Target Cell Recognition Research Articles

Mining HIV controllers for broad and functional antibodies to recognize and eliminate HIV-infected cells.

SUMMARYHIV monoclonal antibodies for viral reservoir eradication strategies will likely need to recognize reactivated infected cells and potently drive Fc-mediated innate effector cell activity. We systematically characterize a library of 185 HIV-envelope-specific antibodies derived from 15 spontaneous HIV controllers (HCs) that selectively exhibit robust serum Fc functionality and compared them to broadly neutralizing antibodies (bNAbs) in clinical development. Within the 10 antibodies with the broadest cell-recognition capability, seven originated from HCs and three were bNAbs. V3-loop-targeting antibodies are enriched among the top cell binders, suggesting the V3-loop may be selectively exposed and accessible on the cell surface. Fc functionality is more variable across antibodies, which is likely influenced by distinct binding topology and corresponding Fc accessibility, highlighting not only the importance of target-cell recognition but also the need to optimize for Fc-mediated elimination. Ultimately, our results demonstrate that this comprehensive selection process can identify monoclonal antibodies poised to eliminate infected cells.

Protein yoga: Conformational versatility of the Hemolysin II C-terminal domain detailed by NMR structures for multiple states.

The C-terminal domain of Bacillus cereus hemolysin II (HlyIIC), stabilizes the trans-membrane-pore formed by the HlyII toxin and may aid in target cell recognition. Initial efforts to determine the NMR structure of HlyIIC were hampered by cis/trans isomerization about the single proline at position 405 that leads to doubling of NMR resonances. We used the mutant P405M-HlyIIC that eliminates the cis proline to determine the NMR structure of the domain, which revealed a novel fold. Here, we extend earlier studies to the NMR structure determination of the cis and trans states of WT-HlyIIC that exist simultaneously in solution. The primary structural differences between the cis and trans states are in the loop that contains P405, and structurally adjacent loops. Thermodynamic linkage analysis shows that at 25 C the cis proline, which already has a large fraction of 20% in the unfolded protein, increases to 50% in the folded state due to coupling with the global stability of the domain. The P405M or P405A substitutions eliminate heterogeneity due to proline isomerization but lead to the formation of a new dimeric species. The NMR structure of the dimer shows that it is formed through domain-swapping of strand β5, the last segment of secondary structure following P405. The presence of P405 in WT-HlyIIC strongly disfavors the dimer compared to the P405M-HlyIIC or P405A-HlyIIC mutants. The WT proline may thus act as a "gatekeeper," warding off aggregative misfolding.

Proteínas importantes para la invasión de Babesia bovis a las células huésped

Introducción. La babesiosis bovina es causada por parásitos Apicomplexa del género Babesia, siendo Babesia bovis la especie asociada con cuadros clínicos más graves de la enfermedad. La invasión de B. bovis a los eritrocitos bovinos implica la interacción entre moléculas de los merozoítos del parásito con receptores de las células huésped. Por ende, conocer las proteínas involucradas en este proceso supone un importante paso para entender la biología del parásito. Objetivo. Describir las principales moléculas implicadas en el proceso de invasión de B. bovis a eritrocitos bovinos. Metodología. Se realizó una búsqueda en NCBI, Medline, LILACS y SciELO usando los términos: “Babesia bovis AND invasion process”, “MSA-1”, “RON2”, “AMA-1”, “moving junction”, “B. bovis AND Vaccine candidates”. 61 publicaciones disponibles en inglés que describen el estudio de las anteriores proteínas y su participación en la invasión los cuales han sido publicados hasta mayo 2020 se revisaron completamente. Resultados: Siendo el proceso de invasión a eritrocitos bovinos clave para la patogénesis de la babesiosis bovina, se hizo una revisión donde se encontraron 3 proteínas de B. bovis que participan en el reconocimiento e invasión a las células diana: MSA-1, AMA-1 y RON2. Sin embargo, los detalles a nivel molecular para las interacciones inter e intramoleculares aún no se han dilucidado por completo. Conclusiones. Conocer las moléculas involucradas en las interacciones parásito-hospedero permitirá comprender cómo ocurre el proceso de invasión de B. bovis a los eritrocitos y así evaluar su futura utilidad como componente de una estrategia de control efectiva contra esta parasitosis.

CAR-T Technology and New Opportunities for Tumor Treatment

As a cause of death malignant neoplasms come in at the second place after cardiovascular disorders. CAR-T (chimeric antigen receptor of T-cells) therapy is an advanced malignant tumor treatment method. The use of CAR-T lymphocytes refers to adoptive immunotherapy. CAR-T technology is based on “extracting” immune cells (T-lymphocytes) and their genetic modification aimed at acquiring antitumor properties and followed by reinfusion. The advantage of CAR-T therapy in comparison to other treatment methods is that for target cell recognition T-lymphocytes are not dependent on major histocompatibility complex class 1 (MHC-I) molecules. The literature data we collected and analyzed show that this is a fundamentally new and effective treatment method of oncohematological diseases including acute lymphoblastic leukemia, chronic lymphocytic leukemia, and non-Hodgkin’s lymphomas. Clinical trials proved the advantage of CAR-T therapy in comparison to other treatment methods applied in this field. The analysis of literature showed that CAR-T therapy can be reasonably regarded as one of the advanced opportunities for malignant tumor treatment.

The subtle interplay between gamma delta T lymphocytes and dendritic cells: is there a role for a therapeutic cancer vaccine in the era of combinatorial strategies?

Human gamma delta (γδ) T cells represent heterogeneous subsets of unconventional lymphocytes with an HLA-unrestricted target cell recognition. γδ T cells display adaptive clonally restricted specificities coupled to a powerful cytotoxic function against transformed/injured cells. Dendritic cells (DCs) are documented to be the most potent professional antigen-presenting cells (APCs) able to induce adaptive immunity and support the innate immune response independently from T cells. Several data show that the cross-talk of γδ T lymphocytes with DCs can play a crucial role in the orchestration of immune response by bridging innate to adaptive immunity. In the last decade, DCs, as well as γδ T cells, have been of increasing clinical interest, especially as monotherapy for cancer immunotherapy, even though with unpredictable results mainly due to immune suppression and/or tumor-immune escape. For these reasons, new vaccine strategies have to be explored to reach cancer immunotherapy's full potential. The effect of DC-based vaccines on γδ T cell is less extensively investigated, and a combinatorial approach using DC-based vaccines with γδ T cells might promote a strong synergy for long-term tumor control and protection against escaping tumor clones. Here, we discuss the therapeutic potential of the interaction between DCs and γδ T cells to improve cancer vaccination. In particular, we describe the most relevant and updated evidence of such combinatorial approaches, including the use of Zoledronate, Interleukin-15, and protamine RNA, also looking towards future strategies such as CAR therapies.

A dual recognition strategy for accurate detection of CTCs based on novel branched PtAuRh trimetallic nanospheres

Accurate detection of circulating tumor cells (CTCs) has a pivotal role in the metastasis monitoring and prognosis of tumor. In this work, an ultrasensitive electrochemical cytosensor was developed based on excellent electrocatalytic materials and a dual recognition strategy. Herein, novel branched PtAuRh trimetallic nanospheres (b-PtAuRh TNS) were synthesized for the first time by a facile one-pot method, which had a huge specific surface area and outstanding catalytic activity. B-PtAuRh TNS linked with aptamers targeting mucin1 (MUC1) were served as signal tags to amplify the signal. As electrode modified material, the nanocomposites of Cabot carbon black (BP2000) and AuNPs were used to improve the electron transfer efficiency of electrode. In addition to using b-PtAuRh TNS labeled anti-MUC1 aptamers as signal probes, anti-EpCAM antibodies were worked as capture probes to achieve dual recognition of target cells. In other words, only cells expressing both MUC1 and EpCAM could produce electrochemical signal. The constructed cytosensor presented a wide linear range (5 - 1 × 106 cells mL−1) and a low detection limit (1 cell mL−1). It was worth noting that the proposed cytosensor could detect CTCs in clinical blood samples. To sum up, the developed cytosensor might become a promising detection platform for cancer diagnosis and tumor metastasis.

Structural requirement of G-quadruplex/aptamer-combined DNA macromolecule serving as efficient drug carrier for cancer-targeted drug delivery

Photodynamic therapy (PDT) as a clinical cancer treatment method has been used to treat carcinomas in different organs, and G-quadruplex-based DNA nanocompartments serving as the carriers of cationic porphyrin photosensitizers, especially combined with cell-targeting aptamers, is considered to offer new opportunities for future cancer treatment. However, the structural features of G-quadruplex/aptamer complexes suitable for the capsulation of photosensitizers and target cell recognition is unexplored so far. In this study, unimolecular (UM), bimolecular (BM) and tetramolecular (TM) G-quadruplex structures were used as the drug loading compartments and grafted onto tumor cell-targeting aptamer Sgc8, constructing several targeting drug delivery vehicles (T-GMVs). Besides the binding affinity of resulting DNA architectures for target cells and cell recognition specificity were explored in a comparative fashion, the drug loading capability and cancer therapy efficacy were evaluated using TMPyP4 as the model porphyrin-based drug. The experimental results show that only TM G-quadruplex structure is suitable to combine with Sgc8 for the development of drug delivery vehicle and the as-prepared T-GMV- TMPyP4 complexes display the desirable cancer therapy efficacy, holding the potential application in the future cancer therapy. More importantly, T-GMV- TMPyP4 is expected to lay the scientific groundwork for the successful development of G-quadruplex-based photosensitizer drug delivery carriers for the targeted cancer therapy.

Selection of tumor‑resistant variants following sustained natural killer cell‑mediated immune stress.

Resistance of tumor cells to cell-mediated cytotoxicity remains an obstacle to the immunotherapy of cancer and its molecular basis is poorly understood. To investigate the acquisition of tumor resistance to cell-mediated cytotoxicity, resistant variants were selected following long-term natural killer (NK) cell selection pressure. It was observed that these variants were resistant to NK cell-mediated lysis, but were sensitive to autologous cytotoxic T lymphocytes or cytotoxic drugs. This resistance appeared to be dependent, at least partly, on an alteration of target cell recognition by NK effector cells, but did not appear to involve any alterations in the expression of KIR, DNAM1 or NKG2D ligands on resistant cells, nor the induction of protective autophagy. In the present study, in order to gain further insight into the molecular mechanisms underlying the acquired tumor resistance to NK cell-mediated cytotoxicity, a comprehensive analysis of the variant transcriptome was conducted. Comparative analysis identified an expression profile of genes that best distinguished resistant variants from parental sensitive cancer cells, with candidate genes putatively involved in NK cell-mediated lysis resistance, but also in adhesion, migration and invasiveness, including upregulated genes, such as POT1, L1CAM or ECM1, and downregulated genes, such as B7-H6 or UCHL1. Consequently, the selected variants were not only resistant to NK cell-mediated lysis, but also displayed more aggressive properties. The findings of the present study emphasized that the role of NK cells may span far beyond the mere killing of malignant cells, and NK cells may be important effectors during cancer immunoediting.

Paths taken towards NK cell-mediated immunotherapy of human cancer-a personal reflection.

The discovery that NK cells are able to specifically recognize cells lacking the expression of self‐MHC class I molecules provided the first insight into NK cell recognition of tumour cells. It started a flourishing field of NK cell research aimed at exploring the molecular nature of NK cell receptors involved in tumour cell recognition. While much of the important early work was conducted in murine experimental model systems, studies of human NK cells rapidly followed. Over the years, human NK cell research has swiftly progressed, aided by new detailed molecular information on human NK cell development, differentiation, molecular specificity, tissue heterogeneity and functional capacity. NK cells have also been studied in many different diseases aside from cancer, including viral diseases, autoimmunity, allergy and primary immunodeficiencies. These fields of research have all, indirectly or directly, provided further insights into NK cell‐mediated recognition of target cells and paved the way for the development of NK cell‐based immunotherapies for human cancer. Excitingly, NK cell‐based immunotherapy now opens up for novel strategies aimed towards treating malignant diseases, either alone or in combination with other drugs. Reviewed here are some personal reflections of select contributions leading up to the current state‐of‐the‐art in the field, with a particular emphasis on contributions from our own laboratory. This review is part of a series of articles on immunology in Scandinavia, published in conjunction with the 50th anniversary of the Scandinavian Society for Immunology.

Cord-Blood Natural Killer Cell-Based Immunotherapy for Cancer.

Natural killer (NK) cells are a predominant part of innate immune cells and play a crucial role in anti-cancer immunity. NK cells can kill target cells nonspecifically, and their recognition of target cells is not restricted by the major histocompatibility complex. NK cells also fight against tumor cells independently of antibodies and prior activation. Of note, umbilical cord blood (UCB) is a rich source of NK cells. Immunotherapies based on UCB-derived NK cells are becoming increasingly researched, and the investigations are producing encouraging results. In recent years, non-modified and modified UCB-derived NK cells have been successfully developed to fight against tumor cells. Herein, UCB-derived NK cell-based immunotherapy is a potential strategy for the treatment of cancer in the future. In this review, we focus on discussing the biological characteristics of UCB-derived NK cells and their application prospects in anti-tumor immunotherapy, including the latest preclinical and clinical researches.

Keeping It Together: Structures, Functions, and Applications of Viral Decoration Proteins.

Decoration proteins are viral accessory gene products that adorn the surfaces of some phages and viral capsids, particularly tailed dsDNA phages. These proteins often play a “cementing” role, reinforcing capsids against accumulating internal pressure due to genome packaging, or environmental insults such as extremes of temperature or pH. Many decoration proteins serve alternative functions, including target cell recognition, participation in viral assembly, capsid size determination, or modulation of host gene expression. Examples that currently have structures characterized to high-resolution fall into five main folding motifs: β-tulip, β-tadpole, OB-fold, Ig-like, and a rare knotted α-helical fold. Most of these folding motifs have structure homologs in virus and target cell proteins, suggesting horizontal gene transfer was important in their evolution. Oligomerization states of decoration proteins range from monomers to trimers, with the latter most typical. Decoration proteins bind to a variety of loci on capsids that include icosahedral 2-, 3-, and 5-fold symmetry axes, as well as pseudo-symmetry sites. These binding sites often correspond to “weak points” on the capsid lattice. Because of their unique abilities to bind virus surfaces noncovalently, decoration proteins are increasingly exploited for technology, with uses including phage display, viral functionalization, vaccination, and improved nanoparticle design for imaging and drug delivery. These applications will undoubtedly benefit from further advances in our understanding of these versatile augmenters of viral functions.

Escape of tumor cells from the NK cell cytotoxic activity

In recent years, NK cells, initially identified as potent cytotoxic effector cells, have revealed an unexpected complexity, both at phenotypic and functional levels. The discovery of different NK cell subsets, characterized by distinct gene expression and phenotypes, was combined with the characterization of the diverse functions NK cells can exert, not only as circulating cells, but also as cells localized or recruited in lymphoid organs and in multiple tissues. Besides the elimination of tumor and virus-infected cells, these functions include the production of cytokines and chemokines, the regulation of innate and adaptive immune cells, the influence on tissue homeostasis. In addition, NK cells display a remarkable functional plasticity, being able to adapt to the environment and to develop a kind of memory. Nevertheless, the powerful cytotoxic activity of NK cells remains one of their most relevant properties, particularly in the antitumor response. In this review, the process of tumor cell recognition and killing mediated by NK cells, starting from the generation of cytolytic granules and recognition of target cell, to the establishment of the NK cell immunological synapse, the release of cytotoxic molecules, and consequent tumor cell death is described. Next, the review focuses on the heterogeneous mechanisms, either intrinsic to tumors or induced by the tumor microenvironment, by which cancer cells can escape the NK cell-mediated attack.

Engineering G-quadruplex aptamer to modulate its binding specificity.

The use of aptamers in bioanalytical and biomedical applications exploits their ability to recognize cell surface protein receptors. Targeted therapeutics and theranostics come to mind in this regard. However, protein receptors occur on both cancer and normal cells; as such, aptamers are now taxed with identifying high vs. low levels of protein expression. Inspired by the flexible template mechanism and elegant control of natural nucleic acid-based structures, we report an allosteric regulation strategy for constructing a structure-switching aptamer for enhanced target cell recognition by engineering aptamers with DNA intercalated motifs (i-motifs) responsive to the microenvironment, such as pH. Structure-switching sensitivity can be readily tuned by manipulating i-motif sequences. However, structure-switching sensitivity is difficult to estimate, making it equally difficult to effectively screen modified aptamers with the desired sensitivity. To address this problem, we selected a fluorescent probe capable of detecting G-quadruplex in complicated biological media.

Abstract 888: Vδ1γδT-Cells: A unique allogeneic cell therapy platform for the treatment of a broad range of malignancies

Abstract The treatment of haematologic malignancies with adoptive cell therapy is largely limited to platforms based on patient-derived, autologous αβ T cells. Although successful, this approach comes with challenges including associated toxicities, risk of relapse, high production costs and a requirement to gene edit cells to avoid graft vs host disease (GvHD) risk if the therapy is to be used in an allogeneic setting. In contrast to αβ T cells, human Vδ1 γδ T cells are a subset of T cells defined by expression of heterodimeric T cell receptors (TCRs) composed of a γ chain paired to a Vδ1 chain. Vδ1 γδ T cell function is highly differentiated from αβ T cells, as target cell recognition is not MHC restricted and γδ T cells are not alloreactive. Allogeneic matching of patients is therefore not required for Vδ1 γδ T cell therapeutic approaches. Instead Vδ1 T cells elicit direct anti-tumour responses via activation of diverse receptor repertoires that recognise multiple ligands upregulated on the surface of transformed cells. These key features of Vδ1 T cell biology therefore confer several advantages when generating an allogeneic cell therapy platform. We have developed a good manufacturing practice (GMP) compliant, scalable process to generate αβ T cell depleted, γδ T cell cultures (93.5% ± 3.7 of live), enriched for Vδ1 T cells (67.9% ± 2.2% of live). The cryopreserved therapeutic product shows good recoverability and proliferation after thawing, an activated innate phenotype and produces high levels of IFNγ and chemokines which can stimulate activation of other cells of the immune system. Post-thaw, Vδ1 T cell product has cytotoxic activity against a variety of malignant leukaemia and lymphoma cells in vitro and the cells home to the bone marrow and mediate anti-tumour activity following IV dosing in an in vivo xenograft model. Importantly, Vδ1 T cells have a good safety profile and do not mediate; cytotoxic activity against healthy tissues, mixed lymphocyte reactions or GvHD in in vivo models. Building on our expertise for expanding Vδ1 T cells, we have now developed scalable transient and stable gene engineering platforms for the generation of Vδ1 T cells expressing chimeric antigen receptors (CAR) and other molecules. Genetically engineered cells show good expansion kinetics and recoverability and viability post-cryopreservation. The cells also maintain a favourable activated phenotype post-thaw characterised by high levels of expression of CD27, NCR receptors (DNAM1, NKG2D and NKp30) and stable CAR expression at the cell surface. We have now used the Vδ1 CAR-T platform to develop and screen CAR constructs designed to generate allogeneic CAR-T cell therapies that exploit the unique safety and activity profile of Vδ1 T cells while additionally potentiating on-target tumour cell killing. Using this approach and tool constructs containing CARs targeting CD19, we have demonstrated potent and enhanced killing of a B-cell tumour cell line without eliciting on-target bystander killing of healthy B cells. In addition, the Vδ1 CAR-T anti-tumour activity is not associated with release of cytokines that may potentiate off-target toxicities. These data demonstrate that Vδ1 T cells have the potential to provide a unique CAR-T platform capable of targeting a broader cancer antigen profile than is possible for conventional CAR-T therapies. Citation Format: Istvan Kovacs, Andre Simoes, Tim Recaldin, Elizabeth Reynolds, Katharina Bergerhoff, Mihil Patel, Rebecca Alade, Victoria Hillerdal, Andrew Hutton, Daniel Fowler, Joanna Kawalkowska, Kalle Soderstrom, Valentino Parravicini, Michael Koslowski, Oliver Nussbaumer, Alice Brown. Vδ1γδT-Cells: A unique allogeneic cell therapy platform for the treatment of a broad range of malignancies [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 888.

The Art and Science of Selecting a CD123-Specific Chimeric Antigen Receptor for Clinical Testing

Chimeric antigen receptor (CAR) T cells targeting CD123, an acute myeloid leukemia (AML) antigen, hold the promise of improving outcomes for patients with refractory/recurrent disease. We generated five lentiviral vectors encoding CD20, which may serve as a target for CAR T cell depletion, and 2nd or 3rd generation CD123-CARs since the benefit of two costimulatory domains is model dependent. Four CARs were based on the CD123-specific single-chain variable fragment (scFv) 26292 (292) and one CAR on the CD123-specific scFv 26716 (716), respectively. We designed CARs with different hinge/transmembrane (H/TM) domains and costimulatory domains, in combination with the zeta (z) signaling domain: 292.CD8aH/TM.41BBz (8.41BBz), 292.CD8aH/TM.CD28z (8.28z), 716.CD8aH/TM.CD28z (716.8.28z), 292.CD28H/TM. CD28z (28.28z), and 292.CD28H/TM.CD28.41BBz (28.28.41BBz). Transduction efficiency, expansion, phenotype, and target cell recognition of the generated CD123-CAR T cells did not significantly differ. CAR constructs were eliminated for the following reasons: (1) 8.41BBz CARs induced significant baseline signaling, (2) 716.8.28z CAR T cells had decreased anti-AML activity, and (3) CD28.41BBz CAR T cells had no improved effector function in comparison to CD28z CAR T cells. We selected the 28.28z CAR since CAR expression on the cell surface of transduced T cells was higher in comparison to 8.28z CARs. The clinical study (NCT04318678) evaluating 28.28z CAR T cells is now open for patient accrual.

Biological characterisation, expression and functional analysis of non-specific cytotoxic cell receptor protein 1 in Nile tilapia (Oreochromis niloticus)

Non-specific cytotoxic cell receptor protein 1 (NCCRP-1) plays a role in recognition of target cell and activation of non-specific cytotoxic cell (NCC). In this study, the full length of Nile tilapia NCCRP-1 (On-NCCRP-1) was cloned. cDNA is composed of 1045 bp with a 90 bp of 5′-Untranslated Regions (UTR), 702 bp open reading frame (ORF) and 253 bp 3′-UTR, encoding 233 amino acids (GenBank accession no: MF162296). The On-NCCRP-1 genomic sequence is 4471 bp in length and contains six exons and five introns. On-NCCRP-1 possesses some inherent conservative domains, such as proline-rich motifs, antigen recognition site, and F-box-related domain. Subcellular localisation and Western blot analysis indicated that On-NCCRP-1 is located in the cell membrane. The transcript of On-NCCRP-1 was detected in all the examined tissues of healthy Nile tilapia by using qRT-PCR, with the highest expression levels in the liver. Following Streptococcus agalactiae challenged in vivo, the On-NCCRP-1 expression was up-regulated significantly in brain, intestines, head kidney and spleen. In the in vitro analysis, the On-NCCRP-1 expression in NCCs was up-regulated significantly from 8 h to 12 h after LPS challenge, and up-regulated significantly at 12 h after challenged with polyI:C. After NCCs were challenged with inactivated S. agalactiae, the On-NCCRP-1 expression was down-regulated significantly after 24 h. NF-кB pathway was strongly activated by the over-expression of On-NCCRP-1 in HEK-293T cells. These results indicate that On-NCCRP-1, as a membrane surface receptor of NCCs, may play an important role in immune response to pathogenic infection in Nile tilapia.

T Cell Receptor Engineered Lymphocytes for Cancer Therapy.

T lymphocytes are capable of specific recognition and elimination of target cells. Physiological antigen recognition is mediated by the T cell receptor (TCR), which is an alpha beta heterodimer comprising the products of randomly rearranged V, D, and J genes. The exquisite specificity and functionality of T cells can be leveraged for cancer therapy: specifically, the adoptive transfer of T cells that express tumor-reactive TCRs can induce regression of solid tumors in patients with advanced cancer. However, the isolation and expression of a tumor antigen-specific TCRs is a highly involved process that requires identifying an immunogenic epitope, ensuring human cells are of the correct haplotype, performing a laborious T cell expansion process, and carrying out downstream TCR sequencing and cloning. Recent advances in single-cell sequencing have begun to streamline this process. This protocol synthesizes and expands upon methodologies to generate, isolate, and engineer human T cells with tumor-reactive TCRs for adoptive cell therapy. Though this process is perhaps more arduous than the alternative strategy of using chimeric antigen receptors (CARs) for engineering, the ability to target intracellular proteins using TCRs substantially increases the types of antigens that can be safely targeted. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Generation of human autologous dendritic cells from monocytes Basic Protocol 2: In vitro priming and expansion of human antigen-specific T cells Basic Protocol 3: Cloning of antigen-specific T cell receptors based on single-cell VDJ sequencing data Basic Protocol 4: Validation of T cell receptor expression and functionality in vitro Basic Protocol 5: Rapid expansion of T cell receptor-transduced T cells and human T cell clones.

A comprehensive review and performance evaluation of bioinformatics tools for HLA class I peptide-binding prediction.

Human leukocyte antigen class I (HLA-I) molecules are encoded by major histocompatibility complex (MHC) class I loci in humans. The binding and interaction between HLA-I molecules and intracellular peptides derived from a variety of proteolytic mechanisms play a crucial role in subsequent T-cell recognition of target cells and the specificity of the immune response. In this context, tools that predict the likelihood for a peptide to bind to specific HLA class I allotypes are important for selecting the most promising antigenic targets for immunotherapy. In this article, we comprehensively review a variety of currently available tools for predicting the binding of peptides to a selection of HLA-I allomorphs. Specifically, we compare their calculation methods for the prediction score, employed algorithms, evaluation strategies and software functionalities. In addition, we have evaluated the prediction performance of the reviewed tools based on an independent validation data set, containing 21101 experimentally verified ligands across 19 HLA-I allotypes. The benchmarking results show that MixMHCpred 2.0.1 achieves the best performance for predicting peptides binding to most of the HLA-I allomorphs studied, while NetMHCpan 4.0 and NetMHCcons 1.1 outperform the other machine learning-based and consensus-based tools, respectively. Importantly, it should be noted that a peptide predicted with a higher binding score for a specific HLA allotype does not necessarily imply it will be immunogenic. That said, peptide-binding predictors are still very useful in that they can help to significantly reduce the large number of epitope candidates that need to be experimentally verified. Several other factors, including susceptibility to proteasome cleavage, peptide transport into the endoplasmic reticulum and T-cell receptor repertoire, also contribute to the immunogenicity of peptide antigens, and some of them can be considered by some predictors. Therefore, integrating features derived from these additional factors together with HLA-binding properties by using machine-learning algorithms may increase the prediction accuracy of immunogenic peptides. As such, we anticipate that this review and benchmarking survey will assist researchers in selecting appropriate prediction tools that best suit their purposes and provide useful guidelines for the development of improved antigen predictors in the future.

Differences in NK and Memory CD8 T cell responses to antigen-nonspecific stimulation by interleukin-15

Abstract Natural killer (NK) cells are innate lymphoid cells that exert immediate functions which can be further augmented and sustained with immunostimulatory cytokines. Memory CD8 T cells, due to expression of CD132 and CD122, can be activated by similar cytokines in the absence of TCR engagement (termed “bystander” activation). This results in activation and proliferation but necessitates high amounts of cytokine as high-affinity IL2R complexes (CD25) are not induced. Interestingly, both cell types can then elicit similar effector functions via NKG2D-mediated target cell recognition. As these cell types can fill a similar immunologic niche, we set out to compare NK and memory CD8 T cell responses following IL-15 exposure in vitro from healthy human donors. Cell analysis was performed by flow cytometry and qRT-PCR. At baseline, CD25 expression is negligible at <5% on both human NK and memory CD8 T cells. Surprisingly, culture with rhIL-15 (10 ng/mL) for 4–6 days resulted in marked CD25 upregulation on CD56+CD3− NK cells but not bystander-activated CD45RA-CD95+ CD8 T cells (72±9.2% vs 11±3.8%, P=0.003) despite comparable expansion. Additionally, cytokine-activated NK cells expressed higher levels of inhibitory receptor TIGIT (85±4% vs 57±2%, P=0.01) and activation marker CD69 (99±1% vs 27±9%, P=0.008). Functionally, NK cells had increased expression of granzyme B compared to bystander-activated CD8 T cells. Thus, although NK and bystander CD8 T cells can fill a similar immunologic niche regarding target cell killing, there are significant differences in expression of critical markers following activation. These differences may have consequences in the regulation of these cell types and impact anti-viral and anti-tumor responses.

Nanoparticles Mimicking Viral Cell Recognition Strategies Are Superior Transporters into Mesangial Cells.

Poor drug availability in the tissue of interest is a frequent cause of therapy failure. While nanotechnology has developed a plethora of nanocarriers for drug transport, their ability to unequivocally identify cells of interest remains moderate. Viruses are the ideal nanosized carriers as they are able to address their embedded nucleic acids with high specificity to their host cells. Here, it is reported that particles endowed with a virus‐like ability to identify cells by three consecutive checks have a superior ability to recognize mesangial cells (MCs) in vivo compared to conventional nanoparticles. Mimicking the initial viral attachment followed by a stepwise target cell recognition process leads to a 5‐ to 15‐fold higher accumulation in the kidney mesangium and extensive cell uptake compared to particles lacking one or both of the viral traits. These results highlight the relevance that the viral cell identification process has on specificity and its application on the targeting strategies of nanomaterials. More so, these findings pave the way for transporting drugs into the mesangium, a tissue that is pivotal in the development of diabetic nephropathy and for which currently no efficient pharmacotherapy exists.