Fast and quantitative mitophagy assessment by flow cytometry using the mito-QC reporter.

Mitochondrial quality control is finely tuned by mitophagy, the selective degradation of mitochondria through autophagy, and mitochondrial biogenesis. Removal of damaged mitochondria is essential to preserve cellular bioenergetics and prevent detrimental events such as sustained mitoROS production, pro-apoptotic cytochrome c release or mtDNA leakage. The array of tools available to study mitophagy is very limited but in constant development. Almost a decade ago, we developed a method to assess mitophagy flux using MitoTracker Deep Red in combination with lysosomal inhibitors. Now, using the novel tandem-fluorescence reporter mito-QC (mCherry-GFP-FIS1101-152) that allows to differentiate between healthy mitochondria (mCherry+GFP+) and mitolysosomes (mCherry+GFP-), we have developed a robust and quantitative method to assess mitophagy by flow cytometry. This approach has been validated in ARPE-19 cells using PINK1/Parkin-dependent (CCCP) and PINK1/Parkin-independent (DFP) positive controls and complementary techniques. Furthermore, we show that the mito-QC reporter can be multiplexed, especially if using spectral flow cytometry, to simultaneously study other cellular parameters such as viability or ROS production. Using this technique, we evaluated and characterized two prospective mitophagy inducers and further dissected their mechanism of action. Finally, using mito-QC reporter mice, we developed a protocol to measure mitophagy levels in the retina ex vivo. This novel methodology will propel mitophagy research forward and accelerate the discovery of novel mitophagy modulators.

Targeted proteomics addresses selectivity and complexity of protein degradation by autophagy.

Macroautophagy/autophagy is a constitutively active catabolic lysosomal degradation pathway, often found dysregulated in human diseases. It is often considered to act in a cytoprotective manner and is commonly upregulated in cells undergoing stress. Its initiation is regulated at the protein level and does not require de novo protein synthesis. Historically, autophagy has been regarded as nonselective; however, it is now clear that different stimuli can lead to the selective degradation of cellular components via selective autophagy receptors (SARs). Due to its selective nature and the existence of multiple degradation pathways potentially acting in concert, monitoring of autophagy flux, i.e. selective autophagy-dependent protein degradation, should address this complexity. Here, we introduce a targeted proteomics approach monitoring abundance changes of 37 autophagy-related proteins covering process-relevant proteins such as the initiation complex and the Atg8-family protein lipidation machinery, as well as most known SARs. We show that proteins involved in autophagosome biogenesis are upregulated and spared from degradation under autophagy-inducing conditions in contrast to SARs, in a cell-line dependent manner. Classical bulk stimuli such as nutrient starvation mainly induce degradation of ubiquitin-dependent soluble SARs and not of ubiquitin-independent, membrane-bound SARs. In contrast, treatment with the iron chelator deferiprone leads to the degradation of ubiquitin-dependent and -independent SARs linked to mitophagy and reticulophagy/ER-phagy. Our approach is automatable and supports large-scale screening assays paving the way to (pre)clinical applications and monitoring of specific autophagy flux.Abbreviation: AMBRA1: autophagy and beclin 1 regulator 1; ATG: autophagy related; BafA1: bafilomycin A1; BNIP1: BCL2 interacting protein 1; BNIP3: BCL2 interacting protein 3; BNIP3L/NIX: BCL2 interacting protein 3-like; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; CCPG1: cell cycle progression 1; CV: coefficients of variations; CCCP: carbonyl cyanide m-chlorophenyl hydrazone; DFP: deferiprone; ER: endoplasmic reticulum; FKBP8: FKBP prolyl isomerase 8; GABARAPL: GABA type A receptor associated protein like; LC: liquid chromatography; LOD: limit of detection; LOQ: limit of quantification; MAP1LC3: microtubule associated protein 1 light chain 3; MS: mass spectrometry; NCOA4: nuclear receptor coactivator 4; NBR1: NBR1 autophagy cargo receptor; NUFIP1: nuclear FMR1 interacting protein 1; OPTN: optineurin; PHB2: prohibitin 2; PNPLA2/ATGL: patatin like phospholipase domain containing 2; POI: protein of interest; PTM: posttranslational modification; PRM: parallel reaction monitoring; RB1CC1/FIP200: RB1 inducible coiled-coil 1; RETREG1/FAM134B: reticulophagy regulator 1; RPS6KB1: ribosomal protein S6 kinase B1; RTN3: reticulon 3; SARs: selective autophagy receptors; SQSTM1/p62: sequestosome 1; STBD1: starch binding domain 1; TAX1BP1: Tax1 binding protein 1; TFEB: transcription factor EB; TNIP1: TNFAIP3 interacting protein 1; TOLLIP: toll interacting protein; ULK1: unc-51 like autophagy activating kinase 1; WBP2: WW domain binding protein 2; WDFY3/Alfy: WD repeat and FYVE domain containing 3; WIPI2: WD repeat domain, phosphoinositide interacting 2.

Specific Cellular and Humoral Response after the Third Dose of Anti-SARS-CoV-2 RNA Vaccine in Patients with Immune-Mediated Rheumatic Diseases on Immunosuppressive Therapy.

OBJECTIVE: Data on cellular and humoral immunogenicity after the third dose of anti-SARS-CoV-2 vaccines in patients with immune-mediated rheumatic diseases (IMRDs) are scarce. Herein, we evaluated the adaptive immune response in IMRD patients treated with different immunosuppressive therapies (conventional synthetic disease-modifying antirheumatic drugs [csDMARDs], biological disease-modifying antirheumatic drugs [bDMARDs], and targeted synthetic disease-modifying antirheumatic drugs [tsDMARDs]) after the booster of the anti-SARS-CoV-2 vaccine to determine whether any drug reduced the vaccine's response. METHODS: A single-center prospective study was conducted, including patients presenting with IMRD and healthy controls (HC). Specific anti-SARS-CoV-2 interferon-gamma (IFN-γ) production was evaluated between 8-12 weeks after the third dose of the SARS-CoV-2 vaccine. In addition, anti-Spike IgG antibody titers were also measured. RESULTS: Samples were obtained from 79 IMRD patients (51 women, 28 men; mean age 57 ± 11.3 years old): 43 rheumatoid arthritis, 10 psoriatic arthritis, 14 ankylosing spondylitis, 10 undifferentiated spondyloarthritis, and 2 inflammatory bowel disease-associated spondyloarthritis (IBD-SpA). In total, 31 HC (mean age 50.9 ± 13.1 years old, 67.7% women) were included in the study. Post-vaccine results displayed positive T-cell immune responses in 68 out of 79 (86.1%) IMRD patients (82.3% of those without prior COVID-19). All HC and IMRDs patients had an antibody response against the SARS-CoV-2 receptor-binding domain; however, the HC response was significantly higher (median of 18,048 AU/mL) than in IMRDs patients (median of 6590.3 AU/mL, p < 0.001). MTX and leflunomide were associated with lower titers of IgG and IFN-γ responses. Among bDMARDs, adalimumab, etanercept, and guselkumab are associated with reduced cellular responses. CONCLUSION: Our preliminary data show that the majority of our IMRD patients develop cellular and humoral responses after the SARS-CoV-2 booster vaccination, emphasizing the relevance of vaccination in this group. However, the magnitude of specific responses was dependent on the immunosuppressive therapy administered. Specific vaccination protocols and personalized decisions about boosters are essential for these patients.

Combined Immune Defect in B-Cell Lymphoproliferative Disorders Is Associated with Severe Infection and Cancer Progression.

B cell chronic lymphoproliferative diseases (B-CLPD) are associated with secondary antibody deficiency and other innate and adaptive immune defects, whose impact on infectious risk has not been systematically addressed. We performed an immunological analysis of a cohort of 83 B-CLPD patients with recurrent and/or severe infections to ascertain the clinical relevance of the immune deficiency expression. B-cell defects were present in all patients. Patients with combined immune defect had a 3.69-fold higher risk for severe infection (p = 0.001) than those with predominantly antibody defect. Interestingly, by Kaplan-Meier analysis, combined immune defect showed an earlier progression of cancer with a hazard ratio of 3.21, than predominantly antibody defect (p = 0.005). When B-CLPD were classified in low-degree, high-degree, and plasma cell dyscrasias, risk of severe disease and cancer progression significantly diverged in combined immune defect, compared with predominantly antibody defect (p = 0.001). Remarkably, an underlying primary immunodeficiency (PID) was suspected in 12 patients (14%), due to prior history of infections, autoimmune and granulomatous conditions, atypical or variegated course and compatible biological data. This first proposed SID classification might have relevant clinical implications, in terms of predicting severe infections and cancer progression, and might be applied to different B-CLPD entities.

Evidence of exhausted lymphocytes after the third anti-SARS-CoV-2 vaccine dose in cancer patients.

Introduction: Evidence is scant regarding the long-term humoral and cellular responses Q7 triggered by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mRNA vaccines in cancer patients after repeated booster doses. The possibility of T-cell exhaustion following these booster doses in this population has not yet been fully studied and remains uncertain. Methods: In this single-center prospective observational study, we explored the specific humoral and cellular response to S1 antigen in 36 patients with solid malignancies at baseline, and after the second and third doses of the mRNA-1273 vaccine. Results: A dual behavior was observed: 24 (66.7%) patients showed partial specific IFN-γ response after the second dose that was further enhanced after the third dose; and 11 (30.5%) already showed an optimal response after the second dose and experienced a marked fall-off of specific IFN-γ production after the third (4 patients negativization), which might suggest T cell exhaustion due to repetitive priming to the same antigen. One (2.8%) patient had persistently negative responses after all three doses. Seroconversion occurred in all patients after the second dose. We then studied circulating exhausted CD8+ T-cells in 4 patients from each of the two response patterns, those with increase and those with decrease in cellular response after the third booster. The patients with decreased cellular response after the booster had a higher expression of PD1+CD8+ and CD57+PD1+CD8+ exhausted T cells compared with those with an increased cellular response both in vivo and in vitro. The proportion of PD1+CD8+ and CD57+PD1+CD8+ exhausted T cells inversely correlated with IFN-γ production. Discussion: Our preliminary data show that the two-dose SARS-CoV-2 vaccine regimen was beneficial in all cancer patients of our study. An additional booster seems to be beneficial in suboptimal vaccine seroconverters, in contrast to maximal responders that might develop exhaustion. Our data should be interpreted with caution given the small sample size and highlight the urgent need to validate our results in other independent and larger cohorts. Altogether, our data support the relevance of immunological functional studies to personalize preventive and treatment decisions in cancer patients.

B-cell haematological malignancies and SARS-CoV-2 infection: Could immunological interventions influence the outcome?

B cell haematological malignancies (HMs) have been described as the worst cancer type for concomitant COVID-19 in terms of mortality, with rates up to 65%. This risk factor for COVID-19 cannot only be explained by comorbidities and advanced age of patients, but aggravated by secondary immunodeficiency (SID). We aimed at evaluating the impact of COVID-19 on 86 HM patients with concomitant SID from a single centre. Only 14 HM patients of 86 (16.28%) patients suffered COVID-19, with mortality rate of 7%. When we considered patients according to B-cell defect only or multiple immune defect overlap (B-T-cell/NK cells/complement), patients with immune defect overlap presented 5.30-fold higher risk of COVID-19 than only B cell defect (95% CI, 1.67-17.0) (p = 0.004). Seven (50%) patients were on active IgRT; while five (36%) had received prior mucosal vaccines for respiratory infections. Our results show that modelling SID in HM may contribute to better prediction of infectious risk and to prompt more targeted and timely preventive therapies.