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Purpose: The purpose of this investigation was to characterize skeletal muscle T-cell accumulation after contraction-induced muscle damage and test the hypothesis that T cells contribute to postdamage muscle protection (i.e., the repeated bout effect) in a way reminiscent of their role in adaptive immunity., Methods: In vivo lengthening contractions were used to model the repeated bout effect and contralateral repeated bout effect in rats. Intramuscular T-cell subsets were characterized by flow cytometry after single and repeated bouts of lengthening contractions, and an adoptive T-cell transfer experiment was done to test whether T cells from muscle damage-experienced rats can confer protection from injury to damage-naive rats., Results: Electrically stimulated lengthening contractions elicited the repeated bout effect, but not the contralateral repeated bout effect. Although leukocytes (CD45+) were scarce in undamaged muscle (2.1% of all cells), substantially more (63% of all cells) were observed after a single bout of lengthening contractions. Within the leukocyte population were several subsets of T cells, including conventional CD4+, CD8+, memory, and regulatory T cells. In contrast, a minimal increase in T cells was observed after a second bout of lengthening contractions. Conventional CD4+ T cells (FoxP3-) were the most abundant subset in muscle after lengthening contractions. Adoptive T-cell transfer from damage-experienced rats did not confer protection to damage-naive recipient rats., Conclusions: The robust T-cell accumulation, particularly the CD4 subset, after contraction-induced damage suggests a role for these cells in muscle repair and adaptation to muscle damaging contractions. Moreover, T cells are unlikely to mediate the protective adaptations of the repeated bout effect in a manner similar to their role in adaptive immunity.

Introduction: Primary biliary cholangitis (PBC) is characterized by lymphocyte cell-induced immune destruction of cholangiole. However, the immunological characteristics of peripheral blood cells in PBC patients remain unknown. This study was designed to reveal the differences in the immunological characteristics between PBC patients and healthy adults., Methods: We performed high-throughput sequencing to determine the TRB-CDR3 and IGH-CDR3 repertoires of T and B cells in 19 healthy controls and 29 PBC patients. Different immunological characteristics, such as distinctive complementarity determining region 3 (TRB-CDR3) lengths, usage bias of V and J segments, and random nucleotide addition were identified in PBC and healthy control (HC) groups., Results: The diversity of TRB-CDR3 was significantly lower in the PBC group compared with the HC group. CDR3 and the N addition length distribution were significantly changed compared with the HC group. It appeared that the PBC group had more short N additions and the HC group had more long N additions in the TRB-CDR3 repertoire. The results also revealed a set of PBC-associated clonotypes compared with the HC group., Conclusion: This study suggested that PBC is a complex autoimmune disease process with evidence of different TRB-CDR3 rearrangements compared with healthy adults that share IGH-CDR3 peptides with some autoimmune diseases. This new insight may contribute to a better understanding of the immune functions of PBC patients and benefit efficient applications of PBC diagnosis and treatments., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier B.V.)

Peripheral T cell lymphomas (PTCLs) are a heterogeneous group of orphan neoplasms. Despite the introduction of anthracycline-based chemotherapy protocols, with or without autologous haematopoietic transplantation and a plethora of new agents, the progression-free survival of patients with PTCLs needs to be improved. The rarity of these neoplasms, the limited knowledge of their driving defects and the lack of experimental models have impaired clinical successes. This scenario is now rapidly changing with the discovery of a spectrum of genomic defects that hijack essential signalling pathways and foster T cell transformation. This knowledge has led to new genomic-based stratifications, which are being used to establish objective diagnostic criteria, more effective risk assessment and target-based interventions. The integration of genomic and functional data has provided the basis for targeted therapies and immunological approaches that underlie individual tumour vulnerabilities. Fortunately, novel therapeutic strategies can now be rapidly tested in preclinical models and effectively translated to the clinic by means of well-designed clinical trials. We believe that by combining new targeted agents with immune regulators and chimeric antigen receptor-expressing natural killer and T cells, the overall survival of patients with PTCLs will dramatically increase.

Ethnopharmacological Relevance: Acanthosicyos naudininus, Gomphocarpus fruticosus, and Cryptolepis decidua are, according to the knowledge of traditional healers, used in Namibia to treat inflammatory disorders such as pain, fever and skin rashes., Aim of the Study: The present study was conducted to evaluate the immunomodulatory effects and the possible underlying mechanisms of action of the plant extracts on peripheral blood mononuclear cells (PBMCs) such as T-lymphocytes., Materials and Methods: Methanolic and EtOAc extracts of A. naudinianus, G. fruticosus and C. decidua were analysed for their immunomodulatory potential. PBMCs were isolated from the blood of healthy donors and incubated with the plant extracts at concentrations 100, 30, 10, 3, 1 and 0.3 μg/mL. Effects on proliferation and viability of activated human lymphocytes were assessed in comparison to ciclosporin A by flow cytometry using carboxyfluorescein succinimidyl ester (CFSE) and WST-1 assay. Flow cytometry by annexin V/propidium iodide (PI) staining was performed to investigate the necrotic/apoptotic effect of the plant extracts on mitogen-activated human lymphocytes. In addition, analysis of the influence of plant extracts on the regulatory mechanisms of T-lymphocytes was performed using activation marker and cytokine production assays. An HPLC-PDA-ELSD-ESIMS profile was recorded for each of the extracts., Results: T-lymphocyte proliferation was inhibited in a dose-dependent manner by the extracts of A. naudinianus, G. fruticosus, and C. decidua in concentrations not causing apoptosis or necrosis. This effect was mediated by inhibition of lymphocyte activation, specifically the suppression of CD25 and CD69 surface receptor expression. Moreover, the extracts suppressed effector functions, as indicated by reduced production of IFN-γ and IL-2. Based on the HPLC profile, possible responsible compound classes could be identified for the extracts of A. naudinianus (cucurbitacins) and C. decidua (indole alkaloids), but not for G. fruticosus., Conclusions: The data show that the extracts of A. naudinianus, G. fruticosus and C. decidua have in vitro immunomodulatory activity and they interfere with the function of immunocompetent cells, suggesting an anti-inflammatory mode-of-action. The present chemical determination and pattern recognition results explain the therapeutic potency. However, further studies to investigate the therapeutic potential of the plants in inflammatory disorders should be done., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2020 Elsevier B.V. All rights reserved.)

T cells in vivo migrate primarily via undirected random walks, but it remains unresolved how these random walks generate an efficient search. Here, we use light sheet microscopy of T cells in the larval zebrafish as a model system to study motility across large populations of cells over hours in their native context. We show that cells do not perform Levy flight; rather, there is substantial cell-to-cell variability in speed, which persists over timespans of a few hours. This variability is amplified by a correlation between speed and directional persistence, generating a characteristic cell behavioral manifold that is preserved under a perturbation to cell speeds, and seen in Mouse T cells and Dictyostelium . Together, these effects generate a broad range of length scales over which cells explore in vivo., Competing Interests: EJ, SQ No competing interests declared, (© 2020, Jerison and Quake.)

Like B cells, T cells can be immortalized through hybridization with lymphoma cells, a technique that has been particularly useful in the study of the T cell receptors (TCR) for antigen. In T cell hybridizations, the AKR mouse strain-derived thymus lymphoma BW5147 is by far the most popular fusion line. However, the full potential of this technology had to await inactivation of the productively rearranged TCR-α and -β genes in the lymphoma. BWα-β-, the TCR-gene deficient variant of the original lymphoma, which has become the fusion line of choice for αβ T cells, is now available with numerous modifications, enabling the investigation of many aspects of TCR-mediated responses and TCR-structure. Unexpectedly, inactivating BW's functional TCR-α gene also rendered the lymphoma more permissive for the expression of TCR-γδ, facilitating the study of γδ T cells, their TCRs, and their TCR-mediated reactivity.

Corticosteroids have been utilised in the assisted reproduction setting with the expectation of suppressing aberrant immune activation and improving fertility in women. However, the effects of corticosteroids on fertility, and on pregnancy and offspring outcomes, are unclear. In this study, mice were administered prednisolone (1 mg/kg) or PBS daily in the pre-implantation phase, and effects on the adaptive immune response, the implantation rate, fetal development and postnatal outcomes were investigated. Prednisolone disrupted the expected expansion of CD4+ T cells in early pregnancy, inhibiting generation of both regulatory T cells (Treg cells) and effector T cells and suppressing IFNG required for T cell functional competence. Prednisolone caused an 8-20% increase in the embryo implantation rate and increased the number of viable pups per litter. In late gestation, fetal and placental weights were reduced in a litter size-dependent manner, and the canonical inverse relationship between litter size and fetal weight was lost. The duration of pregnancy was extended by ~ 0.5 day and birth weight was reduced by ~ 5% after prednisolone treatment. Viability of prednisolone-exposed offspring was comparable to controls, but body weight was altered in adulthood, particularly in male offspring. Thus, while prednisolone given in the pre-implantation phase in mice increases maternal receptivity to implantation and resource investment in fetal growth, there is a trade-off in long-term consequences for fetal development, birth weight and offspring health. These effects are associated with, and likely caused by, prednisolone suppression of the adaptive immune response at the outset of pregnancy., (© The Author(s) 2020. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.)

Breakthroughs in anti-tumor immunity have led to unprecedented advances in immunotherapy, yet it is now clear that the tumor microenvironment (TME) restrains immunity. T cells must substantially increase nutrient uptake to mount a proper immune response and failure to obtain sufficient nutrients or engage the appropriate metabolic pathways can alter or prevent effector T cell differentiation and function. The TME, however, can be metabolically hostile due to insufficient vascular exchange and cancer cell metabolism that leads to hypoxia, depletion of nutrients, and accumulation of waste products. Further, inhibitory receptors present in the TME can inhibit T cell metabolism and alter T cell signaling both directly and through release of extracellular vesicles such as exosomes. This review will discuss the metabolic changes that drive T cells into different stages of their development and how the TME imposes barriers to the metabolism and activity of tumor infiltrating lymphocytes., Competing Interests: AL No competing interests declared, WR Incyte pharmaceutics, through a partnership with Vanderbilt University, our laboratory has a funded project unrelated to the topic of this review. JR Incyte pharmaceutics, through a partnership with Vanderbilt University, our laboratory has a funded project unrelated to the topic of this review; Sitryx, SAB and co-founder, a company with relevant project areas, (© 2020, Lim et al.)

Background: The inactivation of the ciliary proteins polycystin 1 or polycystin 2 leads to autosomal dominant polycystic kidney disease (ADPKD). Although signaling by primary cilia and interstitial inflammation both play a critical role in the disease, the reciprocal interactions between immune and tubular cells are not well characterized. The transcription factor STAT3, a component of the cilia proteome that is involved in crosstalk between immune and nonimmune cells in various tissues, has been suggested as a factor fueling ADPKD progression., Method: To explore how STAT3 intersects with cilia signaling, renal inflammation, and cyst growth, we used conditional murine models involving postdevelopmental ablation of Pkd1 , Stat3 , and cilia, as well as cultures of cilia-deficient or STAT3-deficient tubular cell lines., Results: Our findings indicate that, although primary cilia directly modulate STAT3 activation in vitro , the bulk of STAT3 activation in polycystic kidneys occurs through an indirect mechanism in which primary cilia trigger macrophage recruitment to the kidney, which in turn promotes Stat3 activation. Surprisingly, although inactivating Stat3 in Pkd1 -deficient tubules slightly reduced cyst burden, it resulted in a massive infiltration of the cystic kidneys by macrophages and T cells, precluding any improvement of kidney function. We also found that Stat3 inactivation led to increased expression of the inflammatory chemokines CCL5 and CXCL10 in polycystic kidneys and cultured tubular cells., Conclusions: STAT3 appears to repress the expression of proinflammatory cytokines and restrict immune cell infiltration in ADPKD. Our findings suggest that STAT3 is not a critical driver of cyst growth in ADPKD but rather plays a major role in the crosstalk between immune and tubular cells that shapes disease expression., (Copyright © 2020 by the American Society of Nephrology.)

We studied the formation of proliferative response of thymic lymphocytes to T-cell mitogen in rats exposed to endocrine disrupter DDT during prenatal and postnatal ontogeny. Developmental exposure to the endocrine disruptor was found to attenuate proliferative response during puberty and adulthood due to maintenance of higher proliferation rate of thymic lymphocytes in comparison with age-matched controls. Insufficient proliferative response to mitogens in rats developmentally exposed to the endocrine disrupter increases the risk of impairment of cell-mediated reactions of adaptive immunity.

The promising outcomes of immune-checkpoint based immunotherapies in cancer have provided a proportional perspective ahead of exploiting similar approaches in allotransplantation. Belatacept (CTLA-4-Ig) is an example of costimulation blockers successfully exploited in renal transplantation. Due to the wide range of regulatory molecules characterized in the past decades, some of these molecules might be candidates as immunomodulators in the case of tolerance induction in transplantation. Although there are numerous attempts on the apprehension of the effects of co-signaling molecules on immune response, the necessity for a better understanding is evident. By increasing the knowledge on the biology of co-signaling pathways, some pitfalls are recognized and improved approaches are proposed. The blockage of CD80/CD28 axis is an instance of evolution toward more efficacy. It is now evident that anti-CD28 antibodies are more effective than CD80 blockers in animal models of transplantation. Other co-signaling axes such as PD-1/PD-L1, CD40/CD154, 2B4/CD48, and others discussed in the present review are examples of critical immunomodulatory molecules in allogeneic transplantation. We review here the outcomes of recent experiences with co-signaling molecules in preclinical studies of solid organ transplantation., (© 2019 Wiley Periodicals, Inc.)

Tumour-associated macrophages (TAMs) inhibit the killing effect of T lymphocytes on tumour cells through the immunocheckpoint programmed death ligand-1 (PD-L1)/programmed death-1 (PD-1) axis. TAMs-targeted therapy is a promising approach that could be used to reverse the immunosuppressive tumour microenvironment. Here, we further report CMPB90-1, a novel natural polysaccharide from Cordyceps militaris, could function as an anti-tumour modulator that resets TAMs from a tumour-promoting M2 phenotype to a tumour-killing M1 phenotype. This process involves reversing the functional inhibition of T lymphocytes by inhibiting the PD-L1/PD-1 axis between TAMs and T lymphocytes. Mechanistically, the membrane receptor of CMPB90-1 binding to M2 macrophages was identified by tandem mass spectrometry. CMPB90-1 converts immunosuppressive TAMs via binding to toll-like receptor 2 (TLR2), which causes the release of Ca 2+ and the activation of p38, Akt and NF-κB, or ERK. This process then leads to the polarization of TAMs from M2 phenotype to the M1 phenotype. In vivo experiment shows that CMPB90-1 is able to polarize TAMs into the M1 phenotype and has anti-tumour effects with improved safety. Additionally, the anti-tumour effects of CMPB90-1 in vivo depend on the phenotypic conversion of TAMs. The results demonstrated that CMPB90-1 could be developed as a potential immune-oncology treatment reagent., (Copyright © 2020 Elsevier B.V. All rights reserved.)

We studied the effect of a single intraperitoneal injection of metabolites from Bacillus sp. M3 strain isolated from permafrost (from 5×10 3 to 50×10 3 microbial bodies) on differentiation of T cells in the thymus of F1(CBA/Black-6) mice. On day 21 after the injection of metabolites, a dose-dependent decrease in the level of CD34+CD44+ and an increase in the number of CD34 + CD44-, CD25-TCR + , CD25 + TCR +max , CD4 + CD8-, CD4-CD8 + , and CD44 + TCR + lymphocytes were observed in the thymus. The increase in thymus level of mature (CD25 + TCR +max ) and migration-ready (CD44 + TCR + ) T cells in combination with a moderate decrease in the level of T cell precursors entering the thymus from the bone marrow (CD34 + CD44 + ) can indicate a modulating influence of Bacillus sp. metabolites on functional activity of the thymus aimed at maintenance of the T cell balance in the body.

Purpose of Review: MicroRNAs (miRNAs) are small noncoding RNA molecules that are considered one of the fundamental regulatory mechanisms of gene expression. They are involved in many biologic processes, such as signal transduction, cell proliferation and differentiation, apoptosis and stress responses. The purpose of this review is to present recent knowledge about the role of miRNAs in asthma and outline possible applications of miRNAs., Recent Findings: A core set of miRNAs involved in asthma includes downregulated let-7 family, miR-193b, miR-375 as well as upregulated miR-21, miR-223, miR-146a, miR-142-5p, miR-142-3p, miR-146b and miR-155. Recently it has been shown that most of the involved miRNAs increase secretion of Th2 cytokines, decrease secretion of Th1 cytokines, promote differentiation of T cells towards Th2 or play a role in hyperplasia and hypertrophy of bronchial smooth muscle cells. The profiles of miRNAs correlate with clinical characteristics, including lung function, phenotype and severity of asthma., Summary: Recent publications confirmed crucial regulatory role of miRNAs in the pathomechanism of asthma. Some single miRNAs or their sets hold the promise for their use as asthma biomarkers facilitating diagnosis or prediction of treatment outcomes. They are also possible target of future therapies. The studies in this field are lacking though.

Although antigen-specific priming of antibody responses is impaired during sepsis, there is nevertheless a strong increase in IgM and IgG serum concentrations. Using colon ascendens stent peritonitis (CASP), a mouse model of polymicrobial abdominal sepsis, we observed substantial increases in IgM as well as IgG of all subclasses, starting at day 3 and peaking 2 weeks after sepsis induction. The dominant source of antibody-secreting cells was by far the spleen, with a minor contribution of the mesenteric lymph nodes. Remarkably, sepsis induction in splenectomized mice did not change the dynamics of the serum IgM/IgG reaction, indicating that the marginal zone B cells, which almost exclusively reside in the spleen, are dispensable in such a setting. Hence, in systemic bacterial infection, the function of the spleen as dominant niche of antibody-producing cells can be compensated by extra-splenic B cell populations as well as other lymphoid organs. Depletion of CD4+ T cells did not affect the IgM response, while it impaired IgG generation of all subclasses with the exception of IgG3. Taken together, our data demonstrate that the robust class-switched antibody response in sepsis encompasses both T cell-dependent and -independent components., (Copyright © 2020 Nicolai, Pötschke, Schmoeckel, Darisipudi, van der Linde, Raafat and Bröker.)

Background: Adoptive T-cell therapy (ACT) using autologous tumor-reactive T lymphocytes has considerable potential for cancer immunotherapy. In ACT, T cells are isolated from cancer patients and then stimulated and expanded in vitro by cytokines and costimulatory molecules. 4-1BB is an important costimulatory protein belonging to the TNF receptor superfamily. It is involved in T-cell survival, proliferation and activation. Agonistic anti-4-1BB monoclonal antibodies have been introduced as appropriate tools for ACT., Methods: Here, various single-chain fragment variable (scFv) antibodies were used to activate T cells isolated from peripheral blood via immune magnetic isolation. The T cells were stimulated with IL-2 and anti-CD-3 mAb and then treated with agonistic anti-4-1BB scFvs. The results showed the remarkable effects of anti-41BB scFvs on the functional properties of T cells, including their activation, proliferation and cytokine production. The flow cytometry analysis revealed a considerable increase in the expression of the T-cell activation marker CD69. Moreover, T-cell proliferation was evidenced in treated cells by CFSE labeling compared to the control groups., Result: Anti-4-1BB scFvs significantly increased IFN-γ and IL-2 mRNA and protein expression in T cells, but exhibited no stimulatory effect on IL-4 expression. These findings show that anti-4-1BB scFvs could evoke a Type I immune response., Conclusions: Our results demonstrate that targeting the 4-1BB molecule using agonistic scFvs could be an effective strategy for T-cell stimulation as part of an ACT approach to cancer treatment., Competing Interests: Competing interestsThe authors declare that they have no competing interests., (© The Author(s) 2020.)

Transplant vasculopathy (TV), a hallmark of chronic allograft rejection, is the primary cause of allograft loss after organ transplantation. Because multiple mechanisms are involved in TV pathogenesis, effective therapy for it remains elusive. Here, we identify the role of triptolide, which has a wide spectrum of immuno-suppressive activities, in inhibiting TV development. Murine aortic transplants models were constructed and divided into triptolide-treated and untreated groups. We found that triptolide significantly alleviated intima thickening of allografts by inhibiting multiple pathways. Triptolide significantly reduced infiltration of T lymphocytes and macrophages and inhibited the levels of pro-inflammatory (TNF-α, IL-2, and IL-6) and pro-fibrotic factors (TGF-β, α-SMA, and MMP-9) in the graft. Additionally, triptolide significantly decreased the numbers of IFN-γ-producing T lymphocytes, as well as the expression of IFN-γ and IFN-γ-inducing factor ( CXCL9 and CXCL10 ) in recipient. Moreover, triptolide decreased the numbers of B lymphocytes and plasma cells, as well as the levels of donor specific antibodies (DSAs) in recipient. Furthermore, triptolide not only inhibited vascular smooth muscle cell (VSMC) viability and promoted VSMC apoptosis but also significantly inhibited VSMC migration in vitro . These results emphasize the efficacy of triptolide in inhibiting TV development and provide a basis for developing new treatments to prevent TV-related complications and improve the long-term survival of transplant recipients., (Copyright © 2020 Luo, Liao, Zhang, Zheng, Sun, Han, Yang and Sun.)

The human thymus suffers a transient neonatal involution, recovers and then starts a process of decline between the 1st and 2nd years of life. Age-related morphological changes in thymus were extensively investigated, but the genomic mechanisms underlying this process remain largely unknown. Through Weighted Gene Co-expression Network Analysis (WGCNA) and TF-miRNA-mRNA integrative analysis we studied the transcriptome of neonate and infant thymic tissues grouped by age: 0-30 days (A); 31days-6 months (B); 7-12 months (C); 13-18 months (D); 19-31months (E). Age-related transcriptional modules, hubs and high gene significance (HGS) genes were identified, as well as TF-miRNA-hub/HGS co-expression correlations. Three transcriptional modules were correlated with A and/or E groups. Hubs were mostly related to cellular/metabolic processes; few were differentially expressed (DE) or related to T-cell development. Inversely, HGS genes in groups A and E were mostly DE. In A (neonate) one third of the hyper-expressed HGS genes were related to T-cell development, against one-twentieth in E, what may correlate with the early neonatal depletion and recovery of thymic T-cell populations. This genomic mechanism is tightly regulated by TF-miRNA-hub/HGS interactions that differentially govern cellular and molecular processes involved in the functioning of the neonate thymus and in the beginning of thymic decline., Competing Interests: The authors declare no competing interests.

Cell migration involves dynamic changes in cell shape. Intricate patterns of cell shape can be analyzed and classified using advanced shape descriptors, including spherical harmonics (SPHARM). Though SPHARM have been used to analyze and classify migrating cells, such classification did not exploit SPHARM spectra in their dynamics. Here, we examine whether additional information from dynamic SPHARM improves classification of cell migration patterns. We combine the static and dynamic SPHARM approach with a support-vector-machine classifier and compare their classification accuracies. We demonstrate that the dynamic SPHARM analysis classifies cell migration patterns more accurately than the static one for both synthetic and experimental data. Furthermore, by comparing the computed accuracies with that of a naive classifier, we can identify the experimental conditions and model parameters that significantly affect cell shape. This capability should - in the future - help to pinpoint factors that play an essential role in cell migration.

Recent data suggest that porcine γδ T cells exhibit a similar degree of functional plasticity as human and murine γδ T cells. Due to the high frequency of TCR-γδ + cells in blood and secondary lymphatic organs, the pig is an attractive model to study these cells, especially their combined features of the innate and the adaptive immune system. Using a 5' RACE-like approach, we translated a human/murine NGS library preparation strategy to capture full-length V-(D)-J TRG and TRD clonotypes in swine. After oligo(dT) primed conversion of input RNA, the cDNA population was enriched for full-length V(D)J TCR transcripts with porcine-specific primers including Illumina adaptor sequences as overhangs for Illumina MiSeq analysis. After quality control and processing by FastQC and ea-utils, porcine TRG and TRD sequences were mapped against the human IMGT reference directory. Porcine blood-derived CD2 + and CD2‾ TCR-γδ + cells exhibited two distinct clonotypes Vγ11JγP1 (74.6%) and Vγ10JγP1 (57.7%), respectively. Despite the high TCR-δ diversity among CD2 + cells (39 clonotypes), both subsets shared the same abundant Vδ1DδxJδ4 clonotype at approximately identically frequencies (CD2 + : 31.2%; CD2‾: 37.0%). The flexible nature of this approach will facilitate the assessment of organ-specific phenotypes of γδ T cell subsets alongside with their respective TCR diversity at single cell resolution., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Differences in three-dimensional (3D) chromatin architecture can influence the integrity of topologically associating domains (TADs) and rewire specific enhancer-promoter interactions, impacting gene expression and leading to human disease. Here we investigate the 3D chromatin architecture in T cell acute lymphoblastic leukemia (T-ALL) by using primary human leukemia specimens and examine the dynamic responses of this architecture to pharmacological agents. Systematic integration of matched in situ Hi-C, RNA-seq and CTCF ChIP-seq datasets revealed widespread differences in intra-TAD chromatin interactions and TAD boundary insulation in T-ALL. Our studies identify and focus on a TAD 'fusion' event associated with absence of CTCF-mediated insulation, enabling direct interactions between the MYC promoter and a distal super-enhancer. Moreover, our data also demonstrate that small-molecule inhibitors targeting either oncogenic signal transduction or epigenetic regulation can alter specific 3D interactions found in leukemia. Overall, our study highlights the impact, complexity and dynamic nature of 3D chromatin architecture in human acute leukemia.

Common variable immunodeficiency (CVID) results in defective B cell differentiation and impaired antibody production and is the most common symptomatic primary immunodeficiency. Our aim was to evaluate the correlation among B cell subgroups, κ-deleting recombination excision circle (KREC) copy numbers, and clinical and immunological data of the patients with CVID, and evaluate the patients according to classifications currently available to define the role of KREC copy numbers in the diagnosis of CVID. KREC analysis was performed using a quantitative real-time polymerase chain reaction assay, and B cell subgroups were measured by flow cytometry. The median age of the patients (n = 30) was 25 (6-69) years. Parental consanguinity ratio was 33%. The median age at diagnosis was 15 (4-59), and follow-up period was 6 (1-37) years. CD19 + and CD4 + cell counts at the time of diagnosis were low in 66.7% and 46.7% of the patients, respectively. CD19 + cell counts were positively correlated with KREC copy numbers in patients and healthy controls. CD19 + cell counts and KREC copy numbers were significantly reduced in CVID patients compared to healthy controls as expected. KRECs are quantitative markers for B cell defects. We found low CD4 + cell numbers, recent thymic emigrants, and lymphopenia in some of the patients at diagnosis, which reminds the heterogeneity of CVID's etiology. In this study, a positive correlation was shown between CD19 + cell counts and KREC copy numbers. Low KREC copy numbers indicated B cell deficiency; however, high KREC copy numbers were not sufficient to rule out CVID.

Homeostasis of the lacrimal functional unit is needed to ensure a well-regulated ocular immune response comprising innate and adaptive phases. When the ocular immune system is excessively stimulated and/or immunoregulatory mechanisms are disrupted, the balance between innate and adaptive phases is dysregulated and chronic ocular surface inflammation can result, leading to chronic dry eye disease (DED). According to the Tear Film and Ocular Surface Society Dry Eye Workshop II definition, DED is a multifactorial disorder of the ocular surface characterized by impairment and loss of tear homeostasis (hyperosmolarity), ocular discomfort or pain, and neurosensory abnormalities. Dysregulated ocular immune responses result in ocular surface damage, which is a further contributing factor to DED pathology. Several therapeutics are available to break the vicious circle of DED and prevent chronic disease and progression, including immunosuppressive agents (steroids) and immunomodulators (cyclosporine and lifitegrast). Given the chronic inflammatory nature of DED, each of these agents is commonly used in clinical practice. In this study, we review the immunopathology of DED and the molecular and cellular actions of current topical DED therapeutics to inform clinical decision making.

The transcription factor TCF-1 (encoded by Tcf7 ) plays critical roles in several lineages of hematopoietic cells. In this study, we examined the molecular basis for Tcf7 regulation in T cells, innate lymphoid cells, and migratory conventional dendritic cells that we find express Tcf7 . We identified a 1 kb regulatory element crucial for the initiation of Tcf7 expression in T cells and innate lymphoid cells, but dispensable for Tcf7 expression in Tcf7 -expressing dendritic cells. Within this region, we identified a Notch binding site important for the initiation of Tcf7 expression in T cells but not in innate lymphoid cells. Our work establishes that the same regulatory element is used by distinct transcriptional controllers to initiate Tcf7 expression in T cells and ILCs., (Copyright © 2020 Harly, Kenney, Wang, Ding, Zhao, Awasthi and Bhandoola.)

Membranous nephropathy (MN), a common pathological type of adult nephrotic syndrome, is an antibody-mediated kidney disease. It is widely accepted now that MN is an immune-related disease that involves the whole immune system. In this study, we analyzed the T-cell receptor beta chain (TCRβ) repertoire of the circulating T lymphocytes of MN patients and healthy controls using high-throughput sequencing. We compared multiple aspects of the TCRβ repertoire, including diversity and the Vβ and Jβ genes between MN patients and healthy controls, and we found that the diversities within the VJ cassette combination in the peripheral blood of MN patients were lower than in the healthy controls. We also found the TCRβ repertoire similarity between pre- and post-therapy could reflect the clinical outcome, and two Vβ genes in pre-therapy had the potential to predict the therapeutic effect. These findings indicated the potential of the TCRβ repertoire as non-invasive biomarkers for the prognosis prediction of MN. The characteristics of circulating T-lymphocyte repertoires shed light on MN detection, treatment, and surveillance., (Copyright © 2020 Zhang, Jin, Guan, Li, Su, Xie, Chen, Liu, Pan, Ye, Zhang, Kong and Luo.)

The development of immunocompetence in chicks after hatching is not fully understood. However, detailed knowledge of immunocompetence and maturation processes in day-old chicks (DOCs) and juvenile chickens ( Gallus gallus domesticus ) is necessary to implement enhanced immunization strategies. For viral diseases, this especially includes the development of cellular immunity focusing on T-cell-dependent responses. In the current study, we investigated T-cell subsets in blood and lymphoid tissues of 1-to-21-day-old chickens concerning their cellular composition and localization. We detected an increase of T-cell frequencies in blood and spleen and a shift of the CD8α dimer expression toward a CD8αβ expression on the surface of T cells with increasing age. A relocalization of lymphocytes into antigen presentation structures within the spleen was affirmed. In addition, changes in basal messenger RNA (mRNA) level, with increasing IL2 and IFNγ mRNA levels at different ages were measured. These detected changes suggest an improved T-cell-dependent antiviral response with increasing age in chickens. To confirm this finding on a functional level, we conducted a transfer experiment: adult and, as a negative control, neonatal naïve lymphocytes were transferred into DOCs. Afterward, the protection induced by these transferred cells was verified by a sublethal infection by using a highly pathogenic avian influenza virus with neuraminidase deletion, H5N del. Previous experiments have shown that adult animals survive infection with this virus strain, while naïve DOCs show severe symptoms or even die. As a result, the transfer of adult, but not neonatal lymphocytes, confers protection to DOCs against the infection, demonstrating functional differences in lymphocytes from chicks of different ages. Collectively, these data reveal the inability of chicks to mount an effective, cellular antiviral response in the first 3 wk of life. Therefore, we propose that the observed maturation of both the innate and the adaptive arms of the immune system early in development is mandatory for controlling influenza infection in chickens, as well as for an effective vaccination with replication-competent viral vaccine strains.

The immunomodulatory effects of R-phycoerythrin (R-PE) from Porphyra haitanensis were investigated by a hydrocortisone (HC)-induced immunosuppressive model in the present research. The results showed that R-PE had immunomodulatory potential, such as increasing spleen and thymus indexes, ameliorating spleen morphology, enhancing the phagocytic capability of macrophages, promoting spleen T lymphocyte proliferation, and strengthening serum hemolysin formation. Furthermore, R-PE elevated the CD4 + /CD8 + ratio and up-regulated the levels of cytokines for Th1, Th2, and Th17 and the mRNA expression of transcription factors T-bet, GATA3, and RORγt. Besides, the levels of cytokines for Treg and transcription factor Foxp3 were down-regulated. The levels of T-bet/GATA3 and RORγt/Foxp3 were increased. R-PE activated the phosphorylation of NF-κB through the TLR4-mediated differentiation pathway. These findings suggested that R-PE exerted immunomodulatory activities in the innate and adaptive immune systems through TLR4/NF-κB mediated CD4 + T cell activation and differentiation, which facilitated the further application of R-PE as an immunomodulator.

The European rabbit (Oryctolagus cuniculus) is a good model in biomedicine used in research on several human diseases. The reference values of B and T cells and their subpopu- lations are very important to understand how the adaptive immune system is responding to infectious agents. The aim of this study was to determine values of B and T cells and their subpopulations in Polish mixed-breed rabbits, considering seasons of the year and sex. The study was performed on 200 Polish mixed-breed rabbits and the percentage of B and T lymphocytes was measured cytometrically using mouse anti-rabbit antibodies. The study revealed that the season of the year and sex of the animals affected the percentage of B- and T-cells and their subpopulations in peripheral blood. Statistically significant values of CD19+ B-cells in spring and autumn, of T CD5+ cells in spring and winter, of T CD4+ in spring, summer, autumn and winter, of T CD8+ in winter and of T CD25+ in spring were noted. Generally the highest values were found mainly in warm part of the year, while the lowest in colder months. A statistical significance was also observed between males and females - changes were found in T CD4+ and T CD25+ lymphocytes in spring, T CD8+ cells in winter and higher percentage was generally obtained in females than in males. The only exception was the T CD5+ subpopulation in which no differences were observed between the sexes and throughout the year. This is the first paper on adaptive immune system cell values in the European rabbit of domestic breeds., (Copyright© by the Polish Academy of Sciences.)

Trypanosoma cruzi is a protozoan parasite that infects at least 7 million persons in the world (OMS, 2019). In endemic areas, infection normally occurs by vectorial transmission; however, outside, it normally happens by blood and includes congenital transmission. The persistence of T. cruzi during infection suggests the presence of immune evasion mechanisms and the modulation of the anti-parasite response to a profile incapable of eradicating the parasite. Dendritic cells (DCs) are a heterogeneous population of antigen-presenting cells (APCs) that patrol tissues with a key role in mediating the interface between the innate and adaptive immune response. Previous results from our lab and other groups have demonstrated that T. cruzi modulates the functional properties of DCs, in vitro and in vivo . During vectorial transmission, metacyclic (m) trypomastigotes (Tps) eliminated along with the insect feces reach the mucous membranes or injured skin. When transmission occurs by the hematic route, the parasite stage involved in the infection is the circulating or blood (b) Tp. Here, we studied in vitro the effect of both infective mTp and bTp in two different populations of DCs, bone marrow-derived DCs (BMDCs) and XS106, a cell line derived from epidermal DCs. Results demonstrated that the interaction of both Tps imparts a different effect in the functionality of these two populations of DCs, suggesting that the stage of T. cruzi and DC maturation status could define the immune response from the beginning of the ingress of the parasite, conditioning the course of the infection., (Copyright © 2020 Gutierrez, Lammel, Ramirez, González-Cappa and Poncini.)

The immunological synapse (IS) is a specialized contact area formed between a T cell and an antigen presenting cell (APC). Besides molecules directly involved in antigen recognition such as the TCR/CD3 complex, ion channels important in the membrane potential and intracellular free Ca 2+ concentration control of T cells are also recruited into the IS. These are the voltage-gated Kv1.3 and Ca 2+ -activated KCa3.1 K + channels and the calcium release-activated Ca 2+ channel (CRAC). However, the consequence of this recruitment on membrane potential and Ca 2+ level control is not known. Here we demonstrate that the membrane potential (MP) of murine T cells conjugated with APCs in an IS shows characteristic oscillations. We found that depolarization of the membrane by current injection or by increased extracellular K + concentration produced membrane potential oscillations (MPO) significantly more frequently in conjugated T cells than in lone T cells. Furthermore, oscillation of the free intracellular Ca 2+ concentration could also be observed more frequently in cells forming an IS than in lone cells. We suggest that in the IS the special arrangement of channels and the constrained space between the interacting cells creates a favorable environment for these oscillations, which may enhance the signaling process leading to T cell activation., Competing Interests: The authors declare no conflict of interest.

Here, we developed an unbiased, functional target-discovery platform to identify immunogenic proteins from primary non-small cell lung cancer (NSCLC) cells that had been induced to apoptosis by cisplatin (CDDP) treatment in vitro, as compared with their live counterparts. Among the multitude of proteins identified, some of them were represented as fragmented proteins in apoptotic tumor cells, and acted as non-mutated neoantigens (NM-neoAgs). Indeed, only the fragmented proteins elicited effective multi-specific CD4 + and CD8 + T cell responses, upon a chemotherapy protocol including CDDP. Importantly, these responses further increased upon anti-PD-1 therapy, and correlated with patients' survival and decreased PD-1 expression. Cross-presentation assays showed that NM-neoAgs were unveiled in apoptotic tumor cells as the result of caspase-dependent proteolytic activity of cellular proteins. Our study demonstrates that apoptotic tumor cells generate a repertoire of immunogenic NM-neoAgs that could be potentially used for developing effective T cell-based immunotherapy across multiple cancer patients.

Clathrin-mediated endocytosis (CME) is critical for internalisation of molecules across cell membranes. The FCH domain only 1 (FCHO1) protein is key molecule involved in the early stages of CME formation. The consequences of mutations in FCHO1 in humans were unknown. We identify ten unrelated patients with variable T and B cell lymphopenia, who are homozygous for six distinct mutations in FCHO1. We demonstrate that these mutations either lead to mislocalisation of the protein or prevent its interaction with binding partners. Live-cell imaging of cells expressing mutant variants of FCHO1 provide evidence of impaired formation of clathrin coated pits (CCP). Patient T cells are unresponsive to T cell receptor (TCR) triggering. Internalisation of the TCR receptor is severely perturbed in FCHO1-deficient Jurkat T cells but can be rescued by expression of wild-type FCHO1. Thus, we discovered a previously unrecognised critical role of FCHO1 and CME during T-cell development and function in humans.

The diverse repertoire of T-cell receptors (TCR) plays a key role in the adaptive immune response to infections. Using TCR alpha and beta repertoire sequencing for T-cell subsets, as well as single-cell RNAseq and TCRseq, we track the concentrations and phenotypes of individual T-cell clones in response to primary and secondary yellow fever immunization - the model for acute infection in humans - showing their large diversity. We confirm the secondary response is an order of magnitude weaker, albeit ∼10 days faster than the primary one. Estimating the fraction of the T-cell response directed against the single immunodominant epitope, we identify the sequence features of TCRs that define the high precursor frequency of the two major TCR motifs specific for this particular epitope. We also show the consistency of clonal expansion dynamics between bulk alpha and beta repertoires, using a new methodology to reconstruct alpha-beta pairings from clonal trajectories., Competing Interests: AM, MP, EK, VK, PB, ER, AF, DC, IM, YL, TM No competing interests declared, AW Senior editor, eLife, (© 2020, Minervina et al.)

Beyond its well-admitted role in development and organogenesis, it is now clear that the transcription factor c-Maf has owned its place in the realm of immune-related transcription factors. Formerly introduced solely as a Th2 transcription factor, the role attributed to c-Maf has gradually broadened over the years and has extended to most, if not all, known immune cell types. The influence of c-Maf is particularly prominent among T cell subsets, where c-Maf regulates the differentiation as well as the function of multiple subsets of CD4 and CD8 T cells, lending it a crucial position in adaptive immunity and anti-tumoral responsiveness. Recent research has also revealed the role of c-Maf in controlling Th17 responses in the intestine, positioning it as an essential factor in intestinal homeostasis. This review aims to present and discuss the recent advances highlighting the particular role played by c-Maf in T lymphocyte differentiation, function, and homeostasis., (Copyright © 2020 Imbratta, Hussein, Andris and Verdeil.)

The first steps of human immunodeficiency virus (HIV) infection go through the engagement of HIV envelope (Env) with CD4 and coreceptors (CXCR4 or CCR5) to mediate viral membrane fusion between the virus and the host. New approaches are still needed to better define both the molecular mechanistic underpinnings of this process but also the point of fusion and its kinetics. Here, we have developed a new method able to detect and quantify HIV-1 fusion in single live cells. We present a new approach that employs fluorescence lifetime imaging microscopy (FLIM) to detect Förster resonance energy transfer (FRET) when using the β-lactamase (BlaM) assay. This novel approach allows comparing different populations of single cells regardless the concentration of CCF2-AM FRET reporter in each cell, and more importantly, is able to determine the relative amount of viruses internalized per cell. We have applied this approach in both reporter TZM-bl cells and primary T cell lymphocytes., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Purpose of Review: Acute myeloid leukaemia (AML) is a heterogeneous malignancy for which treatment options remain suboptimal. It is clear that a greater understanding of the biology of the AML niche will enable new therapeutic strategies to be developed in order to improve treatment outcomes for patients., Recent Findings: Recent evidence has highlighted the importance of the bone marrow microenvironment in protecting leukaemia cells, and in particular leukaemic stem cells from chemotherapy-induced cell death. This includes mesenchymal stem cells supporting growth and preventing apoptosis, and altered action and secretion profiles of other niche components including adipocytes, endothelial cells and T cells. Here, we provide a detailed overview of the current understanding of the AML bone marrow microenvironment. Clinical trials of agents that mobilise leukaemic stem cells from the bone marrow are currently ongoing and show early promise. Future challenges will involve combining these novel therapies targeted at the AML niche with conventional chemotherapy treatment.

Aims: The crucial step in the pathogenic events that lead to the development and the progression of multiple sclerosis (MS) is the infiltration of autoreactive T cells in the brain. Data from experimental autoimmune encephalomyelitis (EAE) mice indicate that, together with microglia, T cells are responsible for the enhancement of the glutamatergic transmission in central neurons, contributing to glutamate-mediated excitotoxicity, a pathological hallmark of both EAE and MS brains. Here, we addressed the synaptic role of T cells taken from MS patients., Methods: A chimeric model of human T cells and murine brain slices was established to record, by Patch Clamp technique, the glutamatergic transmission in the presence of T cells isolated from the peripheral blood of healthy subjects (HS), active (a) and nonactive (na) relapsing remitting MS patients. Intracellular staining and flow cytometry were used to assess tumour necrosis factor (TNF) expression in T cells., Results: Chimeric experiments indicated that, compared to HS and naMS, T cells from aMS induced an increase in glutamatergic kinetic properties of striatal neurons. Such alteration, reminiscent of the those induced by EAE T cells, was blocked by incubation of the slices with etanercept, a TNF receptor antagonist. Of note, T cells from aMS expressed more TNF than naMS patients and HS subjects., Conclusion: These data highlight the synaptotoxic potential retained by MS T cells, suggesting that during the inflammatory phase of the disease infiltrating T cells could influence the neuronal activity contributing to the TNF-mediated mechanisms of glutamate excitotoxicity in central neurons., (© 2019 British Neuropathological Society.)

With several different CAR T cell therapies under advanced phases of clinical trials, and the first FDA-approved CAR treatments in 2017 (Yescarta and Kymriah), CAR T cell therapy has become one of the most promising therapies for the treatment of certain types of cancer. This success has bred an opportunity to optimize the production of CAR T cells for easier patient access. CAR T cell therapy is a rather expensive and personalized process that requires expensive measures to collect cells from patients, engineer those cells, and re-infuse the cells into the patient with adequate quality controls at each phase. With this in mind, significant attempts at creating a "universal" CAR T cell are underway in order to create an "off-the-shelf" product that would reduce the expense and time required for traditional CAR T cell treatment. The primary obstacle facing this endeavor is avoiding graft-versus-host disease that accompanies allogeneic transplants between genetically dissimilar individuals. With the advent of CRISPR and TALEN technology, editing the genome of allogeneic cells has become very possible, and several groups have provided initial data analyzing the effects of CAR T cells that have been edited to avoid host rejection and avoid endogenous TCR alloreactivity. These engineered cells not only have to avoid GVHD but also have to retain their anti-tumor efficacy in vivo. Here, we expand on the recent efforts and strides that have been made in the design and testing of universal allogeneic CAR T cells.

β-Catenin/CTNNB1 is critical for leukemia initiation or the stem cell capacity of several hematological malignancies. This review focuses on a general evaluation of β-catenin function in normal T-cell development and T-cell acute lymphoblastic leukemia (T-ALL). The integration of the existing literature offers a state-of-the-art dissection of the complexity of β-catenin function in leukemia initiation and maintenance in both Notch-dependent and independent contexts. In addition, β-catenin mutations are screened for in T-ALL primary samples, and it is found that they are rare and with little clinical relevance. Transcriptional analysis of Wnt family members (Ctnnb1, Axin2, Tcf7, and Lef1) and Myc in different publicly available T-ALL cohorts indicates that the expression of these genes may correlate with T-ALL subtypes and/or therapy outcomes., (© 2019 WILEY Periodicals, Inc.)

Objective: Takayasu arteritis (TAK) is a progressive autoimmune large vessel vasculitis with infiltration of proinflammatory T cells, with a largely unknown etiology. This study was undertaken to explore the involvement of mechanistic target of rapamycin (mTOR) in proinflammatory T cell differentiation and disease progression in TAK., Methods: Ninety-five patients with TAK, 26 patients with small vessel vasculitis, and 40 healthy donors were enrolled. Naive and memory CD4+ T cells were activated with anti-CD3/CD28 beads and analyzed for lineage differentiation. The mTORC1 activity was determined by quantifying intracellular phospho-S6 kinase 1 and phospho-S6 ribosomal protein. Rapamycin and lentiviral regulatory-associated protein of mTOR short hairpin RNA were used to block mTORC1 activity. Human artery-NSG mouse chimeras representing human TAK were established for targeting mTORC1 in disease treatment., Results: TAK CD4+ T cells were selectively prepositioned with hyperactivity of mTORC1 (P < 0.001), resulting in spontaneous maldifferentiation of Th1 and Th17 cells (P < 0.001). Activity of mTORC1 high in circulating CD4+ T cells predicted elevated frequencies of proinflammatory T cells and active disease in TAK patients (P < 0.001). Blockade of mTORC1 with rapamycin efficiently abrogated the maldifferentiation of Th1 and Th17 cells (P < 0.01) and ameliorated vasculitis in humanized TAK chimeras (P < 0.001). Inhibition of mTORC1 using RNA interference technology is sufficient to reduce proinflammatory T cell frequencies (P < 0.01) and restrict TAK disease progression in vivo (P < 0.01)., Conclusion: Our findings indicate that hyperactivity of mTORC1 is a critical cell-intrinsic mechanism underlying spontaneous maldifferentiation of proinflammatory T cells in TAK patients. Targeting mTORC1 is a promising therapeutic strategy against TAK., (© 2019, American College of Rheumatology.)

Objective: This study was aimed at examining the role of Tenascin-C in T cell trafficking in Oral Lichen Planus (OLP)., Design: For the in vivo immunohistochemical analyses, 115 OLP samples were collected from patients and immunostaining was performed. The intensity and distribution of TN-C expression were quantified and correlated with histological analyses of basement membrane integrity and presence of inflammatory infiltrate. For the in vitro study, TN-C and collagen were coated on culture plates and migration of T lymphocytes was assessed., Results: TN-C immunoexpression was increased in terms of both distribution and intensity along the basement membrane zone. These changes were significantly associated with basement membrane duplication (distribution p < 0.002 and intensity p < 0.001) and bands of inflammation (distribution p < 0.002 and intensity p < 0.001) assessed by Chi-square test. T lymphocytes demonstrated significant migration towards TN-C as compared to collagen (n = 3, p < 0.05)., Conclusions: These findings indicate TN-C may have a key role in promoting T cell migration at the epithelial-mesenchymal junction in OLP. These observations suggest TN-C could be a good target for therapeutic intervention, either in itself or synergistically with anti-inflammatory directed strategies in this chronic disease management., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Amitriptyline is prescribed for treating the symptoms of neuroinflammatory disorders including neuropathic pain and fibromyalgia. As amitriptyline has evidence of modulating the neuroimmune interface; the effects of amitriptyline treatment on T-cell phenotype and function were examined in vitro. Peripheral blood mononuclear cells(PBMCs) were isolated and treated with amitriptyline, nortriptyline and a combination of both drugs. Toxicity for T-cells was assessed by Annexin V/Propidium Iodide staining. Activation status and cytokine expression by T-cells post treatment was assessed by flow cytometry. The levels of secreted cytokines, chemokines and neurotrophins were measured by ELISA in the supernatants. There was no significant increase in T-cell death following 24 or 48 h compared to controls. There were significantly lower frequencies of CD8 + T-cells after treatment with amitriptyline, nortriptyline and a combination of both compared to a Vehicle Control(VC)(p<0.001). The frequencies of naive CD8 + CD45RA + cells were significantly lower after amitriptyline, nortriptyline and a combination of both (p<0001). The frequencies of CD27 + CD4 + (p<0.05) and CD27 + CD8 + (p<0.01) T-cells were also significantly lower following combination drug treatment. Significantly lower frequencies of IFN-γ-producing CD8 + T-cells were observed with all treatment combinations(p<0.05) and frequencies of IL-17-producing CD4 + and CD8 + T-cells were significantly lower following amitriptyline treatment (p<0.05). Frequencies of Natural Killer T-cells were significantly higher following treatment with nortriptyline (p<0.05). Significantly higher levels of IL-16 (p<0.001) and lower levels of TNF-β (p<0.05) were observed in supernatants. This data indicates that both amitriptyline and nortriptyline modulate the phenotype and function of T-cells and this may have clinical relevance in the pathologies of its off-label applications., (Copyright © 2019 Elsevier B.V. and ECNP. All rights reserved.)

We recently developed a high throughput T cell receptor β chain (TCRβ) sequencing-based approach to identifying and tracking donor-reactive T cells. To address the role of clonal deletion in liver allograft tolerance, we applied this method in samples from a recent randomized study, ITN030ST, in which immunosuppression withdrawal was attempted within 2 years of liver transplantation. We identified donor-reactive T cell clones via TCRβ sequencing following a pre-transplant mixed lymphocyte reaction and tracked these clones in the circulation following transplantation in 3 tolerant and 5 non-tolerant subjects. All subjects showed a downward trend and significant reductions in donor-reactive TCRβ sequences were detected post-transplant in 6 of 8 subjects, including 2 tolerant and 4 non-tolerant recipients. Reductions in donor-reactive TCRβ sequences were greater than those of all other TCRβ sequences, including 3rd party-reactive sequences, in all 8 subjects, demonstrating an impact of the liver allograft after accounting for repertoire turnover. Although limited by patient number and heterogeneity, our results suggest that partial deletion of donor-reactive T cell clones may be a consequence of liver transplantation and does not correlate with success or failure of early immunosuppression withdrawal. These observations underscore the organ- and/or protocol-specific nature of tolerance mechanisms in humans., (© 2019 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Micropatterned nickel titanium (commonly known as nitinol) thin films with complex designs, high structural resolution and excellent biocompatibility can be cheaply fabricated using magnetron sputtering. Here, we show that these benefits can be leveraged to fabricate micromesh implants that are loaded with tumour-specific human chimeric antigen receptor (CAR)-T cells for the treatment of solid tumours. In a mouse model of non-resectable ovarian cancer, the cell-loaded nitinol thin films spatially conformed to the implantation site, fostered the rapid expansion of T cells, delivered a high density of T cells directly to the tumour and significantly improved animal survival. We also show that self-expandable stents that were coated with T-cell-loaded films and implanted into subcutaneous tumours in mice improved the duration of stent patency by delaying tumour ingrowth. By providing direct access to tumours, CAR-T-cell-loaded micropatterned nitinol thin films can improve the effects of cell-based therapies.

Background: The complex preparation procedures and severe toxicities are two major obstacles facing the wide use of chimeric antigen receptor-modified T (CAR-T) cells in clinical cancer immunotherapy. The nanotechnology-based T cell temporary CAR modification may be a potential approach to solve these problems and make the CAR-T cell-based tumor therapy feasible and broadly applicable., Methods: A series of plasmid DNA-loaded self-assembled nanoparticles (pDNA@SNPs x/y ) prepared from adamantane-grafted polyamidoamine (Ad-PAMAM) dendrimers of different generations (G1 or G5) and cyclodextrin-grafted branched polyethylenimine (CD-PEI) of different molecular weights (800, 2000, or 25,000 Da) were characterized and evaluated. The detailed physicochemical properties, cellular interaction, and cytotoxicity of selected pDNA@SNP G1/800 were systematically investigated. Thereafter, the epidermal growth factor receptor variant III (EGFRvIII) CAR-expression plasmid vector (pEGFRvIII-CAR) was constructed and encapsulated into SNP G1/800 . The resulting pEGFRvIII-CAR@SNP G1/800 was used for Jurkat cell transient transfection, and the EGFRvIII-CAR expressed in transfected cells was measured by flow cytometry and Western blot. Finally, the response of EGFRvIII CAR-positive Jurkat T cell to target tumor cell was evaluated., Results: The pDNA@SNP G1/800 showed the highest efficacy in Jurkat cell gene transfection and exhibited low cytotoxicity. pEGFRvIII-CAR@SNP G1/800 can efficiently deliver pEGFRvIII-CAR into Jurkat T cells, thereby resulting in transient EGFRvIII-CAR expression in transfected cells. EGFRvIII-CAR that is present on the cell membrane enabled Jurkat T cells to recognize and bind specifically with EGFRvIII-positive tumor cells., Conclusion: These results indicated that pEGFRvIII-CAR@SNP G1/800 can effectively achieve T-cell transient CAR modification, thereby demonstrating considerable potential in CAR-T cancer therapy., Competing Interests: The authors report no conflicts of interest in this work., (© 2020 Yu et al.)

Reactive oxygen species (ROS) are central effectors of inflammation and play a key role in cell signaling. Previous reports have described an association between oxidative events and the modulation of innate immunity. However, the role of redox signaling in adaptive immunity is still not well understood. This work is based on a novel investigation of diamide, a specific oxidant of sulfhydryl groups, and it is the first performed in purified T cell tyrosine phosphorylation signaling. Our data show that ex vivo T cells respond to -SH group oxidation with a distinctive tyrosine phosphorylation response and that these events elicit specific cellular responses. The expression of two essential T-cell receptors, CD25 and CD62L, and T-cell cytokine release is also affected in a specific way. Experiments with Syk inhibitors indicate a major contribution of this kinase in these phenomena. This pilot work confirms the presence of crosstalk between oxidation of cysteine residues and tyrosine phosphorylation changes, resulting in a series of functional events in freshly isolated T cells. Our experiments show a novel role of Syk inhibitors in applying their anti-inflammatory action through the inhibition of a ROS-generated reaction., Competing Interests: The authors declare that they have no conflict of interest.