and migration. These signatures were blocked with REGN7257 treatment. In addition, REGN7257 potently blocked activation (i.e. IFNG production) and cytotoxic activity of both human T-cells and NK-cells in in vitro target cell killing assays. Finally, the impact of γc cytokine signaling blockade on lymphocyte populations in vivo were investigated in cynomolgus monkeys in a single dose pharmacokinetic/pharmacodynamic study and a repeat-dose toxicology study. Inhibition of γc cytokine signaling with REGN7257 in cynomolgus monkeys reduced circulating T-cells and NK-cells, without impacting B-cells, granulocytes, platelets or red blood cells. γc cytokine signaling blockade led to a reduction in both peripheral CD4+ and CD8+ T-cell counts with effector memory T-cells being the most impacted T-cell population studied. Similarly to that observed in human T-cell MLR assays, both activated and proliferating T-cells were reduced in monkeys dosed with REGN7257. Taken together, these pharmacological observations highlight the major role for γc cytokine signaling in maintenance of lymphocyte populations (i.e. NK-cells and T-cells), but not other immune cell populations, such as granulocytes, platelets and red blood cells. Furthermore, our data highlight the importance of γc cytokines in driving functions of cynomolgus monkeys and human effector NK and T-cells, opening a potential new route for the management of immune-mediated diseases.

Abstract LB220: The anticalin-antibody bispecific PRS-352/S095025 strongly stimulates human CD4+ T cells in a PD-L1-dependent manner

Abstract Background: OX40, a member of the tumor necrosis factor receptor superfamily, is a costimulatory molecule promoting survival and cytokine release by effector T cells and inhibiting human regulatory T-cell activity. OX40 activation has been explored as a potential treatment for solid malignancies, using mainly agonistic anti-OX40 antibodies. These antibodies rely on Fc gamma receptor (FcγR)-mediated crosslinking to activate the OX40 pathway and have shown so far only limited antitumoral efficacy in clinical settings. One underlying hypothesis is that FcγR crosslinking of these antibodies may not allow optimal activation of the OX40 pathway to fully exploit the potential of this target for immunotherapy of cancer. To overcome this limitation, we designed PRS-352/S095025, a strong PD-L1-dependent OX40 agonistic bispecific molecule, that allows combination of OX40 costimulation and PD-L1 blockade. Methods: We generated an Anticalin protein that binds with high affinity to human OX40. Anticalin® proteins are approximately 18-20 kDa protein therapeutics derived from human lipocalins that can be engineered to bind with high affinity and specificity to different targets. The PRS-352/S095025 bispecific fusion protein was obtained by genetic fusion of OX40-targeting Anticalin protein to a PD-L1-targeting monoclonal antibody with a modified IgG4 backbone, allowing an optimal activation of OX40 in the presence of PD-L1, but not FcγRs. Results: We showed that PRS-352/S095025 retains binding to PD-L1 like the anti PD-L1 antibody backbone alone and binds with high affinity to both human and cynomolgus OX40. We showed that PRS-352/S095025 inhibited the PD-1/PD-L1 pathway, as the parental PD-L1 targeting antibody and an approved anti PD-L1 antibody. In addition to its PD-L1 blocking properties, we showed that PRS-352/S095025 activates OX40, and that this activity is dependent on the expression of PD-L1 and not on FcγR, consistent with the desired mechanism of action. Furthermore, we demonstrated that PRS-352/S095025 activity is superior to both PD-L1 benchmark antibodies and to the combination of clinically relevant OX40 and PD-L1 benchmark antibodies. We could also show that PRS352/S095025 strongly stimulated human CD4 T cells. In vivo, we showed that PRS-352/S095025 has a PK profile similar to that of the PD-L1 antibody alone. Conclusions: We provide here the preclinical characterization of the fusion protein PRS-352/S095025, a bispecific molecule composed of a PD-L1 blocking moiety and an Anticalin protein agonizing OX40. In vitro, this molecule showed the desired MoA, PD-L1 blocking and potent OX40 agonism driven by binding to PD-L1, with superior activity to a clinical stage OX40 agonist. Citation Format: Lucia Pattarini, Marina Pavlidou, Aizea Kastresana Morales, Janet Peper-Gabriel, Matthieu Riviere, Didier Demarles, Alix Scholer-Dahirel, Veronique Blanc, Shane Olwill. The anticalin-antibody bispecific PRS-352/S095025 strongly stimulates human CD4+ T cells in a PD-L1-dependent manner [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 LB220.

Production of Triple-Gene (GGTA1, B2M and CIITA)-Modified Donor Pigs for Xenotransplantation.

Activation of human immune T-cells by swine leukocyte antigens class I (SLA-I) and class II (SLA-II) leads to xenograft destruction. Here, we generated the GGTA1, B2M, and CIITA (GBC) triple-gene-modified Diannan miniature pigs, analyzed the transcriptome of GBC-modified peripheral blood mononuclear cells (PBMCs) in the pig's spleen, and investigated their effectiveness in anti-immunological rejection. A total of six cloned piglets were successfully generated using somatic cell nuclear transfer, one of them carrying the heterozygous mutations in triple genes and the other five piglets carrying the homozygous mutations in GGTA1 and CIITA genes, but have the heterozygous mutation in the B2M gene. The autopsy of GBC-modified pigs revealed that a lot of spot bleeding in the kidney, severe suppuration and necrosis in the lungs, enlarged peripulmonary lymph nodes, and adhesion between the lungs and chest wall were found. Phenotyping data showed that the mRNA expressions of triple genes and protein expressions of B2M and CIITA genes were still detectable and comparable with wild-type (WT) pigs in multiple tissues, but α1,3-galactosyltransferase was eliminated, SLA-I was significantly decreased, and four subtypes of SLA-II were absent in GBC-modified pigs. In addition, even in swine umbilical vein endothelial cells (SUVEC) induced by recombinant porcine interferon gamma (IFN-γ), the expression of SLA-I in GBC-modified pig was lower than that in WT pigs. Similarly, the expression of SLA-II DR and DQ also cannot be induced by recombinant porcine IFN-γ. Through RNA sequencing (RNA-seq), 150 differentially expressed genes were identified in the PBMCs of the pig's spleen, and most of them were involved in immune- and infection-relevant pathways that include antigen processing and presentation and viral myocarditis, resulting in the pigs with GBC modification being susceptible to pathogenic microorganism. Furthermore, the numbers of human IgM binding to the fibroblast cells of GBC-modified pigs were obviously reduced. The GBC-modified porcine PBMCs triggered the weaker proliferation of human PBMCs than WT PBMCs. These findings indicated that the absence of the expression of α1,3-galactosyltransferase and SLA-II and the downregulation of SLA-I enhanced the ability of immunological tolerance in pig-to-human xenotransplantation.

Priming treatment with T-cell redirecting bispecific antibody ERY974 reduced cytokine induction without losing cytotoxic activity in vitro by changing the chromatin state in T cells

CD3 bispecific constructs are anticipated to become an important form of cancer immunotherapy, but they frequently cause cytokine release syndrome (CRS) that is difficult to manage in clinical contexts. A combination of intra-patient dose escalation and immunosuppressive treatment is widely used to mitigate CRS. Studies suggest that CRS after subsequent doses of CD3 bispecific constructs is less severe than after the priming dose, and that step-up dosing reduces cytokine levels in animals and humans. However, the mechanism underlying the reduced cytokine induction after priming treatment with CD3 bispecific constructs is unclear. To understand human T-cell activation and chromatin states after priming treatment with CD3 bispecific construct targeting CD3ɛ and glypican 3 (ERY974), we examined cytokine levels, cytokine mRNA expression, CD3ɛ expression, CD3-mediated signal transduction, T cell activation markers, cytotoxicity against target cells, and chromatin states in T cells after ERY974 priming treatment or negative control. The second ERY974 treatment decreased cytokines on Day 8, and ERY974 priming treatment changed the chromatin state in T cells. CD3ɛ expression, CD3-mediated signal transduction, T cell activation markers, and cytotoxicity were similar between the priming treatment with ERY974 and negative control. The present study suggests that chromatin state changes in T cells after the priming treatment was a pivotal factor in the mitigation of cytokine release after the second ERY974 treatment.

Novel phosphatidylserine-binding molecule enhances antitumor T-cell responses by targeting immunosuppressive exosomes in human tumor microenvironments

BackgroundThe human tumor microenvironment (TME) is a complex and dynamic milieu of diverse acellular and cellular components, creating an immunosuppressive environment, which contributes to tumor progression. We have previously shown...

Abstract 1691: CD137 (4-1BB) costimulation of CD8 T cells is more potent when provided in cis than in trans with respect to CD3-TCR stimulation

Abstract CD137 (4-1BB, TNFSFR9) costimulation provides antigen-primed T cells with increased survival, proliferation and effector functions as well as metabolic advantages. These immunobiological mechanisms are being harnessed for cancer immunotherapy with agonist CD137-binding and crosslinking-inducing agents that elicit CD137 intracellular signaling. In this study, side-by-side comparisons show that provision of CD137 costimulation in-cis with regard to the TCR-CD3-ligating cells is far superior to that provided in-trans in terms of T-cell activation, proliferation, survival, cytokine secretion and mitochondrial fitness in mice and humans. Cis ligation of CD137 relative to the TCR-CD3 complex results in more intense canonical and non-canonical NF-κB signaling and provides a more robust induction of cell-cycle and DNA damage repair gene expression programs. Superiority of cis versus trans CD137-costimulation is readily observed in vivo and is relevant for understanding the immunotherapeutic effects of CAR T cells and CD137 agonistic therapies currently undergoing clinical trials, which provide costimulation either in cis or in trans. Citation Format: Itziar Otano, Arantza Azpilikueta, Javier Glez-Vaz, Peter Ellmark, Sara Fritzell, Gabriela Fernandez-Hoyos, Michelle Hase Nelson, Maite Alvarez, María del Carmen Ochoa, Elixabet Bolaños, Doina Cuculescu, Patricia Jauregui, Sandra Sanchez, Iñaki Etxeberría, María E. Rodriguez-Ruiz, Miguel F. Sanmamed, Alvaro Teijeira, Pedro Berraondo, Igancio Melero. CD137 (4-1BB) costimulation of CD8 T cells is more potent when provided in cis than in trans with respect to CD3-TCR stimulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1691.

IspH inhibitors kill Gram-negative bacteria and mobilize immune clearance.

Isoprenoids are vital to all organisms in supporting core functions of life, like respiration and membrane stability.1 IspH, an enzyme in the methyl erythritol phosphate pathway of isoprenoid synthesis, is essential to gram-negative bacteria, mycobacteria and apicomplexans.2,3 The IspH substrate, HMBPP, is not produced in humans and other metazoans and activates cytotoxic Vγ9Vδ2 T-cells in humans and primates at extremely low concentrations.4-6 We describe novel IspH inhibitors and through structure-guided analog design, refine their potency to nanomolar levels. We have modified these into prodrugs for delivery into bacteria and report that they kill clinical isolates of several multidrug resistant bacterial species such as Acinetobacter, Pseudomonas, Klebsiella, Enterobacter, Vibrio, Shigella, Salmonella, Yersinia, Mycobacterium and Bacillus, while being relatively non-toxic to mammalian cells. Proteomic analysis reveals that bacteria treated with prodrugs resemble those with conditional IspH knockdown. Notably, these prodrugs also cause expansion and activation of human Vγ9Vδ2 T-cells in a humanized mouse model of bacterial infection. These IspH prodrugs synergize direct antibiotic killing with a simultaneous rapid immune response by cytotoxic γδ T-cells, which may limit the rise of antibiotic resistant bacterial populations.

Quantitative and time-resolved miRNA pattern of early human T cell activation

T cells are central to the immune response against various pathogens and cancer cells. Complex networks of transcriptional and post-transcriptional regulators, including microRNAs (miRNAs), coordinate the T cell activation process. Available miRNA datasets, however, do not sufficiently dissolve the dynamic changes of miRNA controlled networks upon T cell activation. Here, we established a quantitative and time-resolved expression pattern for the entire miRNome over a period of 24 h upon human T-cell activation. Based on our time-resolved datasets, we identified central miRNAs and specified common miRNA expression profiles. We found the most prominent quantitative expression changes for miR-155-5p with a range from initially 40 molecules/cell to 1600 molecules/cell upon T-cell activation. We established a comprehensive dynamic regulatory network of both the up- and downstream regulation of miR-155. Upstream, we highlight IRF4 and its complexes with SPI1 and BATF as central for the transcriptional regulation of miR-155. Downstream of miR-155-5p, we verified 17 of its target genes by the time-resolved data recorded after T cell activation. Our data provide comprehensive insights into the range of stimulus induced miRNA abundance changes and lay the ground to identify efficient points of intervention for modifying the T cell response.

Activation of Human γδ T Cells: Modulation by Toll-Like Receptor 8 Ligands and Role of Monocytes.

Background: Human Vγ9Vδ2 γδ T cells can kill a variety of cancer cells and have attracted substantial interest for cancer immunotherapy. Toll-like receptor (TLR) ligands are promising adjuvants for cancer immunotherapy, but TLR7/8 ligand Resiquimod has been shown to inhibit CD4 T-cell activation in a monocyte-dependent manner. Therefore, we studied the modulation of human γδ T-cell activation by TLR7/8 ligands. Methods: Peripheral blood mononuclear cells (PBMC) or purified γδ T cells together with purified monocytes were stimulated with zoledronic acid or phosphoantigens in the absence or presence of various imidazoquinoline TLR7 or TLR8 agonists. Read-out systems included interferon-γ induction and cellular expansion of γδ T cells, as well as viability, cell surface antigen modulation, and IL-1β and TNF-α production of monocytes. Results: TLR8 ligand TL8-506 and TLR7/8 ligand Resiquimod (but not TLR7 ligands) rapidly induced IFN-γ expression in γδ T cells within PBMC, and co-stimulated phosphoantigen-induced IFN-γ expression in γδ T cells. On the other hand, TLR8 ligands potently suppressed γδ T-cell expansion in response to zoledronic acid and phosphoantigen. Purified monocytes secreted large amounts of IL-1β and TNF-α when stimulated with TLR8 ligands but simultaneously underwent substantial cell death after 24 h. Conclusions: TLR8 ligand-activated monocytes potently co-stimulate early γδ T-cell activation but failed to provide accessory cell function for in vitro expansion of γδ T cells.