Search

Antigen presentation via major histocompatibility complex class I (MHC-I) molecules is essential for surveillance by the adaptive immune system. Central to this process is the peptide-loading complex (PLC), which translocates peptides from the cytosol to the endoplasmic reticulum and catalyzes peptide loading and proofreading of peptide-MHC-I (pMHC-I) complexes. Despite its importance, the impact of individual PLC components on the presented pMHC-I complexes is still insufficiently understood. Here, we used stoichiometrically defined antibody-nanobody complexes and engineered soluble T cell receptors (sTCRs) to quantify different MHC-I allomorphs and defined pMHC-I complexes, respectively. Thereby, we uncovered distinct effects of individual PLC components on the pMHC-I surface pool. Knockouts of components of the PLC editing modules, namely tapasin, ERp57, or calreticulin, changed the MHC-I surface composition to a reduced proportion of HLA-A*02:01 presentation compensated by a higher ratio of HLA-B*40:01 molecules. Intriguingly, these knockouts not only increased the presentation of suboptimally loaded HLA-A*02:01 complexes but also elevated the presentation of high-affinity peptides overexpressed in the cytosol. Our findings suggest that the components of the PLC editing module serve a dual role, acting not only as peptide proofreaders but also as limiters for abundant peptides. This dual function ensures the presentation of a broad spectrum of antigenic peptides. [ABSTRACT FROM AUTHOR]

Chlamydia trachomatis (Ct) is the most common cause of genital tract infections as well as preventable blindness worldwide. Pattern recognition receptors such as toll-like receptors (TLRs) represent the initial step in recognizing pathogenic microorganisms and are crucial for the initiation of an appropriate immune response. However, our understanding of TLR-signaling in Chlamydia-infected immune cells is incomplete. For a better comprehension of pathological inflammatory responses, robust models for interrogating TLR-signaling upon chlamydial infections are needed. To analyze the TLR response, we developed and utilized a highly sensitive and selective fluorescent transcriptional cellular reporter system to measure the activity of the transcription factor NF-κB. Upon incubation of the reporter cells with different preparations of Ct, we were able to pinpoint which components of TLRs are involved in the recognition of Ct. We identified CD14 associated with unique characteristics of different serovars as the crucial factor of the TLR4/CD14/MD2 complex for Ct-mediated activation of the NF-κB pathway. Furthermore, we found the TLR4/CD14/MD2 complex to be decisive for the uptake of Ct-derived lipopolysaccharides but not for infection and replication of Ct. Imaging flow cytometry provided information about inclusion formation in myeloid- as well as lymphocytic cells and was highest for Ct L2 with at least 25% of inclusion forming cells. Ct E inclusion formation was eminent in Jurkat cells without CD14 expression (11.1%). Thus, our model enables to determine Ct uptake and signal induction by pinpointing individual components of the recognition and signaling pathways to better understand the immune response towards infectious pathogens. [ABSTRACT FROM AUTHOR]

Simple Summary: Therapies with monoclonal antibodies (mAbs) targeting tumor-associated antigens (TAAs) or immune checkpoint inhibitors (ICIs) have revolutionized cancer treatment. Nevertheless, the inevitable development of resistance and the failure to respond are among this approach's disadvantages, limiting the duration of disease- or progression-free and overall survival. As an alternative to therapeutically efficacious monoclonal antibodies, the concept of active immunization with vaccines has been repeatedly discussed. In particular, mimotopes, representing the B cell epitope of therapeutic mAbs, have been shown to induce immunological memory and effectively produce antibodies with similar functionality to the respective mAbs/ICIs. This review focuses on a new frontier of vaccinations directed against two cancer-relevant targets, addresses concerns about the safety of active immunization targeting PD-1 and discusses limitations and outlooks. The application of monoclonal antibodies (mAbs), targeting tumor-associated (TAAs) or tumor-specific antigens or immune checkpoints (ICs), has shown tremendous success in cancer therapy. However, the application of mAbs suffers from a series of limitations, including the necessity of frequent administration, the limited duration of clinical response and the emergence of frequently pronounced immune-related adverse events. However, the introduction of mAbs has also resulted in a multitude of novel developments for the treatment of cancers, including vaccinations against various tumor cell-associated epitopes. Here, we reviewed recent clinical trials involving combination therapies with mAbs targeting the PD-1/PD-L1 axis and Her-2/neu, which was chosen as a paradigm for a clinically highly relevant TAA. Our recent findings from murine immunizations against the PD-1 pathway and Her-2/neu with peptides representing the mimotopes/B cell peptides of therapeutic antibodies targeting these molecules are an important focus of the present review. Moreover, concerns regarding the safety of vaccination approaches targeting PD-1, in the context of the continuing immune response, as a result of induced immunological memory, are also addressed. Hence, we describe a new frontier of cancer treatment by active immunization using combined mimotopes/B cell peptides aimed at various targets relevant to cancer biology. [ABSTRACT FROM AUTHOR]

The engagement of the herpesvirus entry mediator (HVEM, TNFRSF14) by the B and T lymphocyte attenuator (BTLA) represents a unique interaction between an activating receptor of the TNFR-superfamily and an inhibitory receptor of the Ig-superfamily. BTLA and HVEM have both been implicated in the regulation of human T cell responses, but their role is complex and incompletely understood. Here, we have used T cell reporter systems to dissect the complex interplay of HVEM with BTLA and its additional ligands LIGHT and CD160. Co-expression with LIGHT or CD160, but not with BTLA, induced strong constitutive signaling via HVEM. In line with earlier reports, we observed that in cis interaction of BTLA and HVEM prevented HVEM costimulation by ligands on surrounding cells. Intriguingly, our data indicate that BTLA mediated inhibition is not impaired in this heterodimeric complex, suggesting a dominant role of BTLA co-inhibition. Stimulation of primary human T cells in presence of HVEM ligands indicated a weak costimulatory capacity of HVEM potentially owed to its in cis engagement by BTLA. Furthermore, experiments with T cell reporter cells and primary T cells demonstrate that HVEM antibodies can augment T cell responses by concomitantly acting as checkpoint inhibitors and co-stimulation agonists. [ABSTRACT FROM AUTHOR]

NKG2A has emerged as a new immunotherapy target and its blockade with the novel immune checkpoint inhibitor (ICI) monalizumab can boost both NK cell and CD8+ T cell responses. NKG2A forms heterodimers with CD94 and binds to the human non‐classical MHC class I molecule HLA‐E. HLA‐E forms complexes with a limited set of peptides mainly derived from the leader sequences of the classical MHC class I molecules (HLA‐A, HLA‐B and HLA‐C) and the non‐classical class I paralogue HLA‐G, and it is well established that the interaction between CD94/NKG2x receptors and its ligand HLA‐E is peptide‐sensitive. Here, we have evaluated peptide dependence of NKG2A‐mediated inhibition and the efficiency of interference by monalizumab in a transcriptional T cell reporter system. NKG2A inhibition was mediated by cell‐expressed HLA‐E molecules stably presenting disulfate‐trapped peptide ligands. We show that different HLA‐class I leader peptides mediate varying levels of inhibition. We have used NKG2A/NKG2C chimeric receptors to map the binding site of NKG2A and NKG2C blocking antibodies. Furthermore, we determined the functional EC50 values of blocking NKG2A antibodies and show that they greatly depend on the HLA‐leader peptide presented by HLA‐E. Monalizumab was less effective in augmenting NK cell‐mediated killing of target cells displaying HLA‐G peptide on HLA‐E, than cells expressing HLA‐E complexed with HLA‐A, HLA‐B and HLA‐C peptides. Our results indicate that peptides displayed by HLA‐E molecules on tumour cells might influence the effectivity of NKG2A‐ICI therapy and potentially suggest novel approaches for patient stratification, for example, based on tumoral HLA‐G levels. [ABSTRACT FROM AUTHOR]

Toll-like receptors (TLRs) are primary pattern recognition receptors (PRRs), which recognize conserved microbial components. They play important roles in innate immunity but also in the initiation of adaptive immune responses. Impurities containing TLR ligands are a frequent problem in research but also for the production of therapeutics since TLR ligands can exert strong immunomodulatory properties even in minute amounts. Consequently, there is a need for sensitive tools to detect TLR ligands with high sensitivity and specificity. Here we describe the development of a platform based on a highly sensitive NF-κB::eGFP reporter Jurkat JE6-1 T cell line for the detection of TLR ligands. Ectopic expression of TLRs and their coreceptors and CRISPR/Cas9-mediated deletion of endogenously expressed TLRs was deployed to generate reporter cell lines selectively expressing functional human TLR2/1, TLR2/6, TLR4 or TLR5 complexes. Using well-defined agonists for the respective TLR complexes we could demonstrate high specificity and sensitivity of the individual reporter lines. The limit of detection for LPS was below 1 pg/mL and ligands for TLR2/1 (Pam3CSK4), TLR2/6 (Fsl-1) and TLR5 (flagellin) were detected at concentrations as low as 1.0 ng/mL, 0.2 ng/mL and 10 pg/mL, respectively. We showed that the JE6-1 TLR reporter cells have the utility to characterize different commercially available TLR ligands as well as more complex samples like bacterially expressed proteins or allergen extracts. Impurities in preparations of microbial compounds as well as the lack of specificity of detection systems can lead to erroneous results and currently there is no consensus regarding the involvement of TLRs in the recognition of several molecules with proposed immunostimulatory functions. This reporter system represents a highly suitable tool for the definition of structural requirements for agonists of distinct TLR complexes. [ABSTRACT FROM AUTHOR]

The soluble cytoplasmic tail of CD45 (ct‐CD45) is a cleavage fragment of CD45, that is generated during the activation of human phagocytes. Upon release to the extracellular space, ct‐CD45 binds to human T cells and inhibits their activation in vitro. Here, we studied the potential role of TLR4 as a receptor for ct‐CD45. Treatment of Jurkat TLR4/CD14 reporter cells with ct‐CD45 induced the upregulation of the reporter gene NFκB‐eGFP and could be blocked by inhibitors of TLR4 signaling. Conversely, ct‐CD45 did not promote the NFκB‐controlled eGFP induction in reporter cells expressing TLR1, TLR2, and TLR6 transgenes and did not lead to the activation of the transcription factors NFκB, AP‐1, and NFAT in a Jurkat reporter cell line expressing endogenous TLR5. Moreover, ct‐CD45 binds to recombinant TLR4 in an in vitro assay and this association was reduced in the presence of oxidized 1‐palmitoyl‐2‐arachidonyl‐sn‐glycero‐3‐phosphorylcholine. Blockade of TLR4 with mAb HTA125 partially reversed the ct‐CD45‐mediated inhibition of T‐cell proliferation. Interestingly, targeting of TLR4 with mAb W7C11 also suppressed T‐cell proliferation. In summary, the results of this study demonstrate that ct‐CD45 acts via a noncanonical TLR4 activation pathway on T cells, which modulates TCR signaling. [ABSTRACT FROM AUTHOR]

Therapeutic antibodies that block PD‐1‐mediated inhibition of T cells have revolutionized cancer therapy. Murine cancer models are an essential tool for testing the efficacy of PD‐1 blockers alone or in combination with other treatments. Depending on the isotype of the antibody and the host species, blocking antibodies can also exert cytotoxic activity towards cells expressing the target molecule. In the current issue of the European Journal of Immunology [Eur. J. Immunol. 2021. 51: 1473–1481], Polesso et al. demonstrate that depletion of PD‐1+ T cells by "blocking" PD‐1 antibodies can greatly impact the outcome of preclinical immunotherapy experiments. Whereas some PD‐1 antibodies promoted activation and proliferation of PD‐1‐expressing murine T cells, the authors report that administration of a particular PD‐1 antibody can result in a significant loss of antigen‐specific CD8 T cells in different in vivo models. These findings once more highlight that a comprehensive characterization of antibodies is warranted to avoid misinterpretation of immunotherapy studies. [ABSTRACT FROM AUTHOR]

Costimulatory signals potently promote T‐cell proliferation and effector function. Agonistic antibodies targeting costimulatory receptors of the TNFR family, such as 4‐1BB and CD27, have entered clinical trials in cancer patients. Currently there is limited information how costimulatory signals regulate antigen‐specific but also bystander activation of human CD8 T cells. Engineered antigen presenting cells (eAPC) efficiently presenting several common viral epitopes on HLA‐A2 in combination with MHC class I tetramer staining were used to investigate the impact of costimulatory signals on human CD8 T‐cell responses. CD28 costimulation potently augmented the percentage and number of antigen‐reactive CD8 T cells, whereas eAPC expressing 4‐1BB‐ligand induced bystander proliferation of CD8 T cells and massive expansion of NK cells. Moreover, the 4‐1BB agonist urelumab similarly induced bystander proliferation of CD8 T cells and NK cells in a dose‐dependent manner. However, the promotion of bystander CD8 T‐cell responses is not a general attribute of costimulatory TNF receptor superfamily (TNFRSF) members, since CD27 signals enhanced antigen‐specific CD8 T cells responses without promoting significant bystander activation. Thus, the differential effects of costimulatory signals on the activation of human bystander CD8 T cells should be taken into account when costimulatory pathways are harnessed for cancer immunotherapy. [ABSTRACT FROM AUTHOR]

Therapeutic monoclonal antibodies (mAbs), targeting tumor antigens, or immune checkpoints, have demonstrated a remarkable anti-tumor effect against various malignancies. However, high costs for mono- or combination therapies, associated with adverse effects or possible development of resistance in some patients, warrant further development and modification to gain more flexibility for this immunotherapy approach. An attractive alternative to passive immunization with therapeutic antibodies might be active immunization with mimotopes (B-cell peptides) representing the mAbs' binding epitopes, to activate the patient's own anti-tumor immune response following immunization. Here, we identified and examined the feasibility of inducing anti-tumor effects in vivo following active immunization with a mimotope of the immune checkpoint programmed cell death 1 (PD1), alone or in combination with a Her-2/neu B-cell peptide vaccine. Overlapping peptides spanning the extracellular domains of human PD1 (hPD1) were used to identify hPD1-derived mimotopes, using the therapeutic mAb Nivolumab as a proof of concept. Additionally, for in vivo evaluation in a tumor mouse model, a mouse PD1 (mPD1)-derived mimotope was identified using an anti-mPD1 mAb with mPD1/mPDL-1 blocking capacity. The identified mimotopes were characterized by in vitro assays, including a reporter cell-based assay, and their anti-tumor effects were evaluated in a syngeneic tumor mouse model stably expressing human Her-2/neu. The identified PD1-derived mimotopes were shown to significantly block the mAbs' capacity in inhibiting the respective PD1/PD-L1 interactions. A significant reduction in tumor growth in vivo was observed following active immunization with the mPD1-derived mimotope, associated with a significant reduction in proliferation and increased apoptotic rates in the tumors. Particularly, combined vaccination with the mPD1-derived mimotope and a multiple B-cell epitope Her-2/neu vaccine potentiated the vaccine's anti-tumor effect. Our results suggest active immunization with mimotopes of immune checkpoint inhibitors either as monotherapy or as combination therapy with tumor-specific vaccines, as a new strategy for cancer treatment. [ABSTRACT FROM AUTHOR]

Sensing of pathogens by innate immune cells is essential for the initiation of appropriate immune responses. Toll-like receptors (TLRs), which are highly sensitive for various structurally and evolutionary conserved molecules derived from microbes have a prominent role in this process. TLR engagement results in the activation of the transcription factor NF-κB, which induces the expression of cytokines and other inflammatory mediators. The exquisite sensitivity of TLR signalling can be exploited for the detection of bacteria and microbial contaminants in tissue cultures and in protein preparations. Here we describe a cellular reporter system for the detection of TLR ligands in biological samples. The well-characterized human monocytic THP-1 cell line was chosen as host for an NF-ᴋB-inducible enhanced green fluorescent protein reporter gene. We studied the sensitivity of the resultant reporter cells for a variety of microbial components and observed a strong reactivity towards TLR1/2 and TLR2/6 ligands. Mycoplasma lipoproteins are potent TLR2/6 agonists and we demonstrate that our reporter cells can be used as reliable and robust detection system for mycoplasma contaminations in cell cultures. In addition, a TLR4-sensitive subline of our reporters was engineered, and probed with recombinant proteins expressed in different host systems. Bacterially expressed but not mammalian expressed proteins induced strong reporter activity. We also tested proteins expressed in an E. coli strain engineered to lack TLR4 agonists. Such preparations also induced reporter activation in THP-1 cells highlighting the importance of testing recombinant protein preparations for microbial contaminations beyond endotoxins. Our results demonstrate the usefulness of monocytic reporter cells for high-throughput screening for microbial contaminations in diverse biological samples, including tissue culture supernatants and recombinant protein preparations. Fluorescent reporter assays can be measured on standard flow cytometers and in contrast to established detection methods, like luciferase-based systems or Limulus Amebocyte Lysate tests, they do not require costly reagents. [ABSTRACT FROM AUTHOR]

Immune checkpoint inhibitors, which target coinhibitory T cell molecules to promote anticancer immune responses, are on the rise to become a new pillar of cancer therapy. However, current immune checkpoint-based therapies are successful only in a subset of patients and acquired resistances pose additional challenges. Finding new targets and combining checkpoint inhibitors might help to overcome these limitations. In this study, human T cells stimulated with allogeneic dendritic cells (DCs) were used to compare immune checkpoint inhibitors targeting TIM-3, BTLA, LAG-3, CTLA-4, and TIGIT alone or in combination with a PD-1 antibody. We found that PD-1 blockade bears a unique potency to enhance T cell proliferation and cytokine production. Other checkpoint inhibitors failed to significantly augment T cell responses when used alone. However, antibodies to TIM-3, BTLA, LAG-3, and CTLA-4 enhanced T cell proliferation in presence of a PD-1 antibody. Upregulation of coinhibitory T cell receptors upon PD-1 blockade was identified as a potential mechanism for synergistic effects between checkpoint inhibitors. Donor-specific variation in response to immune checkpoint inhibitors was attributed to the T cells rather than DCs. Additionally, we analyzed the regulation of checkpoint molecules and their ligands on T cells and allogeneic DCs in coculture, which suggested a PD-1 blockade-dependent crosstalk between T cells and APC. Our results indicate that several immune checkpoint inhibitors have the capacity to enhance T cell responses when combined with PD-1 blockade. Additional in vitro studies on human T cells will be useful to identify antibody combinations with the potential to augment T cell responses in cancer patients. [ABSTRACT FROM AUTHOR]

One of the major current trends in cancer immunotherapy is the blockade of immune checkpoint proteins that negatively regulate the immune response. This has been achieved through antibodies blocking PD-1/PD-L1 and CTLA-4/CD80/CD86 interactions. Such antibodies have revolutionized oncological therapy and shown a new way to fight cancer. Additional (negative) immune checkpoints are also promising targets in cancer therapy and there is a demand for inhibitors for these molecules. Our studies are focused on BTLA/HVEM complex, which inhibits T-cell proliferation and cytokine production and therefore has great potential as a new target for cancer treatment. The goal of the presented studies was the design and synthesis of compounds able to block BTLA/HVEM interactions. For that purpose, the N-terminal fragment of glycoprotein D (gD), which interacts with HVEM, was used. Based on the crystal structure of the gD/HVEM complex and MM/GBSA analysis performed on it, several peptides were designed and synthesized as potential inhibitors of the BTLA/HVEM interaction. Affinity tests, ELISA tests, and cellular-based reporter assays were performed on these compounds to check their ability to bind to HVEM and to inhibit BTLA/HVEM complex formation. For leading peptides candidates, all-atom and subsequent docking simulations with a coarse-grained force field were performed to determine their binding modes. To further evaluate their potential as drug candidates, their stability in plasma and their cytotoxicity effects on PBMCs were assessed. Our data indicate that the peptide gD(1-36)(K10C-T29C) is the best candidate as a future drug. It interacts with HVEM protein, blocks the BTLA/HVEM interaction, and is nontoxic to cells. The present study provides a new perspective on the development of BTLA/HVEM inhibitors that disrupt protein interactions. [ABSTRACT FROM AUTHOR]

An amendment to this paper has been published and can be accessed via a link at the top of the paper. [ABSTRACT FROM AUTHOR]