Foxp3 and Toll-like receptor signaling balance Treg cell anabolic metabolism for suppression.

CD4+ effector T cells (Teff cells) and regulatory T cells (Treg cells) undergo metabolic reprogramming to support proliferation and immunological function. Although signaling via the lipid kinase PI(3)K (phosphatidylinositol-3-OH kinase), the serine-threonine kinase Akt and the metabolic checkpoint kinase complex mTORC1 induces both expression of the glucose transporter Glut1 and aerobic glycolysis for Teff cell proliferation and inflammatory function, the mechanisms that regulate Treg cell metabolism and function remain unclear. We found that Toll-like receptor (TLR) signals that promote Treg cell proliferation increased PI(3)K-Akt-mTORC1 signaling, glycolysis and expression of Glut1. However, TLR-induced mTORC1 signaling also impaired Treg cell suppressive capacity. Conversely, the transcription factor Foxp3 opposed PI(3)K-Akt-mTORC1 signaling to diminish glycolysis and anabolic metabolism while increasing oxidative and catabolic metabolism. Notably, Glut1 expression was sufficient to increase the number of Treg cells, but it reduced their suppressive capacity and Foxp3 expression. Thus, inflammatory signals and Foxp3 balance mTORC1 signaling and glucose metabolism to control the proliferation and suppressive function of Treg cells.

Graph-pMHC: graph neural network approach to MHC class II peptide presentation and antibody immunogenicity.

Antigen presentation on MHC class II (pMHCII presentation) plays an essential role in the adaptive immune response to extracellular pathogens and cancerous cells. But it can also reduce the efficacy of large-molecule drugs by triggering an anti-drug response. Significant progress has been made in pMHCII presentation modeling due to the collection of large-scale pMHC mass spectrometry datasets (ligandomes) and advances in machine learning. Here, we develop graph-pMHC, a graph neural network approach to predict pMHCII presentation. We derive adjacency matrices for pMHCII using Alphafold2-multimer and address the peptide-MHC binding groove alignment problem with a simple graph enumeration strategy. We demonstrate that graph-pMHC dramatically outperforms methods with suboptimal inductive biases, such as the multilayer-perceptron-based NetMHCIIpan-4.0 (+20.17% absolute average precision). Finally, we create an antibody drug immunogenicity dataset from clinical trial data and develop a method for measuring anti-antibody immunogenicity risk using pMHCII presentation models. Our model increases receiver operating characteristic curve (ROC)-area under the ROC curve (AUC) by 2.57% compared to just filtering peptides by hits in OASis alone for predicting antibody drug immunogenicity.

mTORC1 and mTORC2 Kinase Signaling and Glucose Metabolism Drive Follicular Helper T Cell Differentiation.

Follicular helper T (Tfh) cells are crucial for germinal center (GC) formation and humoral adaptive immunity. Mechanisms underlying Tfh cell differentiation in peripheral and mucosal lymphoid organs are incompletely understood. We report here that mTOR kinase complexes 1 and 2 (mTORC1 and mTORC2) are essential for Tfh cell differentiation and GC reaction under steady state and after antigen immunization and viral infection. Loss of mTORC1 and mTORC2 in T cells exerted distinct effects on Tfh cell signature gene expression, whereas increased mTOR activity promoted Tfh responses. Deficiency of mTORC2 impaired CD4(+) T cell accumulation and immunoglobulin A production and aberrantly induced the transcription factor Foxo1. Mechanistically, the costimulatory molecule ICOS activated mTORC1 and mTORC2 to drive glycolysis and lipogenesis, and glucose transporter 1-mediated glucose metabolism promoted Tfh cell responses. Altogether, mTOR acts as a central node in Tfh cells by linking immune signals to anabolic metabolism and transcriptional activity.

Application of N-Terminal Site-Specific Biotin and Digoxigenin Conjugates to Clinical Anti-drug Antibody Assay Development.

Biotin- and digoxigenin (DIG)-conjugated therapeutic drugs are critical reagents used for the development of anti-drug antibody (ADA) assays for the assessment of immunogenicity. The current practice of generating biotin and DIG conjugates is to label a therapeutic antibody with biotin or DIG via primary amine groups on lysine or N-terminal residues. This approach modifies lysine residues nonselectively, which can impact the ability of an ADA assay to detect those ADAs that recognize epitopes located at or near the modified lysine residue(s). The impact of the lysine modification is considered greater for therapeutic antibodies that have a limited number of lysine residues, such as the variable heavy domain of heavy chain (VHH) antibodies. In this paper, for the first time, we report the application of site-specifically conjugated biotin- and DIG-VHH reagents to clinical ADA assay development using a model molecule, VHHA. The site-specific conjugation of biotin or DIG to VHHA was achieved by using an optimized reductive alkylation approach, which enabled the majority of VHHA molecules labeled with biotin or DIG at the desirable N-terminus, thereby minimizing modification of the protein after labeling and reducing the possibility of missing detection of ADAs. Head-to-head comparison of biophysical characterization data revealed that the site-specific biotin and DIG conjugates demonstrated overall superior quality to biotin- and DIG-VHHA prepared using the conventional amine coupling method, and the performance of the ADA assay developed using site-specific biotin and DIG conjugates met all acceptance criteria. The approach described here can be applied to the production of other therapeutic-protein- or antibody-based critical reagents that are used to support ligand binding assays.

T Cells Regulate Peripheral Naive Mature B Cell Survival by Cell-Cell Contact Mediated through SLAMF6 and SAP.

The control of lymphoid homeostasis is the result of a very fine balance between lymphocyte production, proliferation, and apoptosis. In this study, we focused on the role of T cells in the maintenance/survival of the mature naive peripheral B cell population. We show that naive B and T cells interact via the signaling lymphocyte activation molecule (SLAM) family receptor, SLAMF6. This interaction induces cell type-specific signals in both cell types, mediated by the SLAM-associated protein (SAP) family of adaptors. This signaling results in an upregulation of the expression of the cytokine migration inhibitory factor in the T cells and augmented expression of its receptor CD74 on the B cell counterparts, consequently enhancing B cell survival. Furthermore, in X-linked lymphoproliferative disease patients, SAP deficiency reduces CD74 expression, resulting in the perturbation of B cell maintenance from the naive stage. Thus, naive T cells regulate B cell survival in a SLAMF6- and SAP-dependent manner.

CCR2 Regulates the Immune Response by Modulating the Interconversion and Function of Effector and Regulatory T Cells.

Chemokines and chemokine receptors establish a complex network modulating immune cell migration and localization. These molecules were also suggested to mediate the differentiation of leukocytes; however, their intrinsic, direct regulation of lymphocyte fate remained unclear. CCR2 is the main chemokine receptor inducing macrophage and monocyte recruitment to sites of inflammation, and it is also expressed on T cells. To assess whether CCR2 directly regulates T cell responses, we followed the fates of CCR2-/- T cells in T cell-specific inflammatory models. Our in vitro and in vivo results show that CCR2 intrinsically mediates the expression of inflammatory T cell cytokines, and its absence on T cells results in attenuated colitis progression. Moreover, CCR2 deficiency in T cells promoted a program inducing the accumulation of Foxp3+ regulatory T cells, while decreasing the levels of Th17 cells in vivo, indicating that CCR2 regulates the immune response by modulating the effector/regulatory T ratio.

Nutritional effects on T-cell immunometabolism.

T cells are highly influenced by nutrient uptake from their environment, and changes in overall nutritional status, such as malnutrition or obesity, can result in altered T-cell metabolism and behavior. In states of severe malnutrition or starvation, T-cell survival, proliferation, and inflammatory cytokine production are all decreased, as is T-cell glucose uptake and metabolism. The altered T-cell function and metabolism seen in malnutrition is associated with altered adipokine levels, most particularly decreased leptin. Circulating leptin levels are low in malnutrition, and leptin has been shown to be a key link between nutrition and immunity. The current view is that leptin signaling is required to upregulate activated T-cell glucose metabolism and thereby fuel T-cell activation. In the setting of obesity, T cells have been found to have a key role in promoting the recruitment of inflammatory macrophages to adipose depots along with the production of inflammatory cytokines that promote the development of insulin resistance leading to diabetes. Deletion of T cells, key T-cell transcription factors, or pro-inflammatory T-cell cytokines prevents insulin resistance in obesity and underscores the importance of T cells in obesity-associated inflammation and metabolic disease. Altogether, T cells have a critical role in nutritional immunometabolism.

Suppression of Glut1 and Glucose Metabolism by Decreased Akt/mTORC1 Signaling Drives T Cell Impairment in B Cell Leukemia.

Leukemia can promote T cell dysfunction and exhaustion that contributes to increased susceptibility to infection and mortality. The treatment-independent mechanisms that mediate leukemia-associated T cell impairments are poorly understood, but metabolism tightly regulates T cell function and may contribute. In this study, we show that B cell leukemia causes T cells to become activated and hyporesponsive with increased PD-1 and TIM3 expression similar to exhausted T cells and that T cells from leukemic hosts become metabolically impaired. Metabolic defects included reduced Akt/mammalian target of rapamycin complex 1 (mTORC1) signaling, decreased expression of the glucose transporter Glut1 and hexokinase 2, and reduced glucose uptake. These metabolic changes correlated with increased regulatory T cell frequency and expression of PD-L1 and Gal-9 on both leukemic and stromal cells in the leukemic microenvironment. PD-1, however, was not sufficient to drive T cell impairment, as in vivo and in vitro anti-PD-1 blockade on its own only modestly improved T cell function. Importantly, impaired T cell metabolism directly contributed to dysfunction, as a rescue of T cell metabolism by genetically increasing Akt/mTORC1 signaling or expression of Glut1 partially restored T cell function. Enforced Akt/mTORC1 signaling also decreased expression of inhibitory receptors TIM3 and PD-1, as well as partially improved antileukemia immunity. Similar findings were obtained in T cells from patients with acute or chronic B cell leukemia, which were also metabolically exhausted and had defective Akt/mTORC1 signaling, reduced expression of Glut1 and hexokinase 2, and decreased glucose metabolism. Thus, B cell leukemia-induced inhibition of T cell Akt/mTORC1 signaling and glucose metabolism drives T cell dysfunction.

Nek7 kinase accelerates microtubule dynamic instability.

The NIMA-related kinases (NRK or Nek) are emerging as conserved and crucial regulators of mitosis and cilia formation. The microtubule (MT) network has long been suspected as a major target of the Neks. However, the underlying mechanism remains unclear. Using the PlusTipTracker software, recently developed by the Danuser group, we followed the consequences of alterations in Nek7 levels on MT dynamic instability. siRNA-mediated downregulation of Nek7 in HeLa cells resulted in lower speeds of MT growth and catastrophe, reduction of the relative time spent in catastrophe, and considerably lowered the overall MT dynamicity. Co-expression of Nek7 with the siRNA treatment rescued the MT phenotypes, while ectopic overexpression of Nek7 yielded inverse characteristics compared to Nek7 downregulation. MT dynamics in mouse embryonic fibroblasts derived from targeted null mutants for Nek7 recapitulated the siRNA downregulation phenotypes. Precise MT dynamic instability is critical for accurate shaping of the mitotic spindle and for cilium formation, and higher MT dynamicity is associated with tumorigenicity. Thus, our results can supply a mechanistic explanation for Nek involvement in these processes.

The cytokine midkine and its receptor RPTPζ regulate B cell survival in a pathway induced by CD74.

Lasting B cell persistence depends on survival signals that are transduced by cell surface receptors. In this study, we describe a novel biological mechanism essential for survival and homeostasis of normal peripheral mature B cells and chronic lymphocytic leukemia cells, regulated by the heparin-binding cytokine, midkine (MK), and its proteoglycan receptor, the receptor-type tyrosine phosphatase ζ (RPTPζ). We demonstrate that MK initiates a signaling cascade leading to B cell survival by binding to RPTPζ. In mice lacking PTPRZ, the proportion and number of the mature B cell population are reduced. Our results emphasize a unique and critical function for MK signaling in the previously described MIF/CD74-induced survival pathway. Stimulation of CD74 with MIF leads to c-Met activation, resulting in elevation of MK expression in both normal mouse splenic B and chronic lymphocytic leukemia cells. Our results indicate that MK and RPTPζ are important regulators of the B cell repertoire. These findings could pave the way toward understanding the mechanisms shaping B cell survival and suggest novel therapeutic strategies based on the blockade of the MK/RPTPζ-dependent survival pathway.

Deficiency of caspase recruitment domain family, member 11 (CARD11), causes profound combined immunodeficiency in human subjects.

BACKGROUND: Profound combined immunodeficiency can present with normal numbers of T and B cells, and therefore the functional defect of the cellular and humoral immune response is often not recognized until the first severe clinical manifestation. Here we report a patient of consanguineous descent presenting at 13 months of age with hypogammaglobulinemia, Pneumocystis jirovecii pneumonia, and a suggestive family history. OBJECTIVE: We sought to identify the genetic alteration in a patient with combined immunodeficiency and characterize human caspase recruitment domain family, member 11 (CARD11), deficiency. METHODS: Molecular, immunologic, and functional assays were performed. RESULTS: The immunologic characterization revealed only subtle changes in the T-cell and natural killer cell compartment, whereas B-cell differentiation, although normal in number, was distinctively blocked at the transitional stage. Genetic evaluation revealed a homozygous deletion of exon 21 in CARD11 as the underlying defect. This deletion abrogated protein expression and activation of the canonical nuclear factor κB (NF-κB) pathway in lymphocytes after antigen receptor or phorbol 12-myristate 13-acetate stimulation, whereas CD40 signaling in B cells was preserved. The abrogated activation of the canonical NF-κB pathway was associated with severely impaired upregulation of inducible T-cell costimulator, OX40, cytokine production, proliferation of T cells, and B cell-activating factor receptor expression on B cells. CONCLUSION: Thus in patients with CARD11 deficiency, the combination of impaired activation and especially upregulation of inducible T-cell costimulator on T cells, together with severely disturbed peripheral B-cell differentiation, apparently leads to a defective T-cell/B-cell cooperation and probably germinal center formation and clinically results in severe immunodeficiency. This report discloses the crucial and nonredundant role of canonical NF-κB activation and specifically CARD11 in the antigen-specific immune response in human subjects.

Reducing Immunogenicity by Design: Approaches to Minimize Immunogenicity of Monoclonal Antibodies.

Monoclonal antibodies (mAbs) have transformed therapeutic strategies for various diseases. Their high specificity to target antigens makes them ideal therapeutic agents for certain diseases. However, a challenge to their application in clinical practice is their potential risk to induce unwanted immune response, termed immunogenicity. This challenge drives the continued efforts to deimmunize these protein therapeutics while maintaining their pharmacokinetic properties and therapeutic efficacy. Because mAbs hold a central position in therapeutic strategies against an array of diseases, the importance of conducting comprehensive immunogenicity risk assessment during the drug development process cannot be overstated. Such assessment necessitates the employment of in silico, in vitro, and in vivo strategies to evaluate the immunogenicity risk of mAbs. Understanding the intricacies of the mechanisms that drive mAb immunogenicity is crucial to improving their therapeutic efficacy and safety and developing the most effective strategies to determine and mitigate their immunogenic risk. This review highlights recent advances in immunogenicity prediction strategies, with a focus on protein engineering strategies used throughout development to reduce immunogenicity.

Nonclinical immunogenicity risk assessment for knobs-into-holes bispecific IgG1 antibodies.

Bispecific antibodies, including bispecific IgG, are emerging as an important new class of antibody therapeutics. As a result, we, as well as others, have developed engineering strategies designed to facilitate the efficient production of bispecific IgG for clinical development. For example, we have extensively used knobs-into-holes (KIH) mutations to facilitate the heterodimerization of antibody heavy chains and more recently Fab mutations to promote cognate heavy/light chain pairing for efficient in vivo assembly of bispecific IgG in single host cells. A panel of related monospecific and bispecific IgG1 antibodies was constructed and assessed for immunogenicity risk by comparison with benchmark antibodies with known low (Avastin and Herceptin) or high (bococizumab and ATR-107) clinical incidence of anti-drug antibodies. Assay methods used include dendritic cell internalization, T cell proliferation, and T cell epitope identification by in silico prediction and MHC-associated peptide proteomics. Data from each method were considered independently and then together for an overall integrated immunogenicity risk assessment. In toto, these data suggest that the KIH mutations and in vitro assembly of half antibodies do not represent a major risk for immunogenicity of bispecific IgG1, nor do the Fab mutations used for efficient in vivo assembly of bispecifics in single host cells. Comparable or slightly higher immunogenicity risk assessment data were obtained for research-grade preparations of trastuzumab and bevacizumab versus Herceptin and Avastin, respectively. These data provide experimental support for the common practice of using research-grade preparations of IgG1 as surrogates for immunogenicity risk assessment of their corresponding pharmaceutical counterparts.

Appendicitis in adults with incidental midgut malrotation: CT findings.

AIM: To report the computed tomography (CT) findings of acute and complicated appendicitis in adults with incidental midgut malrotation. MATERIALS AND METHODS: The medical records and CT studies of eight patients with appendicitis and incidental midgut malrotation who presented to two medical centres between 1998 and 2009 were reviewed. RESULTS: All patients presented with 1-5 days of acute abdominal pain, which was diffuse in two, left-sided in two, lower abdominal in two, and in the right lower quadrant in two patients. The inflamed appendix was right-sided in three, left-sided in three, and in the midline in two patients. Three cases were complicated by a peri-appendicular abscess, and one patient also had a small bowel obstruction. All patients had a complete non-rotation with right-sided duodenum and jejunum, and left-sided colon. All eight patients had an abnormal superior mesenteric artery-superior mesenteric vein (SMA/SMV) relationship and a dysplastic uncinate process of the pancreas. Urgent surgery was performed in six patients and the remaining two were treated conservatively. CONCLUSION: Altered anatomy in malrotation affects the typical clinical and CT findings of acute appendicitis, therefore delaying diagnosis. When CT shows focal inflammation anywhere within the abdomen, along with an abnormal SMA/SMV relationship, the position of the caecum should be ascertained and acute appendicitis ruled out.

The immunogenicity of human-origin therapeutic antibodies are associated with V gene usage.

Therapeutic antibodies can elicit unwanted immune responses in a subset of patients, which leads to the production of anti-drug antibodies (ADA). Some of these ADAs have been reported to effect the pharmacokinetics, efficacy and/or safety of the therapeutic antibodies. The sequence diversity of antibodies are generated by VDJ recombination and mutagenesis. While the antibody generation process can create a large candidate pool for identifying high-affinity antibodies, it also could produce sequences that are foreign to the human immune system. However, it is not clear how VDJ recombination and mutagenesis impact the clinical ADA rate of therapeutic antibodies. In this study, we identified a positive correlation between the clinical ADA rate and the number of introduced mutations in the antibody sequences. We also found that the use of rare V alleles in human-origin antibody therapeutics is associated with higher risk of immunogenicity. The results suggest that antibody engineering projects should start with frameworks that contain commonly used V alleles and prioritize antibody candidates with low number of mutations to reduce the risk of immunogenicity.

Cytokines as regulators of proliferation and survival of healthy and malignant peripheral B cells.

Adaptive immunity depends on the production and maintenance of a pool of mature peripheral lymphocytes throughout life. The signals regulating the survival of mature splenic B cells have become a major focus in recent studies of B cell immunology. Lasting B cell persistence in the periphery is dependent on survival signals that are transduced by cell surface receptors. Cytokines have been shown to play a critical role in maintaining lymphocyte homeostasis. This review focuses on the role of cytokines and their receptors in the regulation of peripheral B cell survival, with an emphasis on those that have received relatively less attention in the literature.

c-Met and its ligand hepatocyte growth factor/scatter factor regulate mature B cell survival in a pathway induced by CD74.

The signals regulating the survival of mature splenic B cells have become a major focus in recent studies of B cell immunology. Durable B cell persistence in the periphery is dependent on survival signals that are transduced by cell surface receptors. In this study, we describe a novel biological mechanism involved in mature B cell homeostasis, the hepatocyte growth factor/scatter factor (HGF)/c-Met pathway. We demonstrate that c-Met activation by HGF leads to a survival cascade, whereas its blockade results in induction of mature B cell death. Our results emphasize a unique and critical function for c-Met signaling in the previously described macrophage migration inhibitory factor/CD74-induced survival pathway. Macrophage migration inhibitory factor recruits c-Met to the CD74/CD44 complex and thereby enables the induction of a signaling cascade within the cell. This signal results in HGF secretion, which stimulates the survival of the mature B cell population in an autocrine manner. Thus, the CD74-HGF/c-Met axis defines a novel physiologic survival pathway in mature B cells, resulting in the control of the humoral immune response.

A BCMA/CD16A bispecific innate cell engager for the treatment of multiple myeloma.

Despite the recent progress, multiple myeloma (MM) is still essentially incurable and there is a need for additional effective treatments with good tolerability. RO7297089 is a novel bispecific BCMA/CD16A-directed innate cell engager (ICE®) designed to induce BCMA+ MM cell lysis through high affinity binding of CD16A and retargeting of NK cell cytotoxicity and macrophage phagocytosis. Unlike conventional antibodies approved in MM, RO7297089 selectively targets CD16A with no binding of other Fcγ receptors, including CD16B on neutrophils, and irrespective of 158V/F polymorphism, and its activity is less affected by competing IgG suggesting activity in the presence of M-protein. Structural analysis revealed this is due to selective interaction with a single residue (Y140) uniquely present in CD16A opposite the Fc binding site. RO7297089 induced tumor cell killing more potently than conventional antibodies (wild-type and Fc-enhanced) and induced lysis of BCMA+ cells at very low effector-to-target ratios. Preclinical toxicology data suggested a favorable safety profile as in vitro cytokine release was minimal and no RO7297089-related mortalities or adverse events were observed in cynomolgus monkeys. These data suggest good tolerability and the potential of RO7297089 to be a novel effective treatment of MM patients.

In vitro immunogenicity prediction: bridging between innate and adaptive immunity.

Development of antidrug antibodies (ADAs) is an undesirable potential outcome of administration of biotherapeutics and involves the innate and adaptive immune systems. ADAs can have detrimental clinical consequences: they can reduce biotherapeutic efficacy or produce adverse events. Because animal models are considered poor predictors of immunogenicity in humans, in vitro assays with human innate and adaptive immune cells are commonly used alternatives that can reveal cell-mediated unwanted immune responses. Multiple methods have been developed to assess the immune cell response following exposure to biotherapeutics and estimate the potential immunogenicity of biotherapeutics. This review highlights the role of innate and adaptive immune cells as the drivers of immunogenicity and summarizes the use of these cells in assays to predict clinical ADA.

Immunogenicity risk assessment for biotherapeutics through in vitro detection of CD134 and CD137 on T helper cells.

Biotherapeutics, which are biologic medications that are natural or bioengineered products of living cells, have revolutionized the treatment of many diseases. However, unwanted immune responses still present a major challenge to their widespread adoption. Many patients treated with biotherapeutics develop antigen-specific anti-drug antibodies (ADAs) that may reduce the efficacy of the therapy or cross-react with the endogenous counterpart of a protein therapeutic, or both. Here, we describe an in vitro method for assessing the immunogenic risk of a biotherapeutic. We found a correlation between clinical immunogenicity and the frequency with which a biotherapeutic stimulated an increase in CD134, CD137, or both cell surface markers on CD4+ T cells. Using high-throughput flow cytometry, we examined the effects of 14 biotherapeutics with diverse rates of clinical immunogenicity on peripheral blood mononuclear cells from 120 donors with diverse human leukocyte antigen class II-encoding alleles. Biotherapeutics with high rates of ADA development in the clinic had higher proportions of CD4+ T cells positive for CD134 or CD137 than biotherapeutics with low clinical immunogenicity. This method provides a rapid and simple preclinical test of the immunogenic potential of a new candidate biotherapeutic or biosimilar. Implementation of this approach during biotherapeutic research and development enables rapid elimination of candidates that are likely to cause ADA-related adverse events and detrimental consequences.