Trimodal single-cell profiling reveals a novel pediatric CD8αα+ T cell subset and broad age-related molecular reprogramming across the T cell compartment.

Age-associated changes in the T cell compartment are well described. However, limitations of current single-modal or bimodal single-cell assays, including flow cytometry, RNA-seq (RNA sequencing) and CITE-seq (cellular indexing of transcriptomes and epitopes by sequencing), have restricted our ability to deconvolve more complex cellular and molecular changes. Here, we profile >300,000 single T cells from healthy children (aged 11-13 years) and older adults (aged 55-65 years) by using the trimodal assay TEA-seq (single-cell analysis of mRNA transcripts, surface protein epitopes and chromatin accessibility), which revealed that molecular programming of T cell subsets shifts toward a more activated basal state with age. Naive CD4+ T cells, considered relatively resistant to aging, exhibited pronounced transcriptional and epigenetic reprogramming. Moreover, we discovered a novel CD8αα+ T cell subset lost with age that is epigenetically poised for rapid effector responses and has distinct inhibitory, costimulatory and tissue-homing properties. Together, these data reveal new insights into age-associated changes in the T cell compartment that may contribute to differential immune responses.

Persistent serum protein signatures define an inflammatory subcategory of long COVID.

Long COVID or post-acute sequelae of SARS-CoV-2 (PASC) is a clinical syndrome featuring diverse symptoms that can persist for months following acute SARS-CoV-2 infection. The aetiologies may include persistent inflammation, unresolved tissue damage or delayed clearance of viral protein or RNA, but the biological differences they represent are not fully understood. Here we evaluate the serum proteome in samples, longitudinally collected from 55 PASC individuals with symptoms lasting ≥60 days after onset of acute infection, in comparison to samples from symptomatically recovered SARS-CoV-2 infected and uninfected individuals. Our analysis indicates heterogeneity in PASC and identified subsets with distinct signatures of persistent inflammation. Type II interferon signaling and canonical NF-κB signaling (particularly associated with TNF), appear to be the most differentially enriched signaling pathways, distinguishing a group of patients characterized also by a persistent neutrophil activation signature. These findings help to clarify biological diversity within PASC, identify participants with molecular evidence of persistent inflammation, and highlight dominant pathways that may have diagnostic or therapeutic relevance, including a protein panel that we propose as having diagnostic utility for differentiating inflammatory and non-inflammatory PASC.

Patients With Low Drug Levels or Antibodies to a Prior Anti-Tumor Necrosis Factor Are More Likely to Develop Antibodies to a Subsequent Anti-Tumor Necrosis Factor.

Therapeutic drug monitoring (TDM) with measurement of serum drug and antidrug antibodies (ADAb) is used widely to confirm therapeutic exposure, rule out immunogenicity, and optimize treatment of biologics in patients with inflammatory bowel diseases.1 A recent genome-wide association study found the variant HLA-DQA1∗05 to increase the risk of development of antibodies against infliximab (IFX) and adalimumab (ADM) 2-fold, regardless of concomitant immunomodulator use.2,3 However, there is currently limited evidence showing whether patients who develop antibodies to 1 anti-tumor necrosis factor (TNF) are prone to develop antibodies to the subsequent anti-TNF. Our aim was to investigate the risk of subsequent antibody development in cases (with ADAb to prior anti-TNF) versus control subjects (without ADAb to prior anti-TNF) using a large cohort of patients with inflammatory bowel diseases who underwent TDM with a drug-tolerant assay.

Longitudinal immune dynamics of mild COVID-19 define signatures of recovery and persistence.

SARS-CoV-2 has infected over 200 million and caused more than 4 million deaths to date. Most individuals (>80%) have mild symptoms and recover in the outpatient setting, but detailed studies of immune responses have focused primarily on moderate to severe COVID-19. We deeply profiled the longitudinal immune response in individuals with mild COVID-19 beginning with early time points post-infection (1-15 days) and proceeding through convalescence to >100 days after symptom onset. We correlated data from single cell analyses of peripheral blood cells, serum proteomics, virus-specific cellular and humoral immune responses, and clinical metadata. Acute infection was characterized by vigorous coordinated innate and adaptive immune activation that differed in character by age (young vs. old). We then characterized signals associated with recovery and convalescence to define and validate a new signature of inflammatory cytokines, gene expression, and chromatin accessibility that persists in individuals with post-acute sequelae of SARS-CoV-2 infection (PASC).

Noninvasive Targeted Crohn Disease Management by Combining Endoscopic Healing Index and Therapeutic Drug Monitoring.

Background and Aims: Therapeutic drug monitoring (TDM) with measurement of serum drug and antidrug antibody concentrations is used to optimize tumor necrosis factor antagonists (anti-TNF). The endoscopic healing index (EHI) is a validated serum-based assay to measure mucosal inflammation in adults with Crohn disease (CD). Our objectives were to evaluate the relationship between EHI and TDM results and to determine the anti-TNF concentration range associated with EHI <20 (consistent with endoscopic remission). Methods: Adult and pediatric patients with CD (N = 1731) were selected retrospectively from a clinical laboratory cohort. Patients were selected if they had an ICD-10 code for CD and if results for EHI and TDM were available within 30 days of each other. The relationship between EHI and TDM results was examined and the anti-TNF concentration range associated with EHI <20 vs >50 was evaluated. Results: Median anti-TNF concentration was higher in patients with EHI <20 vs >50 for infliximab (N = 796): 11.1 vs 3.4 µg/mL and for adalimumab (N = 935): 9.2 vs 5.0 µg/mL (P < 0.0001 both drugs). Patients with antibodies to infliximab (12.8%) or adalimumab (14.9%) had lower anti-TNF concentrations (P < 0.001 both drugs) and higher EHI (P < 0.01 both drugs). The concentration range for infliximab: 5-15 µg/mL (5-9 µg/mL in pediatric patients) and for adalimumab: 5-10 µg/mL (8 µg/mL in pediatric patients) best discriminated EHI <20 vs >50. Conclusions: We report the anti-TNF concentration range associated with EHI <20. Combined testing of EHI and TDM is proposed as a noninvasive approach for treat-to-target management which could improve the ability to monitor disease and optimize anti-TNF therapy.

Serum Ustekinumab Concentrations Are Associated With Remission in Crohn's Disease Defined by a Serum-Based Endoscopic Healing Index.

Background: Optimal ustekinumab levels (UST) in Crohn disease (CD) treatment have not been defined. We set out to define the optimal UST to differentiate between remission and active CD, as defined using the serum-based endoscopic healing index (EHI). Methods: Paired serum UST and EHI tests were analyzed. Remission was defined as EHI <20. Active disease was defined as EHI ≥50. The proportion of patients in remission was compared across UST quartiles. UST in subjects with EHI <20 and EHI ≥50 were compared. An area under receiver operating characteristic curve was generated to identify an optimal UST to differentiate between active disease and remission. Results: A total of 337 unique patients were identified; median UST and EHI were 5.0 µg/mL [interquartile range (IQR) 2.7-9.1] and 37 (IQR 26-53), respectively. EHI <20 (remission) was found in 57 (16.9%) patients. EHI ≥50 (active disease) was found in 97 (28.8%) patients. Higher proportions of subjects were in remission for increasing UST quartiles, P = 0.01. Median UST in patients with EHI <20 and EHI ≥50 were 7.5 µg/mL (IQR 4.6-10.9) and 3.1 µg/mL (IQR 1.8-6.6), respectively, P < 0.001. An UST threshold of 3.75 µg/mL optimally differentiated between active disease and remission (area under the curve 0.725). UST levels >3.75 µg/mL were associated with a lower proportion of subjects with active disease (EHI ≥50; 18.9%) compared with UST levels ≤3.75 µg/mL (45.6%, P < 0.001). Conclusions: Using the EHI, we identified a threshold UST level of 3.75 µg/mL to optimally differentiate between active and quiescent CD. These data suggest that UST serum concentrations of >3.75 µg/mL are optimally associated with endoscopic remission in CD.

Association Between Vedolizumab Levels, Anti-vedolizumab Antibodies, and Endoscopic Healing Index in a Large Population of Patients with Inflammatory Bowel Diseases.

BACKGROUND: The aim of this study was to assess the relationship between serum vedolizumab (VDZ) concentrations and antibodies to VDZ (ATV) in a large cohort of patients with inflammatory bowel diseases. Furthermore, we evaluated the association between serum VDZ concentrations and a novel serum-based biomarker panel designated as the endoscopic healing index (EHI), developed and validated for identifying mucosal inflammation in patients with Crohn's disease (CD). METHODS: Retrospective study where results from patient samples submitted to a commercial clinical laboratory were included. Serum VDZ and ATV levels were analyzed using a drug-tolerant assay. In CD patients for whom both VDZ and EHI were available, VDZ concentrations were correlated with EHI. serum VDZ threshold analysis was performed using ROC curves, and the serum VDZ concentrations that best differentiated EHI < 20 (previously associated with endoscopic remission) were chosen. RESULTS: A total of 9356 patients were included in the VDZ/ATV analysis. Detectable ATV was observed in 2.9% patients with significantly lower serum VDZ concentrations when compared to those with undetectable ATV [3.9 µg/mL (0-9.0) vs. 11.3 µg/mL (5.9-20.6), p < 0.0001]. Of the patients with serum VDZ result, 287 patients had a concomitant EHI test. An inverse correlation was observed between VDZ concentration and EHI (rho = - 0.20, p < 0.001). A serum VDZ concentration ≥ 15.7 µg/ml was best correlated wi th an EHI < 20 [AUROC: 0.67 (95% CI 0.57-0.77)]. CONCLUSIONS: Incidence of ATVs was low, but significantly associated with lower VDZ levels. A serum VDZ concentration threshold of ≥ 15.7 µg/ml was associated with endoscopic remission as defined by an EHI < 20.

Immunogenicity of Tumor Necrosis Factor Antagonists and Effect of Dose Escalation on Anti-Drug Antibodies and Serum Drug Concentrations in Inflammatory Bowel Disease.

BACKGROUND: Infliximab and adalimumab concentrations are associated with important outcomes in inflammatory bowel disease (IBD). Antibodies to infliximab (ATI) and adalimumab (ATA) are associated with reduced drug concentrations and worse outcomes. Because the efficacy of dose escalation to overcome antibodies is unclear, we assessed the impact of this strategy to overcome immunogenicity in IBD. METHODS: Infliximab and adalimumab dosing, drug, and antibody concentrations were extracted from a database of patients with IBD having specimens collected for therapeutic drug monitoring. The primary outcome compared proportions with either infliximab ≥5 µg/mL or adalimumab ≥7.5 µg/mL and undetectable antibodies between dose-escalated and non-escalated patients. Area under the receiver operating characteristic curve analyses determined antibody concentrations below which dose escalation was associated with the primary outcome. RESULTS: The study included 63,176 patients treated with infliximab and 46,429 patients treated with adalimumab. We detected ATI and ATA in 23.6% (n = 14,900) of patients treated with infliximab and 19.6% (n = 9101) of patients treated with adalimumab. In patients with ATI, infliximab dose escalation (n = 453) yielded higher proportions achieving the primary outcome (47.5% vs 30.9%; P < 0.001), greater drug concentration increases (5.9 µg/mL vs 0.2 µg/mL; P < 0.001), and ATI reductions (4.3 U/mL vs 1.9 U/mL; P = 0.002) compared to no escalation (n = 204). An ATI threshold of 8.55 U/mL was associated with achieving the primary outcome with dose escalation (area under the curve = 0.66). For patients with ATI ≤8.55 U/mL (n = 274), higher proportions (59.1% vs 29.6%; P < 0.001) achieved the primary outcome compared with those with ATI >8.55 U/mL (n = 179). No patients treated with adalimumab achieved the primary outcome (0/390), regardless of dose escalation (n = 87). CONCLUSION: Dose escalation increased drug concentrations and eliminated antibodies with infliximab but not adalimumab. Initial ATI ≤8.55 U/mL was associated with increased efficacy of dose escalation using this assay.

Development and Validation of a Test to Monitor Endoscopic Activity in Patients With Crohn's Disease Based on Serum Levels of Proteins.

BACKGROUND & AIMS: Noninvasive tests to measure endoscopic activity in patients with Crohn's disease (CD) have limitations. We aimed to develop a test to identify patients in remission, based on endoscopic analysis, and monitor CD activity based on serum levels of proteins. METHODS: We developed a test to measure 13 proteins in blood (ANG1, ANG2, CRP, SAA1, IL7, EMMPRIN, MMP1, MMP2, MMP3, MMP9, TGFA, CEACAM1, and VCAM1), called the endoscopic healing index [EHI], using samples from 278 patients with CD from a multinational training cohort. We validated the test using 2 independent cohorts of patients with CD: 116 biologic-naive patients with early-stage CD (validation cohort 1) and 195 biologic-exposed patients with chronic CD (validation cohort 2). The ability of the test to identify patients with active disease vs patients in remission (defined as a simple endoscopic score for CD of ≤2 and ≤1 in each segment, or a total CD endoscopic index of severity score <3) was assessed by using area under receiver operating characteristic curve (AUROC) analysis. The diagnostic accuracy of the test was compared with that of measurement of serum C-reactive protein (CRP) and fecal calprotectin. RESULTS: The EHI scores range from 0 to 100 units; higher scores indicate more severe CD activity, based on endoscopy findings. The EHI identified patients in remission with an AUROC of 0.962 in validation cohort 1 (95% confidence interval, 0.942-0.982) and an AUROC of 0.693 in validation cohort 2 (95% confidence interval, 0.619-0.767), regardless of CD location or phenotype. A cutoff value of 20 points identified patients in remission with the highest level of sensitivity (97.1% in validation cohort 1 and 83.2% in validation cohort 2), with specificity values of 69.0% and 36.6%, respectively. A cutoff value of 50 points identified patients in remission with the highest level of specificity (100% in validation cohort 1 and 87.8% in validation cohort 2), with sensitivity values of 37.3% and 30.0%, respectively. The EHI identified patients in remission with a significantly higher AUROC value than the test for CRP (0.876, P < .001 in validation cohort 1 and 0.624, P = .109 in validation cohort 2). In analysis of patients with available FC measurements, the AUROC value for the EHI did not differ significantly from that of measurement of FC (AUROC, 0.950 for EHI vs AUROC, 0.923 for FC; P = .147 in validation cohort 1 and AUROC, 0.803 for EHI vs AUROC, 0.854 for FC; P = .298 in validation cohort 2). CONCLUSIONS: We developed an index called the EHI to identify patients with CD in endoscopic remission based on blood levels of 13 proteins. The EHI identified patients with resolution of endoscopic disease activity, with good overall accuracy, although with variation between the 2 cohorts assessed. The EHI AUROC values were comparable to measurement of FC and higher than measurement of serum CRP. The test might be used in practice to assess endoscopic activity in patients with CD.

Validation of Serum Test for Advanced Liver Fibrosis in Patients With Nonalcoholic Steatohepatitis.

BACKGROUND & AIMS: We analyzed markers of fibrosis in serum samples from patients with nonalcoholic fatty liver disease (NAFLD), assessed by liver biopsy. We used serum levels of markers to develop an algorithm to discriminate patients with advanced fibrosis from those with mild or moderate fibrosis and validated its performance in 2 independent cohorts of patients with NAFLD. METHODS: We performed a retrospective analysis of serum samples from 396 patients with NAFLD and different stages of fibrosis (F0-F4), collected from 2007 through 2017 on the day of liver biopsy (training cohort 1). We measured serum concentrations of alpha-2 macroglobulin (A2M), hyaluronic acid (HA), and TIMP metallopeptidase inhibitor 1 (TIMP1), and used measurements to develop an algorithm that could discriminate patients with NAFLD with advanced fibrosis (F3-F4; 24.1% of cohort) from those with mild or moderate fibrosis (F0-F2; 79.5% of cohort). We validated the algorithm using serum samples collected from a separate 396 patients from the same time period and location (validation cohort 1), as well as 244 patients with NAFLD evaluated at a separate location, from 2011 through 2017, within a median of 11 days of liver biopsy (cohort 2). RESULTS: The algorithm identified patients with advanced fibrosis vs mild or moderate fibrosis in training cohort 1 with an area under the receiver operating characteristic (AUROC) curve of 0.867 (95% CI, 0.827-0.907), 84.8% sensitivity (95% CI, 75.5%-91.0%), and 72.3% specificity (95% CI, 66.9%-77.3%), at a cutoff score of 17. The AUROC for the combined validation cohorts 1 and 2 (n=640) was 0.856 (95% CI, 0.820-0.892), identifying patients with 79.7% sensitivity (95% CI, 71.9%-86.2%) and 75.7% specificity (95% CI, 71.8%-79.4%) at the predetermined cutoff score of 17. The algorithm had negative predictive values that ranged from 92.5% to 94.7% in the validation cohorts; it correctly classified 90.0% of F0 samples, 75.0% of F1 samples, 77.4% of F3 samples, and 94.4% of F4 samples. CONCLUSION: We developed an algorithm that identifies patients with advanced fibrosis from those with mild to moderate fibrosis in patients with NAFLD with an AUROC value of approximately 0.86, based on levels of serum biomarkers. We validated the findings in 2 separate sets of patients with biopsy-proven NAFLD. The algorithm can be used non-invasively to determine risk of advanced fibrosis in patients with NAFLD.