CD40/APC-specific antibodies with three T-cell epitopes loaded in the constant domains induce CD4+ T-cell responses

CD4+ T lymphocytes play a central role in the orchestration and maintenance of the adaptive immune response. Targeting of antigen to antigen presenting cells (APCs) increases peptide loading of major histocompatibility complex (MHC) class II molecules and CD4+ T-cell activation. APCs have been targeted by APC-specific recombinant antibodies (rAbs) with single T-cell epitopes integrated in the constant region of the heavy chain (C(H)). However, the strategy may be improved if several T-cell epitopes could be delivered simultaneously by one rAb. We here demonstrate that a single rAb can be loaded with multiple identical or different T-cell epitopes, integrated as loops between β-strands in C(H) domains. One epitope was inserted in C(H)1, while two were placed in C(H)2 of IgG. T-cell proliferation assays showed that all three peptides were excised from loops and presented on MHC class II to T-cells. Induction of T-cell activation by each epitope in the multi-peptide rAb was as good, or even better, than that elicited by corresponding single-peptide rAbs. Furthermore, following DNA vaccination of mice with plasmids that encode CD40-specific rAbs loaded with either one or three peptides, T-cell responses were induced. Thus, integration of multiple epitopes in C(H) region loops of APC-specific rAbs is feasible and may be utilized in design of multi-vaccines.     

Functional Changes of T-Cell Subsets with Age and CMV Infection

Cytomegalovirus (CMV) latent infection and aging contribute to alterations in the function and phenotype of the T-cell pool. We have demonstrated that CMV-seropositivity is associated with the expansion of polyfunctional CD57+ T-cells in young and middle-aged individuals in response to different stimuli. Here, we expand our results on the effects of age and CMV infection on T-cell functionality in a cohort of healthy middle-aged and older individuals stratified by CMV serostatus. Specifically, we studied the polyfunctional responses (degranulation, IFN-γ and TNF-α production) of CD4+, CD8+, CD8+CD56+ (NKT-like), and CD4−CD8− (DN) T-cells according to CD57 expression in response to Staphylococcal Enterotoxin B (SEB). Our results show that CD57 expression by T-cells is not only a hallmark of CMV infection in young individuals but also at older ages. CD57+ T-cells are more polyfunctional than CD57− T-cells regardless of age. CMV-seronegative individuals have no or a very low percentages of cytotoxic CD4+ T-cells (CD1017a+) and CD4+CD57+ T-cells, supporting the notion that the expansion of these T-cells only occurs in the context of CMV infection. There was a functional shift in T-cells associated with CMV seropositivity, except in the NKT-like subset. Here, we show that the effect of CMV infection and age differ among T-cell subsets and that CMV is the major driving force for the expansion of highly polyfunctional CD57+ T-cells, emphasizing the necessity of considering CMV serology in any study of immunosenescence.     

Monovalent antibody scFv fragments selected to modulate T-cell activation by inhibition of CD86-CD28 interaction

Beside the interaction of the antigen-presenting major histocompatibility complex with the T-cell receptor, a co-stimulatory signal is required for T-cell activation in an immune response. To reduce immune-mediated graft rejection in corneal transplantation, where topical application of drugs in ointments or eye-drops may be possible, we selected single-chain antibody fragments (scFv) with binding affinity to rat CD86 (B7.2) that inhibit the co-stimulatory signal. We produced the IgV-like domain of rat CD86 as a fusion protein in Escherichia coli by refolding from inclusion bodies. This protein was used as a target for phage display selection of scFv from HuCAL-1, a fully artificial human antibody library. Selected binding molecules were shown to specifically bind to rat CD86 and inhibit the interaction of CD86 with CD28 and CTLA4 (CD152) in flow cytometry experiments. In an assay for CD86-dependent co-stimulation, the selected scFv fragment successfully inhibited the proliferation of T-cells induced by CD86-expressing P815 cells.     

Blood-Based Immune Profiling Combined with Machine Learning Discriminates Psoriatic Arthritis from Psoriasis Patients

Psoriasis (Pso) is a chronic inflammatory skin disease, and up to 30% of Pso patients develop psoriatic arthritis (PsA), which can lead to irreversible joint damage. Early detection of PsA in Pso patients is crucial for timely treatment but difficult for dermatologists to implement. We, therefore, aimed to find disease-specific immune profiles, discriminating Pso from PsA patients, possibly facilitating the correct identification of Pso patients in need of referral to a rheumatology clinic. The phenotypes of peripheral blood immune cells of consecutive Pso and PsA patients were analyzed, and disease-specific immune profiles were identified via a machine learning approach. This approach resulted in a random forest classification model capable of distinguishing PsA from Pso (mean AUC = 0.95). Key PsA-classifying cell subsets selected included increased proportions of differentiated CD4+CD196+CD183-CD194+ and CD4+CD196-CD183-CD194+ T-cells and reduced proportions of CD196+ and CD197+ monocytes, memory CD4+ and CD8+ T-cell subsets and CD4+ regulatory T-cells. Within PsA, joint scores showed an association with memory CD8+CD45RA-CD197- effector T-cells and CD197+ monocytes. To conclude, through the integration of in-depth flow cytometry and machine learning, we identified an immune cell profile discriminating PsA from Pso. This immune profile may aid in timely diagnosing PsA in Pso.     

Human CD4+ T-Cell Clone Expansion Leads to the Expression of the Cysteine Peptidase Inhibitor Cystatin F

The existence of CD4+ cytotoxic T cells (CTLs) at relatively high levels under different pathological conditions in vivo suggests their role in protective and/or pathogenic immune functions. CD4+ CTLs utilize the fundamental cytotoxic effector mechanisms also utilized by CD8+ CTLs and natural killer cells. During long-term cultivation, CD4+ T cells were also shown to acquire cytotoxic functions. In this study, CD4+ human T-cell clones derived from activated peripheral blood lymphocytes of healthy young adults were examined for the expression of cytotoxic machinery components. Cystatin F is a protein inhibitor of cysteine cathepsins, synthesized by CD8+ CTLs and natural killer cells. Cystatin F affects the cytotoxic efficacy of these cells by inhibiting the major progranzyme convertases cathepsins C and H as well as cathepsin L, which is involved in perforin activation. Here, we show that human CD4+ T-cell clones express the cysteine cathepsins that are involved in the activation of granzymes and perforin. CD4+ T-cell clones contained both the inactive, dimeric form as well as the active, monomeric form of cystatin F. As in CD8+ CTLs, cysteine cathepsins C and H were the major targets of cystatin F in CD4+ T-cell clones. Furthermore, CD4+ T-cell clones expressed the active forms of perforin and granzymes A and B. The levels of the cystatin F decreased with time in culture concomitantly with an increase in the activities of granzymes A and B. Therefore, our results suggest that cystatin F plays a role in regulating CD4+ T cell cytotoxicity. Since cystatin F can be secreted and taken up by bystander cells, our results suggest that CD4+ CTLs may also be involved in regulating immune responses through cystatin F secretion.     

New Insights in Mechanisms and Therapeutics for Short- and Long-Term Impacts of Hepatic Ischemia Reperfusion Injury Post Liver Transplantation

Liver transplantation has been identified as the most effective treatment for patients with end-stage liver diseases. However, hepatic ischemia reperfusion injury (IRI) is associated with poor graft function and poses a risk of adverse clinical outcomes post transplantation. Cell death, including apoptosis, necrosis, ferroptosis and pyroptosis, is induced during the acute phase of liver IRI. The release of danger-associated molecular patterns (DAPMs) and mitochondrial dysfunction resulting from the disturbance of metabolic homeostasis initiates graft inflammation. The inflammation in the short term exacerbates hepatic damage, leading to graft dysfunction and a higher incidence of acute rejection. The subsequent changes in the graft immune environment due to hepatic IRI may result in chronic rejection, cancer recurrence and fibrogenesis in the long term. In this review, we mainly focus on new mechanisms of inflammation initiated by immune activation related to metabolic alteration in the short term during liver IRI. The latest mechanisms of cancer recurrence and fibrogenesis due to the long-term impact of inflammation in hepatic IRI is also discussed. Furthermore, the development of therapeutic strategies, including ischemia preconditioning, pharmacological inhibitors and machine perfusion, for both attenuating acute inflammatory injury and preventing late-phase disease recurrence, will be summarized in the context of clinical, translational and basic research.     

The Role of CTLA4 and Its Polymorphisms in Solid Organ and Haematopoietic Stem Cell Transplantation

HLA matching, transplantation technique, or underlying disease greatly influences the probability of long-term transplantation success. It has been hypothesised that genetic variation affecting antigen presentation also contributes to the outcomes of both solid organ transplantation and allogeneic haematopoietic stem cell transplantation (AHSCT). Those genes, along with those responsible for innate and adaptive immunity, have become targets of investigation. In this review, we focus on the role of CTLA4 in the process of acute graft rejection and summarise the progress in our understanding of its role in predicting the outcome. We present the results of the latest studies investigating the link between CTLA4 gene variability and AHSCT, as well as organ transplantation outcomes. While some studies found a link between +49 A/G and −318 C/T and transplantation outcomes, comprehensive meta-analyses have failed to present any association. The most recent field reviews suggest that the −1772 T/C (rs733618) CC genotype is weakly associated with a lower risk of acute graft rejection, while +49 A/G might be clinically meaningful when investigated in the context of combinations with other polymorphisms. Studies verifying associations between 12 CTLA4 gene SNPs and AHSCT outcomes present inexplicit results. Some of the most commonly studied polymorphisms in this context include +49 A/G (rs231775) and CT60 A/G (rs3087243). The results signify that, in order to understand the role of CTLA4 and its gene polymorphisms in transplantology, further studies must be conducted.     

Activation of T-Lymphocytes by LDL-Cholesterol

Native LDL-cholesterol can be mechanically stressed by strong vortexing. According to one hypothesis, mechanical shear stress within the vessel can lead to an aggregation of LDL-cholesterol and subsequently to activation of CD4 and CD8 T-lymphocytes. The goal of this study was to determine the proportion of activated CD4 and CD8 T-lymphocytes that is induced by adding unstressed and mechanically stressed LDL-cholesterol to whole blood samples. Whole blood was taken from 12 healthy subjects. All probands fasted for at least 12 h before blood withdrawal. In each case, 1 ml of whole blood from each subject was incubated for 16 h at 32 °C (89.3 °F) with concanavalin A (A), without additive (B), with mechanically stressed LDL-cholesterol (C) or with native LDL-cholesterol (D). Subsequently, the samples were measured by four-color flow cytometry. CD3, CD4, CD8, and CD69 were measured as activity markers. CD69 was plotted against CD4 and CD8, and the proportions of activated CD4 and CD8 T-lymphocytes were determined. Native and vortexed LDL-cholesterol elicited significantly different types of T-cell activation. While native LDL activated CD4 T-cells to only a small extent, mechanically stressed (vortexed) LDL potently activated CD8 T-cells. Purely mechanically-induced changes in LDL-cholesterol may be one mechanism that contributes to the activation of CD8 cells and, as a consequence, the emergence of arteriosclerosis.     

Combinatorial Use of Both Epigenetic and Non-Epigenetic Mechanisms to Efficiently Reactivate HIV Latency

The persistence of latent HIV provirus pools in different resting CD4+ cell subsets remains the greatest obstacle in the current efforts to treat and cure HIV infection. Recent efforts to purge out latently infected memory CD4+ T-cells using latency-reversing agents have failed in clinical trials. This review discusses the epigenetic and non-epigenetic mechanisms of HIV latency control, major limitations of the current approaches of using latency-reversing agents to reactivate HIV latency in resting CD4+ T-cells, and potential solutions to these limitations.     

Tryptophan Metabolism via Kynurenine Pathway: Role in Solid Organ Transplantation

Solid organ transplantation is a gold standard treatment for patients suffering from an end-stage organ disease. Patient and graft survival have vastly improved during the last couple of decades; however, the field of transplantation still encounters several unique challenges, such as a shortage of transplantable organs and increasing pool of extended criteria donor (ECD) organs, which are extremely prone to ischemia-reperfusion injury (IRI), risk of graft rejection and challenges in immune regulation. Moreover, accurate and specific biomarkers, which can timely predict allograft dysfunction and/or rejection, are lacking. The essential amino acid tryptophan and, especially, its metabolites via the kynurenine pathway has been widely studied as a contributor and a therapeutic target in various diseases, such as neuropsychiatric, autoimmune disorders, allergies, infections and malignancies. The tryptophan-kynurenine pathway has also gained interest in solid organ transplantation and a variety of experimental studies investigating its role both in IRI and immune regulation after allograft implantation was first published. In this review, the current evidence regarding the role of tryptophan and its metabolites in solid organ transplantation is presented, giving insights into molecular mechanisms and into therapeutic and diagnostic/prognostic possibilities.     

CD146 Delineates an Interfascicular Cell Sub-Population in Tendon That Is Recruited during Injury through Its Ligand Laminin-α4

The interfascicular matrix (IFM) binds tendon fascicles and contains a population of morphologically distinct cells. However, the role of IFM-localised cell populations in tendon repair remains to be determined. The basement membrane protein laminin-α4 also localises to the IFM. Laminin-α4 is a ligand for several cell surface receptors, including CD146, a marker of pericyte and progenitor cells. We used a needle injury model in the rat Achilles tendon to test the hypothesis that the IFM is a niche for CD146+ cells that are mobilised in response to tendon damage. We also aimed to establish how expression patterns of circulating non-coding RNAs alter with tendon injury and identify potential RNA-based markers of tendon disease. The results demonstrate the formation of a focal lesion at the injury site, which increased in size and cellularity for up to 21 days post injury. In healthy tendon, CD146+ cells localised to the IFM, compared with injury, where CD146+ cells migrated towards the lesion at days 4 and 7, and populated the lesion 21 days post injury. This was accompanied by increased laminin-α4, suggesting that laminin-α4 facilitates CD146+ cell recruitment at injury sites. We also identified a panel of circulating microRNAs that are dysregulated with tendon injury. We propose that the IFM cell niche mediates the intrinsic response to injury, whereby an injury stimulus induces CD146+ cell migration. Further work is required to fully characterise CD146+ subpopulations within the IFM and establish their precise roles during tendon healing.     

Functional Ambivalence of Dendritic Cells: Tolerogenicity and Immunogenicity

Dendritic cells (DCs) are the most potent professional antigen-presenting cells (APCs) and inducers of T cell-mediated immunity. Although DCs play a central role in promoting adaptive immune responses against growing tumors, they also establish and maintain peripheral tolerance. DC activity depends on the method of induction and/or the presence of immunosuppressive agents. Tolerogenic dendritic cells (tDCs) induce immune tolerance by activating CD4⁺CD25⁺Foxp3⁺ regulatory T (Treg) cells and/or by producing cytokines that inhibit T cell activation. These findings suggest that tDCs may be an effective treatment for autoimmune diseases, inflammatory diseases, and infertility.     

The Leukotriene Receptor Antagonist Montelukast Attenuates Neuroinflammation and Affects Cognition in Transgenic 5xFAD Mice

Alzheimer’s disease (AD) is the most common form of dementia. In particular, neuroinflammation, mediated by microglia cells but also through CD8+ T-cells, actively contributes to disease pathology. Leukotrienes are involved in neuroinflammation and in the pathological hallmarks of AD. In consequence, leukotriene signaling—more specifically, the leukotriene receptors—has been recognized as a potential drug target to ameliorate AD pathology. Here, we analyzed the effects of the leukotriene receptor antagonist montelukast (MTK) on hippocampal gene expression in 5xFAD mice, a commonly used transgenic AD mouse model. We identified glial activation and neuroinflammation as the main pathways modulated by MTK. The treatment increased the number of Tmem119+ microglia and downregulated genes related to AD-associated microglia and to lipid droplet-accumulating microglia, suggesting that the MTK treatment targets and modulates microglia phenotypes in the disease model compared to the vehicle. MTK treatment further reduced infiltration of CD8+T-cells into the brain parenchyma. Finally, MTK treatment resulted in improved cognitive functions. In summary, we provide a proof of concept for MTK to be a potential drug candidate for AD and provide novel modes of action via modulation of microglia and CD8+ T-cells. Of note, 5xFAD females showed a more severe pathology, and in consequence, MTK treatment had a more pronounced effect in the females compared to the males. The effects on neuroinflammation, i.e., microglia and CD8+ T-cells, as well as the effects on cognitive outcome, were dose-dependent, therefore arguing for the use of higher doses of MTK in AD clinical trials compared to the approved asthma dose.     

Cyclophilin D Regulates the Nuclear Translocation of AIF,Cardiac Endothelial Cell Necroptosis and Murine Cardiac Transplant Injury

Ischemia-reperfusion injury (IRI) is an inevitable consequence of organ transplant procedure and associated with acute and chronic organ rejection in transplantation. IRI leads to various forms of programmed cell death, which worsens tissue damage and accelerates transplant rejection. We recently demonstrated that necroptosis participates in murine cardiac microvascular endothelial cell (MVEC) death and murine cardiac transplant rejection. However, MVEC death under a more complex IRI model has not been studied. In this study, we found that simulating IRI conditions in vitro by hypoxia, reoxygenation and treatment with inflammatory cytokines induced necroptosis in MVECs. Interestingly, the apoptosis-inducing factor (AIF) translocated to the nucleus during MVEC necroptosis, which is regulated by the mitochondrial permeability molecule cyclophilin D (CypD). Furthermore, CypD deficiency in donor cardiac grafts inhibited AIF translocation and mitigated graft IRI and rejection (n = 7; p = 0.002). Our studies indicate that CypD and AIF play significant roles in MVEC necroptosis and cardiac transplant rejection following IRI. Targeting CypD and its downstream AIF may be a plausible approach to inhibit IRI-caused cardiac damage and improve transplant survival.     

Perforin Expression by CD4+ Regulatory T Cells Increases at Multiple Sclerosis Relapse: Sex Differences

Multiple sclerosis (MS) represents the leading cause of neurological deficit among young adults, affecting women more frequently than men. In MS, the extent of central nervous system lesions is determined by the net balance between self-reactive and regulatory T-cells at any given time, among other factors, as well as by the effect of inflammatory response. Here, we studied both CD4+ and CD8+ T(Reg) in parallel in blood and CSF during MS relapse. A recruitment of both regulatory CD4+ and CD8+ T cells (T(Reg)) within the cerebrospinal fluid (CSF) takes place during MS relapse. Not previously described, the presence of CD4+ T(Reg) in CSF was higher in women than in men, which could account for the sexual dimorphism in the incidence of MS. A direct correlation between plasma oestradiol (E2) and IL-2 levels was observed, in line with a putative circuit of E2 and perforin expression by CD4+ T(Reg) playing a role in MS. Also, serum IFN-alpha was higher in females, with direct correlation with serum E2 levels. This is the first study to analyze perforin expression by CD4+ T(Reg) in MS, which was greatly enhanced in CSF, what points out a relevant role of this molecule in the suppressive effects of the CD4+ T(Reg) in MS, and contributes to the understanding of MS pathophysiology.     

Lipoxygenase Products in the Urine Correlate with Renal Function and Body Temperature but not with Acute Transplant Rejection

Acute transplant rejection is the leading cause of graft loss in the first months after kidney transplantation. Lipoxygenase products mediate pro- and anti-inflammatory actions and thus we aimed to correlate the histological reports of renal transplant biopsies with urinary lipoxygenase products concentrations to evaluate their role as a diagnostic marker. This study included a total of 34 kidney transplant recipients: 17 with an acute transplant rejection and 17 controls. LTE4, LTB4, 12-HETE and 15-HETE concentrations were measured by enzyme immunoassay. Urinary lipoxygenase product concentrations were not significantly changed during an acute allograft rejection. Nevertheless, LTB4 concentrations correlated significantly with the body temperature (P ≤ 0.05) 3 months after transplantation, and 12- and 15-HETE concentrations correlated significantly with renal function (P ≤ 0.05) 2 weeks after transplantation. In conclusion, our data show a correlation for LTB4 with the body temperature 3 months after transplantation and urinary 12- and 15-HETE concentrations correlate positively with elevated serum creatinine concentrations but do not predict acute allograft rejection.     

The Proteomic Landscape of Resting and Activated CD4+ T Cells Reveal Insights into Cell Differentiation and Function

CD4+ T cells (T helper cells) are cytokine-producing adaptive immune cells that activate or regulate the responses of various immune cells. The activation and functional status of CD4+ T cells is important for adequate responses to pathogen infections but has also been associated with auto-immune disorders and survival in several cancers. In the current study, we carried out a label-free high-resolution FTMS-based proteomic profiling of resting and T cell receptor-activated (72 h) primary human CD4+ T cells from peripheral blood of healthy donors as well as SUP-T1 cells. We identified 5237 proteins, of which significant alterations in the levels of 1119 proteins were observed between resting and activated CD4+ T cells. In addition to identifying several known T-cell activation-related processes altered expression of several stimulatory/inhibitory immune checkpoint markers between resting and activated CD4+ T cells were observed. Network analysis further revealed several known and novel regulatory hubs of CD4+ T cell activation, including IFNG, IRF1, FOXP3, AURKA, and RIOK2. Comparison of primary CD4+ T cell proteomic profiles with human lymphoblastic cell lines revealed a substantial overlap, while comparison with mouse CD+ T cell data suggested interspecies proteomic differences. The current dataset will serve as a valuable resource to the scientific community to compare and analyze the CD4+ proteome.     

Spinal Cord T-Cell Infiltration in the Rat Spared Nerve Injury Model: A Time Course Study

The immune system is involved in the development of neuropathic pain. In particular, the infiltration of T-lymphocytes into the spinal cord following peripheral nerve injury has been described as a contributor to sensory hypersensitivity. We used the spared nerve injury (SNI) model of neuropathic pain in Sprague Dawley adult male rats to assess proliferation, and/or protein/gene expression levels for microglia (Iba1), T-lymphocytes (CD2) and cytotoxic T-lymphocytes (CD8). In the dorsal horn ipsilateral to SNI, Iba1 and BrdU stainings revealed microglial reactivity and proliferation, respectively, with different durations. Iba1 expression peaked at D4 and D7 at the mRNA and protein level, respectively, and was long-lasting. Proliferation occurred almost exclusively in Iba1 positive cells and peaked at D2. Gene expression observation by RT-qPCR array suggested that T-lymphocytes attracting chemokines were upregulated after SNI in rat spinal cord but only a few CD2/CD8 positive cells were found. A pronounced infiltration of CD2/CD8 positive T-cells was seen in the spinal cord injury (SCI) model used as a positive control for lymphocyte infiltration. Under these experimental conditions, we show early and long-lasting microglia reactivity in the spinal cord after SNI, but no lymphocyte infiltration was found.