Optimization of Rituximab Therapy in Adult Patients With PLA2R1-Associated Membranous Nephropathy With Artificial Intelligence.

Introduction: Rituximab is a first-line treatment for membranous nephropathy. Nephrotic syndrome limits rituximab exposure due to urinary drug loss. Rituximab underdosing (serum level <2 µg/ml at month-3) is a risk factor for treatment failure. We developed a machine learning algorithm to predict the risk of underdosing based on patients' characteristics at rituximab infusion. We investigated the relationship between the predicted risk of underdosing and the cumulative dose of rituximab required to achieve remission. Methods: Rituximab concentrations were measured at month-3 in 92 sera from adult patients with primary membranous nephropathy, split into a training (75%) and a testing set (25%). A forward-backward machine-learning procedure determined the best combination of variables to predict rituximab underdosing in the training data set, which was tested in the test set. The performances were evaluated for accuracy, sensitivity, and specificity in 10-fold cross-validation training and test sets. Results: The best variables combination to predict rituximab underdosing included age, gender, body surface area (BSA), anti-phospholipase A2 receptor type 1 (anti-PLA2R1) antibody titer on day-0, serum albumin on day-0 and day-15, and serum creatinine on day-0 and day-15. The accuracy, sensitivity, and specificity were respectively 79.4%, 78.7%, and 81.0% (training data set), and 79.2%, 84.6% and 72.7% (testing data set). In both sets, the algorithm performed significantly better than chance (P < 0.05). Patients with an initial high probability of underdosing experienced a longer time to remission with higher rituximab cumulative doses required to achieved remission. Conclusion: This algorithm could allow for early intensification of rituximab regimen in patients at high estimated risk of underdosing to increase the likelihood of remission.

Lipid droplets control mitogenic lipid mediator production in human cancer cells.

OBJECTIVES: Polyunsaturated fatty acids (PUFAs) are structural components of membrane phospholipids and precursors of oxygenated lipid mediators with diverse functions, including the control of cell growth, inflammation and tumourigenesis. However, the molecular pathways that control the availability of PUFAs for lipid mediator production are not well understood. Here, we investigated the crosstalk of three pathways in the provision of PUFAs for lipid mediator production: (i) secreted group X phospholipase A2 (GX sPLA2) and (ii) cytosolic group IVA PLA2 (cPLA2α), both mobilizing PUFAs from membrane phospholipids, and (iii) adipose triglyceride lipase (ATGL), which mediates the degradation of triacylglycerols (TAGs) stored in cytosolic lipid droplets (LDs). METHODS: We combined lipidomic and functional analyses in cancer cell line models to dissect the trafficking of PUFAs between membrane phospholipids and LDs and determine the role of these pathways in lipid mediator production, cancer cell proliferation and tumour growth in vivo. RESULTS: We demonstrate that lipid mediator production strongly depends on TAG turnover. GX sPLA2 directs ω-3 and ω-6 PUFAs from membrane phospholipids into TAG stores, whereas ATGL is required for their entry into lipid mediator biosynthetic pathways. ATGL controls the release of PUFAs from LD stores and their conversion into cyclooxygenase- and lipoxygenase-derived lipid mediators under conditions of nutrient sufficiency and during serum starvation. In starving cells, ATGL also promotes the incorporation of LD-derived PUFAs into phospholipids, representing substrates for cPLA2α. Furthermore, we demonstrate that the built-up of TAG stores by acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1) is required for the production of mitogenic lipid signals that promote cancer cell proliferation and tumour growth. CONCLUSION: This study shifts the paradigm of PLA2-driven lipid mediator signalling and identifies LDs as central lipid mediator production hubs. Targeting DGAT1-mediated LD biogenesis is a promising strategy to restrict lipid mediator production and tumour growth.

Both Humoral and Cellular Immune Responses to SARS-CoV-2 Are Essential to Prevent Infection: a Prospective Study in a Working Vaccinated Population from Southern France.

COVID-19 vaccines have significantly decreased the number of severe cases of the disease, but the virus circulation remains important, and questions about the need of new vaccination campaigns remain unanswered. The individual's protection against SARS-CoV-2 infection is most commonly measured by the level and the neutralizing capacity of antibodies produced against SARS-CoV-2. T cell response is a major contributor in viral infection, and several studies have shown that cellular T cell response is crucial in fighting off SARS-CoV-2 infection. Actually, no threshold of protective immune response against SARS-CoV2 infection has been identified. To better understand SARS-CoV-2-mediated immunity, we assessed both B cell (measuring anti-Spike IgG titer and neutralization capacity) and T cell (measuring IFNγ release assay after specific SARS-CoV2 stimulation) responses to SARS-CoV-2 vaccination with or without virus encounter in a cohort of 367 working volunteers. Vaccinated individuals who had previously been infected had a stronger and more lasting immunity in comparison to vaccinated individuals naive to infection whose immunity started to decline 3 months after vaccination. IFNγ release ≥ 0.285 IU/mL and anti-Spike IgG antibodies ≥ 244 BAU/mL were associated with a sufficient immune response following vaccination preventing future infections. Individuals with comorbidities had a lower chance of reaching the protective thresholds of T cell and B cell responses as identified in multivariate analysis. A combined B cell and T cell analysis of immune responses to determine protective thresholds after SARS-CoV-2 vaccination will allow us to identify individuals in need of a booster vaccine dose, particularly in comorbid subjects.

Breakthrough infections due to SARS-CoV-2 Delta variant: relation to humoral and cellular vaccine responses.

Introduction: COVID-19 vaccines are expected to provide effective protection. However, emerging strains can cause breakthrough infection in vaccinated individuals. The immune response of vaccinated individuals who have experienced breakthrough infection is still poorly understood. Methods: Here, we studied the humoral and cellular immune responses of fully vaccinated individuals who subsequently experienced breakthrough infection due to the Delta variant of SARS-CoV-2 and correlated them with the severity of the disease. Results: In this study, an effective humoral response alone was not sufficient to induce effective immune protection against severe breakthrough infection, which also required effective cell-mediated immunity to SARS-CoV-2. Patients who did not require oxygen had significantly higher specific (p=0.021) and nonspecific (p=0.004) cellular responses to SARS-CoV-2 at the onset of infection than those who progressed to a severe form. Discussion: Knowing both humoral and cellular immune response could allow to adapt preventive strategy, by better selecting patients who would benefit from additional vaccine boosters. Trial registration numbers: https://clinicaltrials.gov, identifier NCT04355351; https://clinicaltrials.gov, identifier NCT04429594.

Immune response to BNT162b2 SARS-CoV-2 vaccine in patients living with HIV: The COVIH-DAPT study.

Introduction: Data on immune response to SARS-CoV-2 vaccine in patients living with HIV (PLWH) over a period longer than 3 months are currently limited. We measured the immune response after BNT162b2 vaccination against SARS-CoV-2 in this population. Methods: We prospectively enrolled PLWH on successful antiretroviral therapy, initiating vaccination with two doses of the BNT162b2 SARS-CoV-2 vaccine administered at six-week interval. SARS-CoV-2 humoral and cellular responses and lymphocyte cell subsets were recorded at inclusion and 6 weeks (W6), 3 months (M3) and 6 months (M6) later. Humoral, humoral strong and cellular responders were defined by IgG titers >10, ≥264BAU/mL and IFN-γ T cell release, respectively. Results: Nineteen subjects without SARS-CoV-2 infection were included (74% men, mean age 51 years, CD4 nadir 399/mm3). All subjects were humoral responders, their antibody titer peak reached at M3. Strong responders' rates were 63% and 21% at M3 and M6, respectively. CD19+CD10+ B cells had increased significantly at W6 then decreased at M3, while CD19+CD27+ B cells remained unchanged. Rates of patients with a cellular response increased from 39% at W6 to 69% at M6. Cellular responders had significantly higher CD3+, CD4+ and CD8+ Effector Memory cells at inclusion (p=0.048, p=0.024, p=0.012, respectively) and CD4+ Terminally Differentiated Effector Memory cells at M3 (p=0.044). Discussion: PLWH have a robust immune response after SARS-CoV-2 vaccination, but a rapid decline in humoral response from 3 months onwards, due to a blunted memory B cell response. Analysis of lymphocyte subsets may help identify optimal times for vaccine boosters.

Untargeted plasma metabolomic fingerprinting highlights several biomarkers for the diagnosis and prognosis of coronavirus disease 19.

Objectives: The COVID-19 pandemic has been a serious worldwide public health crisis since 2020 and is still challenging healthcare systems. New tools for the prognosis and diagnosis of COVID-19 patients remain important issues. Design: Here, we studied the metabolome of plasma samples of COVID-19 patients for the identification of prognosis biomarkers. Patients: Plasma samples of eighty-six SARS-CoV-2-infected subjects and 24 healthy controls were collected during the first peak of the COVID-19 pandemic in France in 2020. Main results: Plasma metabolome fingerprinting allowed the successful discrimination of healthy controls, mild SARS-CoV-2 subjects, and moderate and severe COVID-19 patients at hospital admission. We found a strong effect of SARS-CoV-2 infection on the plasma metabolome in mild cases. Our results revealed that plasma lipids and alterations in their saturation level are important biomarkers for the detection of the infection. We also identified deoxy-fructosyl-amino acids as new putative plasma biomarkers for SARS-CoV-2 infection and COVID-19 severity. Finally, our results highlight a key role for plasma levels of tryptophan and kynurenine in the symptoms of COVID-19 patients. Conclusion: Our results showed that plasma metabolome profiling is an efficient tool for the diagnosis and prognosis of SARS-CoV-2 infection.

Low baseline IFN-γ response could predict hospitalization in COVID-19 patients.

The SARS-CoV-2 infection has spread rapidly around the world causing millions of deaths. Several treatments can reduce mortality and hospitalization. However, their efficacy depends on the choice of the molecule and the precise timing of its administration to ensure viral clearance and avoid a deleterious inflammatory response. Here, we investigated IFN-γ, assessed by a functional immunoassay, as a predictive biomarker for the risk of hospitalization at an early stage of infection or within one month prior to infection. Individuals with IFN-γ levels below 15 IU/mL were 6.57-times more likely to be hospitalized than those with higher values (p<0.001). As confirmed by multivariable analysis, low IFN-γ levels, age >65 years, and no vaccination were independently associated with hospitalization. In addition, we found a significant inverse correlation between low IFN-γ response and high level of IL-6 in plasma (Spearman's rho=-0.38, p=0.003). Early analysis of the IFN-γ response in a contact or recently infected subject with SARS-CoV-2 could predict hospitalization and thus help the clinician to choose the appropriate treatment avoiding severe forms of infection and hospitalization.

Toxic Occupational Exposures and Membranous Nephropathy.

BACKGROUND AND OBJECTIVES: Membranous nephropathy is a rare autoimmune kidney disease whose increasing prevalence in industrialized countries pleads for the involvement of an environmental factor in the development of the disease. In addition, the predominance of men in membranous nephropathy, classically attributed to biologic or genetic differences between men and women, could also be due to different occupational exposures. To support this hypothesis, we sought to describe the toxic occupational exposures of patients with membranous nephropathy. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: In this observational epidemiologic study, we compared the occupations and toxic occupational exposures of 100 patients with membranous nephropathy with those of the general population, consisting of two cohorts of 26,734,000 and 26,500 French workers. We then compared the characteristics of patients exposed to an occupational toxic substance with those of unexposed patients. RESULTS: Patients with membranous nephropathy worked more frequently in the construction sector than the general population (33% versus 7%, P<0.001). This difference remained significant by age and sex. They were also more frequently exposed to toxic substances, such as asbestos (16% versus 5%, P<0.001), lead (9% versus 1%, P<0.001), or organic solvents (37% versus 15%, P<0.001), than the general population. The predominance of men in the subgroup of patients occupationally exposed to toxic substances was not observed in unexposed individuals (organic solvents: 80% men versus 41%, P<0.001; asbestos: 90% men versus 55%, P=0.004). In addition, patients with phospholipase A2 receptor 1 (PLA2R1) epitope spreading were more frequently exposed to asbestos and organic solvents than patients without epitope spreading (32% versus 7%, P=0.02 and 74% versus 43%, P=0.02, respectively), with a dose-dependent effect. CONCLUSIONS: Patients with membranous nephropathy were more frequently exposed to certain occupational toxic substances, such as asbestos and organic solvents, than the general population. This occupational exposure was more frequent in men and in patients with PLA2R1 epitope spreading. CLINICAL TRIAL REGISTRY NAME AND REGISTRATION NUMBER: Immunopathological Analysis in a French National Cohort of Membranous Nephropathy (IHMN), NCT04326218. PODCAST: This article contains a podcast at https://www.asn-online.org/media/podcast/CJASN/2022_10_25_CJN02930322.mp3.

Air pollution exposure induces a decrease in type II interferon response: A paired cohort study.

BACKGROUND: While air pollution is a major issue due to its harmful effects on human health, few studies focus on its impact on the immune system and vulnerability to viral infections. The lockdown declared following the COVID-19 pandemic represents a unique opportunity to study the large-scale impact of variations in air pollutants in real life. We hypothesized that variations in air pollutants modify Th1 response represented by interferon (IFN) γ production. METHODS: We conducted a single center paired pilot cohort study of 58 participants, and a confirmation cohort of 320 participants in Nice (France), with for each cohort two samplings at six months intervals. We correlated the variations in the production of IFNγ after non-specific stimulation of participants' immune cells with variations in key regulated pollutants: NO2, O3, PM2.5, and PM10 and climate variables. Using linear regression, we studied the effects of variations of each pollutant on the immune response. FINDINGS: In the pilot cohort, IFNγ production significantly decreased by 25.7% post-lockdown compared to during lockdown, while NO2 increased significantly by 46.0%. After the adjustment for climate variations during the study period (sunshine and temperature), we observed a significant effect of NO2 variation on IFNγ production (P=0.03). In the confirmation cohort IFNγ decreased significantly by 47.8% and after adjustment for environmental factors and intrinsic characteristics we observed a significant effect of environmental factors: NO2, PM10, O3, climatic conditions (sunshine exposure, relative humidity) on variation in IFNγ production (P=0.005, P<0.001, P=0.001, P=0.002 and P<0.001 respectively) but not independently from the BMI at inclusion and the workplace P=0.007 and P<0.001 respectively). INTERPRETATION: We show a weakening of the antiviral cellular response in correlation with an increase of pollutants exposition. FUNDING: Agence Nationale de la Recherche, Conseil Départemental des Alpes-Maritimes and Region Sud.

Advances in the Management of Primary Membranous Nephropathy and Rituximab-Refractory Membranous Nephropathy.

Primary membranous nephropathy (pMN) is an auto-immune disease characterized by auto-antibodies targeting podocyte antigens resulting in activation of complement and damage to the glomerular basement membrane. pMN is the most common cause of nephrotic syndrome in adults without diabetes. Despite a very heterogeneous course of the disease, the treatment of pMN has for many years been based on uniform management of all patients regardless of the severity of the disease. The identification of prognostic markers has radically changed the vision of pMN and allowed KDIGO guidelines to evolve in 2021 towards a more personalized management based on the assessment of the risk of progressive loss of kidney function. The recognition of pMN as an antibody-mediated autoimmune disease has rationalized the use immunosuppressive drugs such as rituximab. Rituximab is now a first line immunosuppressive therapy for patients with pMN with proven safety and efficacy achieving remission in 60-80% of patients. For the remaining 20-40% of patients, several mechanisms may explain rituximab resistance: (i) decreased rituximab bioavailability; (ii) immunization against rituximab; and (iii) chronic glomerular damage. The treatment of patients with rituximab-refractory pMN remains controversial and challenging. In this review, we provide an overview of recent advances in the management of pMN (according to the KDIGO 2021 guidelines), in the understanding of the pathophysiology of rituximab resistance, and in the management of rituximab-refractory pMN. We propose a treatment decision aid based on immunomonitoring to identify failures related to underdosing or immunization against rituximab to overcome treatment resistance.

Kappa Free Light Chains, Soluble Interleukin-2 Receptor, and Interleukin-6 Help Explore Patients Presenting With Brain White Matter Hyperintensities.

Introduction: Many patients are referred to multiple sclerosis (MS) tertiary centers to manage brain white matter hyperintensities (WMH). Multiple diagnoses can match in such situations, and we lack proper tools to diagnose complex cases. Objective: This study aimed to prospectively analyze and correlate with the final diagnosis, cerebrospinal fluid (CSF) interleukin (IL)-1ß, soluble IL-2 receptor (CD25), IL-6, IL-10, and kappa free light chains (KFLC) concentrations in patients presenting with brain WMH. Methods: All patients over 18 years addressed to our MS tertiary center for the diagnostic workup of brain WMH were included from June 1, 2020, to June 1, 2021. Patients were separated into three groups-MS and related disorder (MSARD), other inflammatory neurological disorder (OIND), and non-inflammatory neurological disorder (NIND) groups-according to clinical presentation, MRI characteristics, and biological workup. Results: A total of 176 patients (129 women, mean age 45.8 ± 14.7 years) were included. The diagnosis was MSARD (n = 88), OIND (n = 35), and NIND (n = 53). Median CSF KFLC index and KFLC intrathecal fraction (IF) were higher in MSARD than in the OIND and NIND groups; p < 0.001 for all comparisons. CSF CD25 and IL-6 concentrations were higher in the OIND group than in both the MSARD and NIND groups; p < 0.001 for all comparisons. KFLC index could rule in MSARD when compared to NIND (sensitivity, 0.76; specificity, 0.91) or OIND (sensitivity, 0.73; specificity, 0.76). These results were similar to those with oligoclonal bands (sensitivity, 0.59; specificity, 0.98 compared to NIND; sensitivity, 0.59; specificity, 0.88 compared to OIND). In contrast, elevated CSF CD25 and IL-6 could rule out MSARD when compared to OIND (sensitivity, 0.58 and 0.88; specificity, 0.95 and 0.74, respectively). Discussion: Our results show that, as OCBs, KFLC biomarkers are helpful tools to rule in MSARD, whereas elevated CSF CD25 and IL-6 rule out MSARD. Interestingly, CSF IL-6 concentration could help identify neuromyelitis optica spectrum disorder, myelin oligodendrocyte glycoprotein antibody-associated disease, and central nervous system (CNS) vasculitis. These results need to be confirmed within more extensive and multicentric studies. Still, they sustain that KFLC, CSF CD25, and CSF IL-6 could be reliable biomarkers in brain WMH diagnostic workup for differentiating MSARD from other brain inflammatory MS mimickers.

Impact of Isolation Time of COVID-19 Patients in Intensive Care Unit on Healthcare Workers Contamination and Nursing Care Intensity.

Background: The optimal isolation time of COVID-19 patients in intensive care unit (ICU) is debated. We investigated the impact of two different COVID-19 patient isolation time strategies on healthcare workers (HCW) contamination, intensity of nursing care and potential associated adverse events. Methods: We prospectively included all consecutive COVID-19 patients and HCW in our ICU in the first two pandemic waves (March to May 2020 and August to November 2020). Specific isolation measures for COVID-19 patients were released after two negative RT-PCR assays in the first wave and 14 days after the onset of symptoms in the second wave. Contamination of HCW was assessed at the end of each pandemic wave by combining both a RT-PCR assay and a serological test. Results: Overall, 117 COVID-19 patients and 73 HCW were included. Despite an earlier release from isolation after ICU admission in the second than in the first wave [6 (4-8) vs. 15 (11-19) days, p < 0.01], the proportion of HCW with a positive serological test (16 vs. 17%, p = 0.94) or with a positive RT-PCR assay (3 vs. 5%, p = 0.58) was not different between the two waves. Although a lower nurse-to-bed ratio, the intensity of nursing care was higher in the second than in the first wave. A longer isolation time was associated with accidental extubation (OR = 1.18, 95%CI:1.07-1.35, p = 0.005) but neither with ventilator-associated pneumonia nor with dysglycemia. Conclusion: A shorter isolation time of COVID-19 patients in ICU was not associated with higher HCW contamination, while a longer isolation time seemed to be associated with higher accidental extubation.

Humoral and cellular responses after a third dose of SARS-CoV-2 BNT162b2 vaccine in patients with lymphoid malignancies.

Patients with hematological malignancies have impaired immune response after two doses of BNT162b2 (Pfizer/BioNTech) vaccine against SARS-CoV-2. Here, in this observational study (registration number HDH F20210324145532), we measure SARS-CoV-2 anti-Spike antibodies, neutralizing antibodies and T-cell responses after immune stimulation with a third dose (D3) of the same vaccine in patients with chronic lymphocytic leukemia (n = 13), B cell non-Hodgkin lymphoma (n = 14), and multiple myeloma (n = 16)). No unexpected novel side effects are reported. Among 25 patients with positive anti-S titers before D3, 23 (92%) patients increase their anti-S and neutralizing antibody titer after D3. All 18 (42%) initially seronegative patients remain negative. D3 increases the median IFN-γ secretion in the whole cohort and induces IFN-γ secretion in a fraction of seronegative patients. Our data thus support the use of a third vaccine dose amongst patients with lymphoid malignancies, even though some of them will still have vaccine failure.

Rituximab Immunomonitoring Predicts Remission in Membranous Nephropathy.

Primary membranous nephropathy (pMN) is an autoimmune kidney disease and a common cause of nephrotic syndrome in adults. Rituximab is becoming a first line therapy for patients with persistent nephrotic syndrome with proven safety and efficacy, achieving remission in 60%-80% of cases. For the remaining 20%-40% of patients there is an urgent need to identify early biomarkers of resistance to rituximab to adapt therapeutic management. In nephrotic patients, rituximab is found in the blood more transiently than in other autoimmune diseases without proteinuria, due to rituximab wasting in the urine. However, rituximab immunomonitoring is not routinely performed. We evaluated the predictive value of serum rituximab levels in patients with pMN three months after rituximab injection (month-3) on clinical remission rates six months (month-6) and 12 months (month-12) after injection and investigated predictive factors for serum rituximab levels at month-3. Sixty-eight patients treated with rituximab between July 2015 and January 2020 from two French nephrology centers were included. We identified residual rituximab levels at month-3 as a novel early predictor of remission at month-6 (p <0.0001) and month-12 (p = 0.001). Reduced likelihood of remission in patients with undetectable rituximab at month-3 was associated with lower serum albumin and higher anti-PLA2R1 titers at baseline and with lower serum albumin, higher proteinuria, higher CD19+ counts and higher anti-PLA2R1 titers during follow-up. In multivariate analysis, high baseline proteinuria and undetectable rituximab levels at month-3 were independent risk factors for treatment failure at month-6 and high baseline weight and undetectable rituximab levels at month-3 were independent risk factors for treatment failure at month-12. We identified serum albumin at baseline as a predictive factor for serum rituximab levels at month-3. Patients with serum albumin below 22.5 g/L at baseline had an 8.66-fold higher risk of having undetectable rituximab levels at month-3. Therefore, rituximab immunomonitoring in pMN patients treated with rituximab would allow the detection of patients at risk of treatment failure as early as month-3. Studies are needed to assess whether patients with low residual rituximab levels at month-3 may benefit from an early additional course of rituximab.

Baseline and early functional immune response is associated with subsequent clinical outcomes of PD-1 inhibition therapy in metastatic melanoma patients.

BACKGROUND: Despite significant progress with antiprogrammed cell death protein 1 (PD-1) therapy, a substantial fraction of metastatic melanoma patients show upfront therapy resistance. Biomarkers for outcome are missing and the association of baseline immune function and clinical outcome remains to be determined. We assessed the in vitro nonspecific stimulation of immune response at baseline and during anti-PD-1 therapy for metastatic melanoma. METHODS: Previously untreated metastatic melanoma patients received nivolumab and radiotherapy as part of the multicentric phase II trial NIRVANA (NCT02799901). The levels of Th1, Th2 and Th17 cytokines on in vitro non-specific stimulation of innate and adaptive immune cells were measured in patient sera before treatment, and at week 2 and week 6 after the beginning of the treatment, and correlated with tumorous response, progression-free survival (PFS) and occurrence of immune-related adverse events (irAEs). The results in melanoma patients were compared with those of a cohort of 9 sex and age-matched healthy donors. RESULTS: Seventeen patients were enrolled in this ancillary study. Median follow-up was 16 months (2.2-28.4). The 12-month PFS rate was 67.7%. The incidence of irAEs of any grade was 58.8%. Without in vitro stimulation no differences in cytokines levels were observed between responders and non-responders. On in vitro stimulation, metastatic patients had lower Th1 cytokine levels than healthy donors at baseline for tumor necrosis factor-α and interferon-γ (IFN-γ) (1136 pg/mL vs 5558 pg/mL, p<0.0001; and 3894 pg/mL vs 17 129 pg/mL, p=0.02, respectively). Responders exhibited increasing cytokine levels from baseline to week 6. Non-responders had lower interleukin 17A (IL-17A) levels at baseline than responders (7 pg/mL vs 32 pg/mL, p=0.03), and lower IFN-γ levels at week 6 (3.3 ng/mL vs 14.5 ng/mL, p=0.03). A lower level of IL-17A at week 2 and a lower level of IFN-γ at week 6 correlated with worse PFS (p=0.04 and p=0.04 respectively). At baseline, patients who developed irAEs had higher IL-6 levels (19.3 ng/mL vs 9.2 ng/mL, p=0.03) and higher IL-17A levels (52.5 pg/mL vs 2.5 pg/mL, p=0.009) than those without irAEs. CONCLUSIONS: Our findings indicate that cytokine levels after in vitro non-specific stimulation could be a promising biomarker to predict the outcome of PD-1 inhibition therapy.