Referral rates and barriers to lung transplantation based on pulmonary function criteria in interstitial lung diseases: a retrospective cohort study.

BACKGROUND: Interstitial lung diseases (ILD) unresponsive to medical therapy often require lung transplantation (LTx), which prolongs quality of life and survival. Ideal timing for referral for LTx remains challenging, with late referral associated with significant morbidity and mortality. Among other criteria, patients with ILD should be considered for LTx if forced vital capacity (FVC) is less than 80% or diffusion capacity for carbon monoxide (DLCO) is less than 40%. However, data on referral rates are lacking. OBJECTIVES: To evaluate referral rates for LTx based on pulmonary function tests (PFTs) and identify barriers associated with non-referral. DESIGN: A single-center retrospective cohort study. METHODS: The study consisted of ILD patients who performed PFT between 2014 and 2020. Patients with FVC < 80% or a DLCO < 40% were included in the study. Patients with absolute contraindications to LTx were excluded. Referral rates were computed, and a comparison was made between referred and non-referred subjects. RESULTS: Out of 114 ILD patients meeting criteria for referral to LTx, 35 were referred (30.7%), and 7 proceeded to undergo LTx. Median time from PFT to referral for assessment was 255 days [interquartile range (IQR) 35-1077]. Median time from referral to LTx was 89 days (IQR 59-143). Referred patients were younger (p = 0.003), had lower FVC (p < 0.001), DLCO (p < 0.001), and a higher rate of pulmonary hypertension (p = 0.04). Relatively better PFT, and older age, were significantly associated with non-referral of patients. CONCLUSION: There is under-referral of ILD patients who are eligible for LTx, which is associated with severe disease and missed opportunities for LTx. Further research is required to validate these findings.

Lung transplants: addressing referral gaps for lung disease patientsPatients with severe lung diseases that are unresponsive to medical treatments often require lung transplants to enhance their quality of life and survival. Determining the optimal timing for considering a transplant is challenging, as delaying it can lead to complications. Our study aimed to assess how frequently individuals with lung problems, particularly interstitial lung diseases, were referred for lung transplants based on lung function tests. We conducted a retrospective analysis of medical records for patients with lung diseases who underwent lung function tests between 2014 and 2020. We selected patients whose test results indicated impaired lung function, excluding those who were ineligible for lung transplants due to other medical reasons. Subsequently, we examined the number of patients referred for a lung transplant and compared them to those who were not referred. Our findings revealed that out of 114 patients eligible for a lung transplant, only 35 were referred, representing a referral rate of approximately 31%. Among these, only 7 patients actually underwent the transplant procedure. The time elapsed between the lung function test and the referral for a transplant assessment was notably long, averaging around 255 days. Additionally, once referred, patients waited an average of 89 days for the transplant assessment. Referred patients tended to be younger and had more severe lung disease, characterized by lower lung function test results and a higher likelihood of pulmonary hypertension. Conversely, patients who were not referred generally enjoyed better overall health and were older. This discrepancy highlights the missed opportunities for patients to improve their health and quality of life through lung transplantation. Further research is essential to verify the accuracy of these findings, but this study represents a crucial step toward ensuring that individuals with lung diseases receive the appropriate care they require.

Persistent Severe Acute Respiratory Syndrome Coronavirus 2 Pneumonia in Patients Treated With Anti-CD20 Monoclonal Antibodies.

We report 8 cases of persistent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pneumonia in patients previously treated with anti-CD20 monoclonal antibodies. Polymerase chain reaction of nasopharyngeal swabs for SARS-CoV-2 was negative in most cases; viral cell cultures confirmed that viable SARS-Co-2 virus was present. Four patients were treated with anti-SARS-CoV-2 hyperimmune globulins with rapid resolution of disease.

Soluble HLA peptidome of pleural effusions is a valuable source for tumor antigens.

BACKGROUND: Soluble human leucocyte antigen (sHLA) molecules, released into the plasma, carry their original peptide cargo and provide insight into the protein synthesis and degradation schemes of their source cells and tissues. Other body fluids, such as pleural effusions, may also contain sHLA-peptide complexes, and can potentially serve as a source of tumor antigens since these fluids are drained from the tumor microenvironment. We explored this possibility by developing a methodology for purifying and analyzing large pleural effusion sHLA class I peptidomes of patients with malignancies or benign diseases. METHODS: Cleared pleural fluids, cell pellets present in the pleural effusions, and the primary tumor cells cultured from cancer patients' effusions, were used for immunoaffinity purification of the HLA molecules. The recovered HLA peptides were analyzed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) and the resulting LC-MS/MS data were analyzed with the MaxQuant software tool. Selected tumor antigen peptides were tested for their immunogenicity potential with donor peripheral blood mononuclear cells (PBMCs) in an in vitro assay. RESULTS: Mass spectrometry analysis of the pleural effusions revealed 39,669 peptides attributable to 11,305 source proteins. The majority of peptides identified from the pleural effusions were defined as HLA ligands that fit the patients' HLA consensus sequence motifs. The membranal and soluble HLA peptidomes of each individual patient correlated to each other. Additionally, soluble HLA peptidomes from the same patient, obtained at different visits to the clinic, were highly similar. Compared with benign effusions, the soluble HLA peptidomes of malignant pleural effusions were larger and included HLA peptides derived from known tumor-associated antigens, including cancer/testis antigens, lung-related proteins, and vascular endothelial growth factor pathway proteins. Selected tumor-associated antigens that were identified by the immunopeptidomics were able to successfully prime CD8+ T cells. CONCLUSIONS: Pleural effusions contain sHLA-peptide complexes, and the pleural effusion HLA peptidome of patients with malignant tumors can serve as a rich source of biomarkers for tumor diagnosis and potential candidates for personalized immunotherapy.

Data on the identification of VRK2 as a mediator of PD-1 function.

Therapeutic programmed cell death protein 1 (PD-1) blockade enhances T cell mediated anti-tumor immunity, but many patients do not respond, and a significant proportion develops inflammatory toxicities. To develop better therapeutics and to understand the signaling pathways downstream of PD-1 we performed phosphoproteomic interrogation of PD-1 to identify key mediators of PD-1 signaling. Hereby, supporting data of the research article "VRK2 inhibition synergizes with PD-1 blockade to improve T cell responses" are presented. In the primary publication, we proposed that VRK2 is a unique therapeutic target and that combination of VRK2 inhibitors with PD-1 blockade may improve cancer immunotherapy. Here, we provide data on the effect of other kinases on PD-1 signaling utilizing shRNA knockdown of the different kinases in Jurkat T cells. In addition, we used VRK2 inhibition by a pharmacologic approach in the MC38 tumor mouse model, to show the combined outcome of anti PD-1 treatment with VRK2 inhibition. These data provide additional targets downstream PD-1 and point toward methods of testing the effect of the inhibition of these targets on tumor progression in vivo.

Transmembrane adaptor protein PAG is a mediator of PD-1 inhibitory signaling in human T cells.

The inhibitory receptor PD-1 is expressed on T cells to inhibit select functions when ligated. The complete signaling mechanism downstream of PD-1 has yet to be uncovered. Here, we discovered phosphoprotein associated with glycosphingolipid-enriched microdomains 1 (PAG) is phosphorylated following PD-1 ligation and associate this with inhibitory T cell function. Clinical cohort analysis correlates low PAG expression with increased survival from numerous tumor types. PAG knockdown in T cells prevents PD-1-mediated inhibition of cytokine secretion, cell adhesion, CD69 expression, and ERK204/187 phosphorylation, and enhances phosphorylation of SRC527 following PD-1 ligation. PAG overexpression rescues these effects. In vivo, PAG contributes greatly to the growth of two murine tumors, MC38 and B16, and limits T cell presence within the tumor. Moreover, PAG deletion sensitizes tumors to PD-1 blockade. Here PAG is established as a critical mediator of PD-1 signaling and as a potential target to enhance T cell activation in tumors.

Baseline spirometry parameters as predictors of airway hyperreactivity in adults with suspected asthma.

BACKGROUND: Methacholine challenge tests (MCTs) are used to diagnose airway hyperresponsiveness (AHR) in patients with suspected asthma where previous diagnostic testing has been inconclusive. The test is time consuming and usually requires referral to specialized centers. Simple methods to predict AHR could help determine which patients should be referred to MCTs, thus avoiding unnecessary testing. Here we investigated the potential use of baseline spirometry variables as surrogate markers for AHR in adults with suspected asthma. METHODS: Baseline spirometry and MCTs performed between 2013 and 2019 in a large tertiary center were retrospectively evaluated. Receiver-operating characteristic curves for the maximal expiratory flow-volume curve indices (angle ß, FEV1, FVC, FEV1/FVC, FEF50%, FEF25-75%) were constructed to assess their overall accuracy in predicting AHR and optimal cutoff values were identified. RESULTS: A total of 2983 tests were analyzed in adults aged 18-40 years. In total, 14% of all MCTs were positive (PC20 ≤ 16 mg/ml). All baseline spirometry parameters were significantly lower in the positive group (p < 0.001). FEF50% showed the best overall accuracy (AUC = 0.688) and proved to be useful as a negative predictor when applying FEF50% ≥ 110% as a cutoff level. CONCLUSIONS: This study highlights the role of FEF50% in predicting AHR in patients with suspected asthma. A value of ≥ 110% for baseline FEF50% could be used to exclude AHR and would lead to a substantial decrease in MCT referrals.

Mesencephalic astrocyte-derived neurotrophic factor is secreted from interferon-γ-activated tumor cells through ER calcium depletion.

The most successful immunotherapeutic agents are blocking antibodies to either programmed cell death-1 (PD-1), an inhibitory receptor expressed on T lymphocytes, or to its ligand, programmed cell death-ligand 1 (PD-L1). Nevertheless, many patients do not respond, and additional approaches, specifically blocking other inhibitory receptors on T cells, are being explored. Importantly, the source of the ligands for these receptors are often the tumor cells. Indeed, cancer cells express high levels of PD-L1 upon stimulation with interferon-γ (IFN-γ), a major cytokine in the tumor microenvironment. The increase in PD-L1 expression serves as a negative feedback towards the immune system, and allows the tumor to evade the attack of immune cells. A potential novel immunoregulator is mesencephalic astrocyte-derived neurotrophic factor (MANF), an endoplasmic reticulum (ER)-resident protein that is secreted from pancreatic beta cells upon cytokines activation, and can induce an alternatively activated macrophage phenotype (M2), and thus may support tumor growth. While MANF was shown to be secreted from pancreatic beta cells, its IFN-γ-induced secretion from tumor cells has never been assessed. Here we found that IFN-γ induced MANF secretion from diverse tumor cell-lines-melanoma cells, colon carcinoma cells and hepatoma cells. Mechanistically, there was no increase in MANF RNA or intracellular protein levels upon IFN-γ stimulation. However, IFN-γ induced ER calcium depletion, which was necessary for MANF secretion, as Dantrolene, an inhibitor of ER calcium release, prevented its secretion. Thus, MANF is secreted from IFN-γ-stimulated tumor cells, and further studies are required to assess its potential as a drug target for cancer immunotherapy.

VRK2 inhibition synergizes with PD-1 blockade to improve T cell responses.

Therapeutic programmed cell death protein 1 (PD-1) blockade enhances T cell mediated anti-tumor immunity but many patients do not respond and a significant proportion develops inflammatory toxicities. To develop better therapeutics and to understand the signaling pathways downstream of PD-1 we performed phosphoproteomic analysis of PD-1 and identified vaccinia related kinase 2 (VRK2) as a key mediator of PD-1 signaling. Using genetic and pharmacological approaches, we discovered that VRK2 is required for PD-1-induced phosphorylation of the protein p21 activated kinase 2 (PAK2), and for the inhibition of IL-2, IL-8, and IFN-γ secretion. Moving into in vivo syngeneic tumor models, pharmacologic inhibition of VRK2 in combination with PD-1 blockade enhanced tumor clearance through T cell activation. This study suggests that VRK2 is a unique therapeutic target and that combination of VRK2 inhibitors with PD-1 blockade may improve cancer immunotherapy.

Weaning of Severe COVID-19 Mechanically Ventilated Patients: Experience within a Dedicated Unit in Israel.

BACKGROUND: Patients diagnosed with coronavirus disease-19 (COVID-19) who deteriorate to respiratory failure and require mechanical ventilation may later need to be weaned from the ventilator and undergo a rehabilitation process. The rate of weaning COVID-19 patients from mechanical ventilation is unknown. OBJECTIVES: To present our experience with ventilator weaning of COVID-19 patients in a dedicated facility. METHODS: A retrospective cohort study was conducted of 18 patients hospitalized in a COVID-19 dedicated ventilator weaning unit. RESULTS: Eighteen patients were hospitalized in the dedicated unit between 6 April and 19 May 2020. Of these, 88% (16/18) were weaned and underwent decannulation, while two patients deteriorated and were re-admitted to the intensive care unit. The average number of days spent in our department was 12. There was no statistically significant correlation between patient characteristics and time to weaning from ventilation or with the time to decannulation. CONCLUSIONS: Despite the high mortality of COVID-19 patients who require mechanical ventilation, most of the patients in our cohort were weaned in a relatively short period of time. Further large-scale studies are necessary to assess the cost effectiveness of dedicated COVID-19 departments for ventilator weaning.

Early Blood-based Liquid Biopsy in Patients with Treatment-naïve Metastatic Adenocarcinoma of the Lung: A Case Series.

BACKGROUND: Guidelines recommend testing for multiple biomarkers in non-small cell lung cancer (NSCLC) tumors. Blood-based liquid biopsy analyzing cell-free DNA (cfDNA) could be used in addition to tumor biopsy genotyping, especially if tissue/time are limiting. OBJECTIVES: To investigate the clinical utility of early cfDNA analysis (Guardant360® CDx) in treatment-naïve NSCLC patients. METHODS: A prospective cohort of treatment-naïve patients with metastatic NSCLC who underwent tumor and cfDNA analysis between 12/2018 and 2/2019 were included. RESULTS: Ten patients were included: 6 males, median age 70.5 years (range 48-87), 8 prior smokers. Liquid biopsy was sent when cancer cells were detected in the biopsy specimen. Median time from diagnosis to receiving the report on the last biomarker from the tumor biopsy was 20 days (range 9-34); median time from blood draw to receiving the cfDNA findings was 9 days (range 7-12). The median difference between the cfDNA and the tumor analysis reports was 20 days (range 9-28). Actionable biomarkers were identified in four patients by both the biopsy analysis and the cfDNA analysis (2cases with EGFR mutations, one with ROS1 fusion, and one with EML4-ALK fusion for whom the biopsy analysis also identified an EGFR mutation not detected in the cfDNA analysis). Overall, eight patients received treatment (2 died before treatment initiation). Three patients received biomarker-based treatment (1 osimertinib, 1 alectinib, and 1 crizotinib). CONCLUSIONS: These findings suggest that cfDNA analysis should be ordered by the pulmonologists early in the evaluation of patients with NSCLC, which might complement the tumor biopsy.

Quantitative phosphoproteomic analysis reveals involvement of PD-1 in multiple T cell functions.

Programmed cell death protein 1 (PD-1) is a critical inhibitory receptor that limits excessive T cell responses. Cancer cells have evolved to evade these immunoregulatory mechanisms by upregulating PD-1 ligands and preventing T cell-mediated anti-tumor responses. Consequently, therapeutic blockade of PD-1 enhances T cell-mediated anti-tumor immunity, but many patients do not respond and a significant proportion develop inflammatory toxicities. To improve anti-cancer therapy, it is critical to reveal the mechanisms by which PD-1 regulates T cell responses. We performed global quantitative phosphoproteomic interrogation of PD-1 signaling in T cells. By complementing our analysis with functional validation assays, we show that PD-1 targets tyrosine phosphosites that mediate proximal T cell receptor signaling, cytoskeletal organization, and immune synapse formation. PD-1 ligation also led to differential phosphorylation of serine and threonine sites within proteins regulating T cell activation, gene expression, and protein translation. In silico predictions revealed that kinase/substrate relationships engaged downstream of PD-1 ligation. These insights uncover the phosphoproteomic landscape of PD-1-triggered pathways and reveal novel PD-1 substrates that modulate diverse T cell functions and may serve as future therapeutic targets. These data are a useful resource in the design of future PD-1-targeting therapeutic approaches.

High-intensity interval training accelerates oxygen uptake kinetics and improves exercise tolerance for individuals with cystic fibrosis.

BACKGROUND: Exercise training provides benefits for individuals with cystic fibrosis; however, the optimal program is unclear. High-intensity interval training is safe and effective for improving 'functional capacity' in these individuals with peak rate of O2 uptake typically referenced. The ability to adjust submaximal rate of oxygen uptake (V̇O2 kinetics) might be more important for everyday function because maximal efforts are usually not undertaken. Moreover, the ability of high-intensity training to accelerate V̇O2 kinetics for individuals with cystic fibrosis could be enhanced with O2 supplementation during training. METHODS: Nine individuals with cystic fibrosis completed incremental cycling to limit of tolerance followed by 8 weeks of high-intensity interval cycling (2 sessions per week x ~ 45 min per session) either with (n = 5; O2+) or without (AMB) oxygen supplementation (100%). Each session involved work intervals at 70% of peak work rate followed by 60 s of recovery at 35%. For progression, duration of work intervals was increased according to participant tolerance. RESULTS: Both groups experienced a significant increase in work-interval duration over the course of the intervention (O2+, 1736 ± 141 v. 700 ± 154 s; AMB, 1463 ± 598 v. 953 ± 253 s; P = 0.000); however, the increase experienced by O2+ was greater (P = 0.027). During low-intensity constant-work-rate cycling, the V̇O2 mean response time was shortened post compared to pre training (O2+, 34 ± 11 v. 44 ± 9 s; AMB, 39 ± 14 v. 45 ± 17 s; P = 0.000) while during high-intensity constant-work-rate cycling, time to exhaustion was increased (O2+, 1628 ± 163 v. 705 ± 133 s; AMB, 1073 ± 633 v. 690 ± 348 s; P = 0.002) and blood [lactate] response was decreased (O2+, 4.5 ± 0.9 v. 6.3 ± 1.4 mmol. L- 1; AMB, 4.5 ± 0.6 v. 5.2 ± 1.4 mmol. L- 1; P = 0.003). These positive adaptations were similar regardless of gas inspiration during training. CONCLUSION: Eight weeks of high-intensity interval training for patients with cystic fibrosis accelerated V̇O2 kinetics and increased time to exhaustion. This provides some evidence that these patients may benefit from this type of exercise. TRIAL REGISTRATION: This study was retrospectively registered in the ISRTCN registry on 22/06/2019 (#ISRCTN13864650).

Specific overexpression of 15-lipoxygenase in endothelial cells promotes cancer cell death in an in vivo Lewis lung carcinoma mouse model.

PURPOSE: Lipoxygenases (LOX) have been implicated in carcinogenesis, however both pro- and anti-carcinogenic effects have been reported in different cancer models. Using transgenic mice, which specifically overexpress human 15-lipoxygenase (ALOX15) in endothelial cells (EC), we previously demonstrated significant inhibition of tumor development. In the Lewis lung carcinoma (LLC) model, the primary tumor developed similarly in both wild type (WT) and ALOX15 overexpressing mice. However, metastases development was significantly inhibited in the transgenic mice. Here, we explored the molecular basis for the anti-metastatic effect of endothelial cell specific ALOX15 overexpression. MATERIALS/METHODS: We used ALOX15 overexpressing mice, and in-vitro cell model to evaluate the molecular effect of ALOX15 on EC and LLC cells. RESULTS: When LLC cells were injected in WT and ALOX15 overexpressing mice, we observed a higher degree of apoptosis and necrosis in primary and metastatic tumors of ALOX15 overexpressing animals. These anti-carcinogenic and anti-metastatic effects were paralleled by augmented expression of cyclin-dependent kinase inhibitor 1A (CDKN1A; p21) and of the peroxisome proliferators-activated receptor (PPAR)γ and by downregulation of the steady state concentrations of connexin26 mRNA. Consistent with these in vivo effects, ALOX15 overexpression in LLC and HeLa cancer cells in vitro significantly reduced cell viability in culture. In contrast, similar treatment of non-cancerous B2B epithelial cells did not impact cell viability. CONCLUSIONS: Taken together, our data suggests that endothelial cell specific overexpression of ALOX15 promotes apoptosis and necrosis in primary and metastatic tumors in mice, by upregulation of P21 and PPARγ expression in adjacent cancer cells.

Correlation of body composition by computerized tomography and metabolic parameters with survival of nivolumab-treated lung cancer patients.

PURPOSE: Weight loss is a well-recognized prognostic parameter for survival of lung cancer patients. Computerized-tomography (CT)-based analysis of body composition and blood-based metabolic evaluation are promising prognostic tools. We aimed to assess the correlation between albumin, body mass index (BMI), skeletal muscle mass index (SMI), fat-free mass index (FFMI), fat mass index (FMI) and weight change, as well as their correlation with survival of lung cancer patients on nivolumab treatment. METHODS: Data were retrospectively collected. Weight was measured at a diagnosis of stage 4 disease and before start of nivolumab. Albumin levels were measured before starting nivolumab. BMI, SMI, FFMI, and FMI were evaluated from CT scans performed at start of nivolumab. Overall survival (OS) was from starting of nivolumab to death or censured at last follow-up. Statistical analysis was done to identify correlation between the various factors and between those factors and survival. RESULTS: Forty-six patients with advanced non-small cell lung cancer (NSCLC) were included. Median follow-up was 22 months. Pathology was Adenocarcinoma/Squamous/non-other specified in 25/15/6 respectively. All patients received nivolumab as an advanced-line treatment for stage IV NSCLC. We observed a significant correlation of weight loss (P=0.01, HR=2.85) and albumin (P=0.043, HR=0.34) with OS in multivariate analysis. A significant correlation was found between BMI to SMI, FFMI, FMI, and weight change. CONCLUSION: Weight loss and low albumin levels are significant negative prognostic factors for NSCLC patients on immunotherapy. CT-based parameters of body composition remain to be proven as more reliable than standard clinical parameters.

SLAMF6 clustering is required to augment T cell activation.

The signaling lymphocytic activation molecule (SLAM) family is comprised of nine distinct receptors that are expressed exclusively on hematopoietic cells. Most of these transmembrane receptors are homotypic by nature and downstream signaling occurs when cells that express the same SLAM receptor interact. Previous studies have determined that anti-SLAMF6 antibodies can have a therapeutic effect in autoimmunity and cancer. However, little is known about the role of SLAMF6 in the adaptive immune responses and in order to utilize SLAMF6 interventional approaches, a better understanding of the biology of this receptor in T cell is warranted. Accordingly, the objective of our study was to investigate both functionally and structurally the role of SLAMF6 in T cell receptor (TCR) mediated responses. Biochemical and genetic experiments revealed that SLAMF6 was required for productive TCR downstream signaling. Interestingly, SLAMF6 ectodomain was required for its function, but not for its recruitment to the immunological synapse. Flow-cytometry analysis demonstrated that tyrosine 308 of the tail of SLAMF6 was crucial for its ability to enhance T cell function. Imaging studies revealed that SLAMF6 clustering, specifically with the TCR, resulted in dramatic increase in downstream signaling. Mechanistically, we showed that SLAMF6 enhanced T cell function by increasing T cell adhesiveness through activation of the small GTPase Rap1. Taken together SLAMF6 is an important regulator of T cell activation where both its ectodomain and its endodomain contribute differentially to T cell functions. Additional studies are underway to better evaluate the role of anti-SLAMF6 approaches in specific human diseases.

Tumor-Infiltrating Lymphocytes-Location for Prognostic Evaluation.

Tumor-infiltrating lymphocytes (TIL) are crucial for the success of immunotherapy, and their density can predict prognosis. It has now been shown that computer-based analysis of the spatial organization of TILs adds prognostic value in early-stage non-small cell lung cancer, possibly improving the treatment algorithm to prevent recurrence.See related article by Corredor et al., p. 1526.

EF Hand Domain Family Member D2 Is Required for T Cell Cytotoxicity.

Programmed cell death 1 (PD-1) is a major coinhibitory receptor and a member of the immunological synapse (IS). To uncover proteins that regulate PD-1 recruitment to the IS, we searched for cytoskeleton-related proteins that also interact with PD-1 using affinity purification mass spectrometry. Among these proteins, EF hand domain family member D2 (EFHD2), a calcium binding adaptor protein, was functionally and mechanistically analyzed for its contribution to PD-1 signaling. EFHD2 was required for PD-1 to inhibit cytokine secretion, proliferation, and adhesion of human T cells. Interestingly, EFHD2 was also required for human T cell-mediated cytotoxicity and for mounting an antitumor immune response in a syngeneic murine tumor model. Mechanistically, EFHD2 contributed to IS stability, lytic vesicles trafficking, and granzyme B secretion. Altogether, EFHD2 is an important regulator of T cell cytotoxicity and further studies should evaluate its role in T cell-mediated inflammation.

Co-immunoprecipitation Assay for Studying Functional Interactions Between Receptors and Enzymes.

Receptor-associated enzymes are the major mediators of cellular activation. These enzymes are regulated, at least in part, by physical interactions with cytoplasmic tails of the receptors. The interactions often occur through specific protein domains and result in activation of the enzymes. There are several methods to study interactions between proteins. While co-immunoprecipitation is commonly used to study domains that are required for protein-protein interactions, there are no assays that document the contribution of specific domains to activity of the recruited enzymes at the same time. Accordingly, the method described here combines co-immunoprecipitation and an on-bead enzymatic activity assay for simultaneous evaluation of interactions between proteins and associated enzymatic activation. The goal of this protocol is to identify the domains that are critical for physical interactions between a protein and enzyme and the domains that are obligatory for complete activation of the enzyme. The importance of this assay is demonstrated, as certain receptor protein domains contribute to the binding of the enzyme to the cytoplasmic tail of the receptor, while other domains are necessary to regulate the function of the same enzyme.

Affinity purification mass spectrometry analysis of PD-1 uncovers SAP as a new checkpoint inhibitor.

Programmed cell death-1 (PD-1) is an essential inhibitory receptor in T cells. Antibodies targeting PD-1 elicit durable clinical responses in patients with multiple tumor indications. Nevertheless, a significant proportion of patients do not respond to anti-PD-1 treatment, and a better understanding of the signaling pathways downstream of PD-1 could provide biomarkers for those whose tumors respond and new therapeutic approaches for those whose tumors do not. We used affinity purification mass spectrometry to uncover multiple proteins associated with PD-1. Among these proteins, signaling lymphocytic activation molecule-associated protein (SAP) was functionally and mechanistically analyzed for its contribution to PD-1 inhibitory responses. Silencing of SAP augmented and overexpression blocked PD-1 function. T cells from patients with X-linked lymphoproliferative disease (XLP), who lack functional SAP, were hyperresponsive to PD-1 signaling, confirming its inhibitory role downstream of PD-1. Strikingly, signaling downstream of PD-1 in purified T cell subsets did not correlate with PD-1 surface expression but was inversely correlated with intracellular SAP levels. Mechanistically, SAP opposed PD-1 function by acting as a molecular shield of key tyrosine residues that are targets for the tyrosine phosphatase SHP2, which mediates PD-1 inhibitory properties. Our results identify SAP as an inhibitor of PD-1 function and SHP2 as a potential therapeutic target in patients with XLP.

The tyrosine phosphatase SHP-1 promotes T cell adhesion by activating the adaptor protein CrkII in the immunological synapse.

The adaptor protein CrkII regulates T cell adhesion by recruiting the guanine nucleotide exchange factor C3G, an activator of Rap1. Subsequently, Rap1 stimulates the integrin LFA-1, which leads to T cell adhesion and interaction with antigen-presenting cells (APCs). The adhesion of T cells to APCs is critical for their proper function and education. The interface between the T cell and the APC is known as the immunological synapse. It is characterized by the specific organization of proteins that can be divided into central supramolecular activation clusters (c-SMACs) and peripheral SMACs (p-SMACs). Through total internal reflection fluorescence (TIRF) microscopy and experiments with supported lipid bilayers, we determined that activated Rap1 was recruited to the immunological synapse and localized to the p-SMAC. C3G and the active (dephosphorylated) form of CrkII also localized to the same compartment. In contrast, inactive (phosphorylated) CrkII was confined to the c-SMAC. Activation of CrkII and its subsequent movement from the c-SMAC to the p-SMAC depended on the phosphatase SHP-1, which acted downstream of the T cell receptor. In the p-SMAC, CrkII recruited C3G, which led to Rap1 activation and LFA-1-mediated adhesion of T cells to APCs. Functionally, SHP-1 was necessary for both the adhesion and migration of T cells. Together, these data highlight a signaling pathway in which SHP-1 acts through CrkII to reshape the pattern of Rap1 activation in the immunological synapse.