Irritable bowel syndrome (IBS) is a gut‐brain disorder in which symptoms are shaped by serotonin acting centrally and peripherally. The serotonin transporter gene SLC6A4 has been implicated in IBS ...pathophysiology, but the underlying genetic mechanisms remain unclear. We sequenced the alternative P2 promoter driving intestinal SLC6A4 expression and identified single nucleotide polymorphisms (SNPs) that were associated with IBS in a discovery sample. Identified SNPs built different haplotypes, and the tagging SNP rs2020938 seems to associate with constipation‐predominant IBS (IBS‐C) in females. rs2020938 validation was performed in 1978 additional IBS patients and 6,038 controls from eight countries. Meta‐analysis on data from 2,175 IBS patients and 6,128 controls confirmed the association with female IBS‐C. Expression analyses revealed that the P2 promoter drives SLC6A4 expression primarily in the small intestine. Gene reporter assays showed a functional impact of SNPs in the P2 region. In silico analysis of the polymorphic promoter indicated differential expression regulation. Further follow‐up revealed that the major allele of the tagging SNP rs2020938 correlates with differential SLC6A4 expression in the jejunum and with stool consistency, indicating functional relevance. Our data consolidate rs2020938 as a functional SNP associated with IBS‐C risk in females, underlining the relevance of SLC6A4 in IBS pathogenesis.
Introduction: The crosstalk of the gut microbiome and the human host can influence T-cell immune responses and has emerged as a modulator in cancer immunotherapy (Gopalakrishnan et al., 2018). ...Individual bacteria isolated from human fecal specimen were also shown to shape systemic and gut mucosal T cell repertoires (Geva-Zatorsky et al., 2017), and several commensal members of the human gut microbiome were recently associated with the kinetics of the reconstitution of peripheral immune cells after allo-HCT (Schluter et al., 2020). However, in patients treated with CAR T-cells a link between gut microbiome configurations and T-cell characteristics has not been described yet. Recently, we and others could associate low CAR T-cell expansion and dysfunction with treatment failure. Here, we hypothesize that certain gut microbes or even intestinal monodomination of facultative pathogens may correlate with CAR T-cell expansion and expression profile of immune checkpoint molecules which might impact treatment outcome.
Methods: Patients with relapsed / refractory Diffuse-Large B-Cell Lymphoma (DLBCL) were treated with the CD19 specific CAR T-cell products Axicabtagene-Ciloleucel or Tisagenlecleucel at our institution from April 2019 to May 2021. Within this time-period, peripheral blood and fecal biospecimens from 23 patients were collected sequentially before, during and after CAR T-cell transfusion (specific time points: before lymphodepleting chemotherapy, day of CAR T-cell transfusion, day 7, day 14). CAR T-cells and the expression profile of immune checkpoint molecules (PD-1, TIM-3, LAG-3, 2B4) were studied by multiparameter flow cytometry. CAR T-cell peak expansion was assessed relatively (% CAR + T-cells of CD3+ T-cells) and absolutely (/ul). In addition, effector : target (E:T) ratios were estimated as absolute peak expansion of CAR T-cells (/ul) per tumor volume (as sum of the product of diameters based on Lugano criteria with up to 6 target lesions in cm 3). 16S rRNA gene sequencing was performed on 83 stool samples. Responder (R, complete or partial remission) were retrospectively assessed compared to Non-Responder (NR, stable or progressive disease) according to response assessment with PET-CT three months after CAR T-cell transfusion.
Results: Higher alpha diversity (i.e., within-sample diversity) prior to CAR T-cell transfusion correlated positively with a favorable E:T ratio (CAR peak expansion/ul : tumor volume in cm 3, p=0.04, r=0.608; Fig. 1). Interestingly, the abundances of certain taxa prior to CAR T-cell transfusion correlated with the expansion of CAR T-cells in vivo. In particular we found a positive association with Pediococcus (p=0.018, r=0.751, FDR corrected) and with Anaerovoracaceae (E:T ratio, p=0.027, r=0.84, FDR corrected). Next, we studied the expression profile of immune checkpoint molecules on CAR T-cells in the context of microbial taxa in R vs NR patients. Notably, we observed a positive association between the frequency of CD8+ CAR T-cells devoid of inhibitory molecules (PD-1, TIM-3, LAG-3 and 2B4) and the relative abundance of the intestinal microbial genus Dorea (p=0.039, r=0.416: see Fig. 1). Furthermore, we found a higher relative abundance of Dorea (p=0.043) prior to transfusion in R compared to NR patients as well as significant positive associations of Dorea (p=0.0.008, r=0.54, not FDR corrected) and Pediococcus (p=0.047, r=0.462, not FDR corrected) with progression free survival .
Conclusion: For the first time, we describe associations between the compositional changes of the gut microbiome and kinetics as well as immune characteristics of CAR T-cell therapy in patients with relapsed / refractory DLBCL. In the future, our findings need to be validated in a larger patient cohort and might serve as a predictive biomarker for microbiome-based patient assessment and microbiome-targeted therapeutic approaches to identify benefiting patients and to improve outcome.
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Blumenberg: Kite/Gilead: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; BMS/Celgene: Research Funding; Janssen: Research Funding. von Bergwelt: MSD Sharpe & Dohme: Honoraria, Research Funding, Speakers Bureau; Novartis: Honoraria, Research Funding, Speakers Bureau; Roche: Honoraria, Research Funding, Speakers Bureau; Kite/Gilead: Honoraria, Research Funding, Speakers Bureau; BMS: Honoraria, Research Funding, Speakers Bureau; Astellas: Honoraria, Research Funding, Speakers Bureau; Mologen: Honoraria, Research Funding, Speakers Bureau; Miltenyi: Honoraria, Research Funding, Speakers Bureau. Buecklein: Amgen: Consultancy, Honoraria; BMS / Celgene: Consultancy, Research Funding; Kite / Gilead: Consultancy, Honoraria, Other: Congress and travel support , Research Funding; Janssen: Consultancy; Miltenyi: Research Funding; Novartis: Consultancy, Other: Congress and travel support , Research Funding, Speakers Bureau; Pfizer: Consultancy, Honoraria, Speakers Bureau. Subklewe: Klinikum der Universität München: Current Employment; Takeda: Speakers Bureau; BMS/Celgene: Consultancy, Research Funding, Speakers Bureau; Gilead: Consultancy, Research Funding, Speakers Bureau; MorphoSys: Research Funding; Novartis: Consultancy, Research Funding, Speakers Bureau; Roche: Research Funding; Seattle Genetics: Consultancy, Research Funding; Janssen: Consultancy; Pfizer: Consultancy, Speakers Bureau; Miltenyi: Research Funding; Amgen: Consultancy, Research Funding, Speakers Bureau.
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Viktoria Blumenberg and Maria-Luisa Schubert contributed equally.
Introduction: The CD19 specific chimeric antigen receptor (CAR) T-cell products Axicabtagene-Ciloleucel and Tisagenlecleucel are ...approved for the treatment of refractory/relapsed B-cell precursor ALL (BCP-ALL) and Diffuse Large B-cell lymphoma (DLBCL). Despite high response rates, long term remission is only achieved in a subgroup if patients. In addition, CAR T cell therapy is accompanied by potentially severe immune related toxicities including cytokine release syndrome (CRS) or neurotoxicity (ICANS). Therefore, we need to identify biological mechanisms of treatment resistance and toxicity occurring in the host in addition to improve the CAR T product itself. The impact of the gut microbiome on T-cell based immunotherapies such as checkpoint inhibition or allogeneic hematopoietic stem cell transplant has been shown, but it´s role in mediating anti-tumor responses and the occurrence of immunotoxicities of CAR T-cell therapy has not yet been reported so far.
Methods: Patients with r/r BCP-ALL and DLBCL were treated with the commercially available CD19 specific CAR T-cell products Axicabtagene-Ciloleucel or Tisagenlecleucel or in-house manufactured CD19-targeted CD28-4-1BB-CD3ζ CAR T-cells at both our institutions. Fecal biospecimens from 33 patients were collected sequentially before, during and after CAR T transfusion (specific time points: before lymphodepleting chemotherapy, day of CAR T-cell transfusion and in 7 day intervals up to day 28). 16S rRNA sequencing and shotgun metagenome sequencing has been performed on 137 stool samples. Sequencing results and clinical metadata are integrated into a patient-centered “hospitalome” including infections and immunotoxicities as well as concomitant anti-infective, immunosuppressive agents and treatment response. Patients having received any type of anti-infective medication exceeding prophylaxis on the day of CAR T cell transfusion or up to two weeks prior were distinguished from patients without prior anti-infective medication or only receiving anti-infective drugs from day 1 after CAR T-cell transfusion.
Results: Patients receiving anti-infectives up to two weeks prior to CAR T-cell transfusion display a significantly lower response rate compared to patients, who have been treated with antibiotic and / or -mycotic treatment after day 0 (Tab. 1). Before CAR T-cell transfusion patients showed a heterogeneous, but largely diverse gut microbial taxa (Shannon index median: 4.2.). After CAR T-cell transfusion the alpha diversity (i.e. within-sample diversity) decreases with the nadir at day 14 (Shannon index, median 2.4), which depended significantly on broad-spectrum antibiotic administration (Fig. 1-2). Furthermore, loss of diversity correlated significantly with the occurrence of CRS (p<0.001, univariate GLM with Shannon diversity index vs. occurrence of CRS, irrespective of grade). Diversity loss was not associated with clinical response at day 90 or the development of ICANS. Age had no effect on microbiome changes and no inter-center effect was detected. Assessment of beta diversity (i.e. variation between samples) demonstrated compositional changes of the microbiome in CAR T-cell patients with approximately 25% of all samples displaying a mono-domination of the microbiome by Enterococcus spp. (i.e., rel. abundance > 30% per sample). The expansion of enterococci was again found in samples of patients, who received antibiotic treatment.
Conclusion: The gut microbiome of CAR T-cell patients undergoes large and diverse compositional changes, whereas altered diversity is significantly associated with administration of anti-infectives prior to CAR T-cell transfusion and the occurrence of CRS. The assessment of the gut microbial taxa of CAR T-cell patients might serve as a predictive biomarker for immunotoxicity and, eventually, treatment response.
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Blumenberg:Gilead: Consultancy, Research Funding; Novartis: Research Funding; Celgene: Research Funding. Schubert:Kite / Gilead: Consultancy; Takeda: Consultancy. Buecklein:Amgen: Consultancy; Pfizer: Consultancy; Celgene: Research Funding; Novartis: Research Funding; Gilead: Consultancy, Research Funding. Müller-Tidow:Pfizer: Research Funding, Speakers Bureau; Daiichi Sankyo: Research Funding; BiolineRx: Research Funding; Janssen-Cilag GmbH: Speakers Bureau. Dreger:Gilead: Consultancy, Speakers Bureau; AbbVie: Consultancy, Speakers Bureau; AstraZeneca: Consultancy; Roche: Consultancy, Speakers Bureau; Neovii: Research Funding; Riemser: Consultancy, Research Funding, Speakers Bureau; Janssen: Consultancy; Novartis: Consultancy, Speakers Bureau. Schmitt:Apogenix: Research Funding; Hexal: Other: Travel grants , Research Funding; Novartis: Other: educational activities and conferences, Research Funding; Kite: Other: Travel grants, educational activities and conferences; MSD: Membership on an entity’s Board of Directors or advisory committees, Other: PI of clinical trials on letermovir; TolerogenixX Ltd: Other: Co-Founder and shareholder. Subklewe:Roche AG: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; AMGEN: Consultancy, Honoraria, Research Funding; Janssen: Consultancy; Pfizer: Consultancy, Honoraria; Gilead Sciences: Consultancy, Honoraria, Research Funding; Seattle Genetics: Research Funding; Morphosys: Research Funding; Celgene: Consultancy, Honoraria.
The prevalence of metabolic-dysfunction-associated steatotic liver disease (MASLD) is alarmingly high; it is estimated to affect up to a quarter of the global population, making it the most common ...liver disorder worldwide. MASLD is characterized by excessive hepatic fat accumulation and is commonly associated with comorbidities such as obesity, dyslipidemia, and insulin resistance; however, it can also manifest in lean individuals. Therefore, it is crucial to develop effective therapies for this complex condition. Currently, there are no approved medications for MASLD treatment, so there is a pressing need to investigate alternative approaches. Extensive research has characterized MASLD as a multifaceted disease, frequently linked to metabolic disorders that stem from dietary habits. Evidence suggests that changes in the gut microbiome play a fundamental role in the development and progression of MASLD from simple steatosis to steatohepatitis and even hepatocellular carcinoma (HCC). In this review, we critically examine the literature on the emerging field of gut-microbiota-based therapies for MASLD and metabolic-dysfunction-associated steatohepatitis (MASH), including interventions such as fecal microbiota transplantation (FMT), probiotics, prebiotics, short-chain fatty acids, antibiotics, metabolic pathway targeting, and immune checkpoint kinase blockade.
Single-nucleotide polymorphisms (SNPs) of the serotonin type 3 receptor subunit (HTR3) genes have been associated with psychosomatic symptoms, but it is not clear whether these associations exist in ...irritable bowel syndrome (IBS).BACKGROUNDSingle-nucleotide polymorphisms (SNPs) of the serotonin type 3 receptor subunit (HTR3) genes have been associated with psychosomatic symptoms, but it is not clear whether these associations exist in irritable bowel syndrome (IBS).To assess the association of HTR3 polymorphisms with depressive, anxiety, and somatization symptoms in individuals with IBS.AIMTo assess the association of HTR3 polymorphisms with depressive, anxiety, and somatization symptoms in individuals with IBS.In this retrospective study, 623 participants with IBS were recruited from five specialty centers in Germany, Sweden, the United States, the United Kingdom, and Ireland. Depressive, anxiety, and somatization symptoms and sociodemographic characteristics were collected. Four functional SNPs - HTR3A c.-42C>T, HTR3B c.386A>C, HTR3C c.489C>A, and HTR3E c.*76G>A - were genotyped and analyzed using the dominant and recessive models. We also performed separate analyses for sex and IBS subtypes. SNP scores were calculated as the number of minor alleles of the SNPs above. The impact of HTR3C c.489C>A was tested by radioligand-binding and calcium influx assays.METHODSIn this retrospective study, 623 participants with IBS were recruited from five specialty centers in Germany, Sweden, the United States, the United Kingdom, and Ireland. Depressive, anxiety, and somatization symptoms and sociodemographic characteristics were collected. Four functional SNPs - HTR3A c.-42C>T, HTR3B c.386A>C, HTR3C c.489C>A, and HTR3E c.*76G>A - were genotyped and analyzed using the dominant and recessive models. We also performed separate analyses for sex and IBS subtypes. SNP scores were calculated as the number of minor alleles of the SNPs above. The impact of HTR3C c.489C>A was tested by radioligand-binding and calcium influx assays.Depressive and anxiety symptoms significantly worsened with increasing numbers of minor HTR3C c.489C>A alleles in the dominant model (F depressive = 7.475, P depressive = 0.006; F anxiety = 6.535, P anxiety = 0.011). A higher SNP score (range 0-6) was linked to a worsened depressive symptoms score (F = 7.710, P-linear trend = 0.006) in IBS. The potential relevance of the HTR3C SNP was corroborated, showing changes in the expression level of 5-HT3AC variant receptors.RESULTSDepressive and anxiety symptoms significantly worsened with increasing numbers of minor HTR3C c.489C>A alleles in the dominant model (F depressive = 7.475, P depressive = 0.006; F anxiety = 6.535, P anxiety = 0.011). A higher SNP score (range 0-6) was linked to a worsened depressive symptoms score (F = 7.710, P-linear trend = 0.006) in IBS. The potential relevance of the HTR3C SNP was corroborated, showing changes in the expression level of 5-HT3AC variant receptors.We have provided the first evidence that HTR3C c.489C>A is involved in depressive and anxiety symptoms in individuals with IBS. The SNP score indicated that an increasing number of minor alleles is linked to the worsening of depressive symptoms in IBS.CONCLUSIONWe have provided the first evidence that HTR3C c.489C>A is involved in depressive and anxiety symptoms in individuals with IBS. The SNP score indicated that an increasing number of minor alleles is linked to the worsening of depressive symptoms in IBS.
Irritable bowel syndrome (IBS) is a gut-brain disorder of multifactorial origin. Evidence of disturbed serotonergic function in IBS accumulated for the 5-HT
3
receptor family. 5-HT
3
Rs are encoded ...by
HTR3
genes and control GI function, and peristalsis and secretion, in particular. Moreover, 5-HT
3
R antagonists are beneficial in the treatment of diarrhea predominant IBS (IBS-D). We previously reported on functionally relevant SNPs in
HTR3A
c.-42C > T (rs1062613),
HTR3C
p.N163K (rs6766410), and
HTR3E
c.*76G > A (rs56109847 = rs62625044) being associated with IBS-D, and the
HTR3B
variant p.Y129S (rs1176744) was also described within the context of IBS. We performed a multi-center study to validate previous results and provide further evidence for the relevance of
HTR3
genes in IBS pathogenesis. Therefore, genotype data of 2682 IBS patients and 9650 controls from 14 cohorts (Chile, Germany (2), Greece, Ireland, Spain, Sweden (2), the UK (3), and the USA (3)) were taken into account. Subsequent meta-analysis confirmed
HTR3E
c.*76G > A (rs56109847 = rs62625044) to be associated with female IBS-D (OR = 1.58; 95% CI (1.18, 2.12)). Complementary expression studies of four GI regions (jejunum, ileum, colon, sigmoid colon) of 66 IBS patients and 42 controls revealed only
HTR3E
to be robustly expressed. On top,
HTR3E
transcript levels were significantly reduced in the sigma of IBS patients (
p
= 0.0187); more specifically, in those diagnosed with IBS-D (
p
= 0.0145). In conclusion, meta-analysis confirmed rs56109847 = rs62625044 as a risk factor for female IBS-D. Expression analysis revealed reduced
HTR3E
levels in the sigmoid colon of IBS-D patients, which underlines the relevance of
HTR3E
in the pathogenesis of IBS-D.
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