Editorial: Methods in T cell biology: 2022

T cells are crucial players of immune responses by their ability to specifically recognize foreign, potentially harmful, antigens. To perform such specific recognition, T cells express at their plasma membrane an antigen receptor called the T cell receptor (TCR) (1–3). The TCR expressed at the plasma membrane in the majority of T cells is composed by a TCR-a and TCR-b heterodimer, which is able to recognize a complex formed by an antigenic peptide coupled to an MHC molecule (pMHC complex). After specific recognition by a pMHC complex the TCR triggers multiple intracellular signals leading to proliferation, target cell killing, cytokine secretion, and differentiation into effector T cells. Although our understanding of the molecular mechanisms governing activation of T cells has dramatically grown in recent years, there is still much to lear

A Story of Kinases and Adaptors: The Role of Lck, ZAP-70 and LAT in Switch Panel Governing T-Cell Development and Activation

Specific antigen recognition is one of the immune system’s features that allows it to mount intense yet controlled responses to an infinity of potential threats. T cells play a relevant role in the host defense and the clearance of pathogens by means of the specific recognition of peptide antigens presented by antigen-presenting cells (APCs), and, to do so, they are equipped with a clonally distributed antigen receptor called the T-cell receptor (TCR). Upon the specific engagement of the TCR, multiple intracellular signals are triggered, which lead to the activation, proliferation and differentiation of T lymphocytes into effector cells. In addition, this signaling cascade also operates during T-cell development, allowing for the generation of cells that can be helpful in the defense against threats, as well as preventing the generation of autoreactive cells. Early TCR signals include phosphorylation events in which the tyrosine kinases Lck and ZAP70 are involved. The sequential activation of these kinases leads to the phosphorylation of the transmembrane adaptor LAT, which constitutes a signaling hub for the generation of a signalosome, finally resulting in T-cell activation. These early signals play a relevant role in triggering the development, activation, proliferation and apoptosis of T cells, and the negative regulation of these signals is key to avoid aberrant processes that could generate inappropriate cellular responses and disease. In this review, we will examine and discuss the roles of the tyrosine kinases Lck and ZAP70 and the membrane adaptor LAT in these cellular processes. © 2023 by the authors

Increased Protein Stability and Interleukin-2 Production of a LAT(G131D)Variant With Possible Implications for T Cell Anergy

The adaptor LAT plays a crucial role in the transduction of signals coming from the TCR/CD3 complex. Phosphorylation of some of its tyrosines generates recruitment sites for other cytosolic signaling molecules. Tyrosine 132 in human LAT is essential for PLC-gamma activation and calcium influx generation. It has been recently reported that a conserved glycine residue preceding tyrosine 132 decreases its phosphorylation kinetics, which constitutes a mechanism for ligand discrimination. Here we confirm that a LAT mutant in which glycine 131 has been substituted by an aspartate (LAT(G131D)) increases phosphorylation of Tyr132, PLC-gamma activation and calcium influx generation. Interestingly, the LAT(G131D)mutant has a slower protein turnover while being equally sensitive to Fas-mediated protein cleavage by caspases. Moreover, J.CaM2 cells expressing LAT(G131D)secrete greater amounts of interleukin-2 (IL-2) in response to CD3/CD28 engagement. However, despite this increased IL-2 secretion, J.CaM2 cells expressing the LAT(G131D)mutant are more sensitive to inhibition of IL-2 production by pre-treatment with anti-CD3, which points to a possible role of this residue in the generation of anergy. Our results suggest that the increased kinetics of LAT Tyr132 phosphorylation could contribute to the establishment of T cell anergy, and thus constitutes an earliest known intracellular event responsible for the induction of peripheral tolerance

A Novel, LAT/Lck Double Deficient T Cell Subline J.CaM1.7 for Combined Analysis of Early TCR Signaling

Intracellular signaling through the T cell receptor (TCR) is essential for T cell development and function. Proper TCR signaling requires the sequential activities of Lck and ZAP-70 kinases, which result in the phosphorylation of tyrosine residues located in the CD3 ITAMs and the LAT adaptor, respectively. LAT, linker for the activation of T cells, is a transmembrane adaptor protein that acts as a scaffold coupling the early signals coming from the TCR with downstream signaling pathways leading to cellular responses. The leukemic T cell line Jurkat and its derivative mutants J.CaM1.6 (Lck deficient) and J.CaM2 (LAT deficient) have been widely used to study the first signaling events upon TCR triggering. In this work, we describe the loss of LAT adaptor expression found in a subline of J.CaM1.6 cells and analyze cis-elements responsible for the LAT expression defect. This new cell subline, which we have called J.CaM1.7, can re-express LAT adaptor after Protein Kinase C (PKC) activation, which suggests that activation-induced LAT expression is not affected in this new cell subline. Contrary to J.CaM1.6 cells, re-expression of Lck in J.CaM1.7 cells was not sufficient to recover TCR-associated signals, and both LAT and Lck had to be introduced to recover activatory intracellular signals triggered after CD3 crosslinking. Overall, our work shows that the new LAT negative J.CaM1.7 cell subline could represent a new model to study the functions of the tyrosine kinase Lck and the LAT adaptor in TCR signaling, and their mutual interaction, which seems to constitute an essential early signaling event associated with the TCR/CD3 complex.This research was funded by Consejeria de Salud de Andalucia, Junta de Andalucia (grant PI-0055-2017 to E.A.), and Fundacion Biomedica Cadiz Proyectos INIBICA 2019 (grant LI19/I14NCO15 to E.A. and M.M.A.-E.)

Immune modulation by the hepatitis C virus core protein

Hepatitis C virus (HCV) infection is currently the most important cause of chronic viral hepatitis in the world and one of the most frequent indications for liver transplantation. HCV uses different strategies to evade the innate and adaptive immune response, and this evasion plays a key role in determining viral persistence. Several HCV viral proteins have been described as immune modulators. In this review, we will focus on the effect of HCV nucleocapsid core protein in the function of immune cells and its correlation with the findings observed in HCV chronically infected patients. Effects on immune cell function related to both extracellular and intracellular HCV core localization will be considered. This review provides an updated perspective on the mechanisms involved in HCV evasion related to one single HCV protein, which could become a key tool in the development of new antiviral strategies able to control and/or eradicate HCV infection.Ministerio de Educación y Ciencia (España) (SAF2009-09449); Consejería de Salud de la Junta de Andalucía (SAS 111206

Expression of Non-T Cell Activation Linker (NTAL) in Jurkat Cells Negatively Regulates TCR Signaling: Potential Role in Rheumatoid Arthritis

T lymphocytes are key players in adaptive immune responses through the recognition of peptide antigens through the T Cell Receptor (TCR). After TCR engagement, a signaling cascade is activated, leading to T cell activation, proliferation, and differentiation into effector cells. Delicate control of activation signals coupled to the TCR is needed to avoid uncontrolled immune responses involving T cells. It has been previously shown that mice deficient in the expression of the adaptor NTAL (Non-T cell activation linker), a molecule structurally and evolutionarily related to the transmembrane adaptor LAT (Linker for the Activation of T cells), develop an autoimmune syndrome characterized by the presence of autoantibodies and enlarged spleens. In the present work we intended to deepen investigation into the negative regulatory functions of the NTAL adaptor in T cells and its potential relationship with autoimmune disorders. For this purpose, in this work we used Jurkat cells as a T cell model, and we lentivirally transfected them to express the NTAL adaptor in order to analyze the effect on intracellular signals associated with the TCR. In addition, we analyzed the expression of NTAL in primary CD4+ T cells from healthy donors and Rheumatoid Arthritis (RA) patients. Our results showed that NTAL expression in Jurkat cells decreased calcium fluxes and PLC-γ1 activation upon stimulation through the TCR complex. Moreover, we showed that NTAL was also expressed in activated human CD4+ T cells, and that the increase of its expression was reduced in CD4+ T cells from RA patients. Our results, together with previous reports, suggest a relevant role for the NTAL adaptor as a negative regulator of early intracellular TCR signaling, with a potential implication in RA.14 página

A Stretch of Negatively Charged Amino Acids of Linker for Activation of T-Cell Adaptor Has a Dual Role in T-Cell Antigen Receptor Intracellular Signaling

The adaptor protein linker for activation of T cells (LAT) has an essential role transducing activatory intracellular signals coming from the TCR/CD3 complex. Previous reports have shown that upon T-cell activation, LAT interacts with the tyrosine kinase Lck, leading to the inhibition of its kinase activity. LAT-Lck interaction seemed to depend on a stretch of negatively charged amino acids in LAT. Here, we have substituted this segment of LAT between amino acids 113 and 126 with a non-charged segment and expressed the mutant LAT (LAT-NIL) in J.CaM2 cells in order to analyze TCR signaling. Substitution of this segment in LAT prevented the activation-induced interaction with Lck. Moreover, cells expressing this mutant form of LAT showed a statistically significant increase of proximal intracellular signals such as phosphorylation of LAT in tyrosine residues 171 and 191, and also enhanced ZAP70 phosphorylation approaching borderline statistical significance (p = 0.051). Nevertheless, downstream signals such as Ca2+ influx or MAPK pathways were partially inhibited. Overall, our data reveal that LAT-Lck interaction constitutes a key element regulating proximal intracellular signals coming from the TCR/CD3 complex.Consejería de Salud de Andalucía, Junta de Andalucía (grants PI-0365-2013 and PI-0055-2017); Instituto de Salud Carlos III (grant PI16-00784 from the “Plan Estatal de I+D+I 2013–2016/FEDER”

Tuberculosis prophylaxis with levofloxacin in liver transplant patients is associated with a high incidence of tenosynovitis: safety analysis of a multicenter randomized trial

This work was supported by the Ayudas para el fomento de la investigacion clinica independiente [EC 10-120] and Programa Intramural Consorcio de Apoyo a la Investigación Biomédica en Red 2010. Other funding sources: National R&D&I Plan 2008–2011 and the Instituto de Salud Carlos III (ISCIII), Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Economía y Competitividad, Spanish Network for Research in Infectious Diseases [RD06/0008, RD12/0015] - co-financed by European Development Regional Fund “A way to achieve Europe” ERDF. Consorcio de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas is financed by the ISCIII.Background: It is necessary to develop a safe alternative to isoniazid for tuberculosis prophylaxis in liver transplant recipients. This study was designed to investigate the efficacy and safety of levofloxacin. Methods: An open-label, prospective, multicenter, randomized study was conducted to compare the efficacy and safety of levofloxacin (500 mg q24h for 9 months) initiated in patients awaiting liver transplantation and isoniazid (300 mg q24h for 9 months) initiated post-transplant when liver function was stabilized. Efficacy was measured by tuberculosis incidence at 18 months after transplantation. All adverse events related to the medication were recorded. Results: CONSORT guidelines were followed in order to present the results. The safety committee suspended the study through a safety analysis when 64 patients had been included (31 in the isoniazid arm and 33 in the levofloxacin arm). The reason for suspension was an unexpected incidence of severe tenosynovitis in the levofloxacin arm (18.2%). Although the clinical course was favorable in all cases, tenosynovitis persisted for 7 weeks in some patients. No patients treated with isoniazid, developed tenosynovitis. Only 32.2% of patients randomized to isoniazid (10/31) and 54.5% of patients randomized to levofloxacin (18/33, P = .094) completed prophylaxis. No patient developed tuberculosis during the study follow-up (median 270 days). Conclusions: Levofloxacin prophylaxis of tuberculosis in liver transplant candidates is associated with a high incidence of tenosynovitis that limits its potential utility.Ayudas para el fomento de la investigación clínica independiente [EC 10-120]Programa Intramural Consorcio de Apoyo a la Investigación Biomédica en Red 2010National R&D&I Plan 2008–2011Instituto de Salud Carlos III (ISCIII)Ministerio de Economía y Competitividad RD06/0008, RD12/0015European Development Regional Fun

Oral fosfomycin for the treatment of lower urinary tract infections among kidney transplant recipients—Results of a Spanish multicenter cohort

Preliminary results of this study were presented at the 29th European Congress of Clinical Microbiology and Infectious Diseases (ECCMID), held in Amsterdam, The Netherlands, from 13 to 16 April, 2019 (oral communication O‐0699).Oral fosfomycin may constitute an alternative for the treatment of lower urinary tract infections (UTIs) in kidney transplant recipients (KTRs), particularly in view of recent safety concerns with fluroquinolones. Specific data on the efficacy and safety of fosfomycin in KTR are scarce. We performed a retrospective study in 14 Spanish hospitals including KTRs treated with oral fosfomycin (calcium and trometamol salts) for posttransplant cystitis between January 2005 and December 2017. A total of 133 KTRs developed 143 episodes of cystitis. Most episodes (131 [91.6%]) were produced by gram‐negative bacilli (GNB), and 78 (54.5%) were categorized as multidrug resistant (including extended‐spectrum β‐lactamase‐producing Enterobacteriaceae [14%] or carbapenem‐resistant GNB [3.5%]). A median daily dose of 1.5 g of fosfomycin (interquartile range [IQR]: 1.5‐2) was administered for a median of 7 days (IQR: 3‐10). Clinical cure (remission of UTI‐attributable symptoms at the end of therapy) was achieved in 83.9% (120/143) episodes. Among those episodes with follow‐up urine culture, microbiological cure at month 1 was achieved in 70.2% (59/84) episodes. Percutaneous nephrostomy was associated with a lower probability of clinical cure (adjusted odds ratio: 10.50; 95% confidence interval: 0.98‐112.29; P = 0.052). In conclusion, fosfomycin is an effective orally available alternative for treating cystitis among KTRs.This study was supported by Plan Nacional de I+D+i 2013‐2016 and Instituto de Salud Carlos III, Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Ciencia, Innovación y Universidades, Spanish Network for Research in Infectious Diseases (REIPI RD16/0016)—cofinanced by the European Development Regional Fund “A way to achieve Europe”; the Group for Study of Infection in Transplantation and the Immunocompromised Host (GESITRA‐IC) of the Spanish Society of Clinical Microbiology and Infectious Diseases (SEIMC); and the Spanish Network for Research in Renal Diseases (REDInREN RD16/0009). MFR holds a research contract “Miguel Servet” (CP 18/00073) from the Spanish Ministry of Science, Innovation and Universities, Instituto de Salud Carlos III

Bronchoscopist's perception of the quality of the single-use bronchoscope (Ambu aScope4™) in selected bronchoscopies: a multicenter study in 21 Spanish pulmonology services

Background: The disposable bronchoscope is an excellent alternative to face the problem of SARS-CoV-2 and other cross infections, but the bronchoscopist’s perception of its quality has not been evaluated. Methods: To evaluate the quality of the Ambu-aScope4 disposable bronchoscope, we carried out a cross-sectional study in 21 Spanish pulmonology services. We use a standardized questionnaire completed by the bronchoscopists at the end of each bronchoscopy. The variables were described with absolute and relative frequencies, measures of cen‑ tral tendency and dispersion depending on their nature. The existence of learning curves was evaluated by CUSUM analysis. Results: The most frequent indications in 300 included bronchoscopies was bronchial aspiration in 69.3% and the median duration of these was 9.1 min. The route of entry was nasal in 47.2% and oral in 34.1%. The average score for ease of use, image, and aspiration quality was 80/100. All the planned techniques were performed in 94.9% and the bronchoscopist was satisfed in 96.6% of the bronchoscopies. They highlighted the portability and immediacy of the aScope4TM to start the procedure in 99.3%, the possibility of taking and storing images in 99.3%. The CUSUM analysis showed average scores>70/100 from the frst procedure and from the 9th procedure more than 80% of the scores exceeded the 80/100 score