Colonic spatial single-cell proteomics and murine models link mitochondrial dysfunction to dimeric IgA-secreting plasma cell deficiency in Crohn’s disease

Domain: neuroscience
Published: 2026-02-12
Editorial outcome: major_revision

Abstract
Secretory IgA (SIgA) is critical for maintaining the intestinal barrier. A dysregulated B-cell compartment and altered Ig secretion have been well documented in Crohn’s disease (CD) patients, although their origin is unknown. To unravel the role of mucosal humoral immunity in CD pathogenesis, we in-depth phenotype colonic plasma cell (PC) differentiation in CD at the single-cell level, linked to ex vivo functional characterization and experimental mouse models with a congenital mitochondrial defect under glucose-free high-protein dietary intervention. Here, we demonstrate that despite expanded colonic B cells, CD patients in remission present significantly diminished mucosal dimeric IgA and fecal SIgA. Colonic plasmablasts and immature CD19+CD45+ PCs are increased at the expense of the mature CD19-CD45- phenotype. Accordingly, CD-derived ex vivo differentiated PCs display impaired maturation into dimeric IgA-secreting PCs. In this study, patient-derived data from colonic RNA-seq, spatial single-cell proteomics, and plasma metabolomics are combined with data from both mouse models and highlight the crucial role of mitochondrial oxidative phosphorylation in colonic IgA+-PC differentiation, suggesting promising directions for future therapeutic strategies.

Introduction
Crohn’s disease (CD) is one of the major entities of inflammatory bowel disease (IBD) with globally rising incidence rates, particularly in newly industrialized countries1. It is characterized by chronic, relapsing transmural inflammation, which can affect any portion of the gastrointestinal tract2.

ManystudiesrevealedanimpairedintestinalbarrierfunctioninCD patients, resulting in the chronic translocation of luminal, commensal bacteria or their products across the intestinal mucosa, thereby triggering an aberrant immune response3. An integral part of the mucosal barrier is the secretory IgA (SIgA)-mediated humoral immunity priming gut-associated lymphoid tissues (GALTs) such as Peyer’s patches (PPs) or lymphoid follicles (LFs).

In the human intestine, next to immature IgA+CD138- plasmablasts (PBs), three distinct IgA+CD138+ subsets of plasma cells (PCs) have been identified: proliferating, early CD19+CD45+, intermediate CD19-CD45+, and terminal mature CD19-CD45-CD138+ PCs4. Secreted joining (J) chain-coupled dimeric IgA1 and IgA2 (dIgA) is transported across the epithelium by the polymeric Ig receptor (pIgR)5 and anchored as SIgA to the mucus layer. In turn, it affects enteric bacteria and fungi through mechanisms such as immune exclusion/inclusion, antigen neutralization, enchained growth, and functional modulation6–8. Numerous studies have demonstrated that deficiencies in the production or function of IgA lead to intestinal dysbiosis and exacerbate spontaneous or trigger-induced inflammation in mouse models9,10. Similarly, selective IgA deficiency (SIgAD), the most common primary immunodeficiency in humans, is associated with a >5-fold increased prevalence of CD11.

Dynamic metabolic adaptations accompany the development of the B-cell lineage. While activated, proliferating B cells mainly rely on aerobic glycolysis, differentiation into Ig-secreting PCs highly depends on mitochondrial oxidative phosphorylation (OXPHOS)12. Although less well studied compared to ulcerative colitis (UC), CD has been associated with mitochondrial dysfunction13, potentially affecting this switch in bioenergetics.

As early as the 1980s, several publications indicated that B cells and humoral immunity were involved in CD pathology14,15. In 1984, Danis et al. showed an altered profile of Ig classes secreted by cultivated rectal biopsies of active CD patients, with increased IgM but diminished IgA secretion compared to non-IBD16. MacDermott et al. reported that cultivated peripheral blood mononuclear cells (PBMNCs) from CD patients displayed highly increased spontaneous IgA secretion15. In contrast, isolated lamina propria mononuclear cells (LPMNCs) from CD patients exhibited decreased IgA secretion and an increased percentage of monomeric IgA1 compared to non-IBD controls15. Similarly, another study verified comparable serum IgA levels but substantially reduced SIgA in fluids of the uninvolved jejunum of CD patients17. Complementing the expansion of the B-cell proportion in inflamed ileum18, our previous study revealed increased ileal IgM+ B cells in CD remission (CDrem)19. Recently, an enrichment of IgA+Ki-67+ PBs and IgG+ PCs and a proportional depletion of mature IgA+CD19- PCs were observed in the inflamed colonic lamina propria (LP) of IBD patients20,21. However, in the past decades, findings linked to B cells have been attributed a bystander role in the etiopathogenesis of IBD.

Here, we aimed to build on previous observations of a skewed B-cell response in CD patients and employ state-of-the-art techniques to characterize the colonic humoral immunity in CD. We demonstrate an expansion of B cells, PBs, and immature CD19+CD45+ PCs in the colonic mucosa, accompanied by impaired terminal CD19-CD45- PC differentiation and reduced dIgA secretion in these patients. Furthermore, our data suggest that a compromised mitochondrial function may underlie the defective terminal maturation of IgA-secreting PCs.

Results
Colonic CD27+CD38+CD138- B cells are expanded in CDrem patients
Colon biopsies were collected from CDrem patients, as well as from age- and sex-matched non-IBD controls during colonoscopy (detailed information is given in Supplementary Tables 1–3). The colonic immune cell compartment was characterized using RNA sequencing (RNA-seq), two independent spatial single-cell proteomics (SSCP) experiments, and quantification of B-cell marker transcripts in isolated LPMNCs by RT-qPCR (Fig. 1a). Although principal component analysis (PCA) did not reveal a clear separation of CDrem patients from non-IBD controls (Supplementary Fig. 1a), differential gene expression (DGE) analysis revealed significantly increased expression of various Ig gene segments of the heavy and light chain in the colonic mucosa of CDrem patients (Fig. 1b). Correspondingly, gene set enrichment analysis (GSEA) identified B cell-related pathways as significantly enriched within the top 40 REACTOME pathways (Fig. 1c). Transcripts highly expressed in B lymphocytes, plasmablasts (PBs) and PCs, such as CD79A (part of the B-cell receptor complex), MZB1, CD27, CD38, and TNFRSF17 (BCMA), as well as human leukocyte antigen (HLA) transcripts, involved in the major histocomatibility complex (MHC) class II antigen presentation, were significantly upregulated in CDrem (Fig. 1d). Notably, the expression of SDC1 (CD138), a common marker of PCs but not PBs, was not increased in CDrem. In line with this, SSCP using colonic formalin-fixed paraffin-embedded (FFPE) tissues revealed a trend towards a higher percentage of CD19+ (P=0.0931) (non-IBD: 6.5%, interquartile range (IQR): 3.6–10.4% versus CDrem: 16.3%, IQR: 9.9–22.6%), and significantly increased percentages of CD27+ (non-IBD: 22.9%, IQR: 19.6–25.6% versus CDrem: 39.1%, IQR: 29.2–45.0%) and CD38+ cells (non-IBD: 22.0%, IQR: 18.3–33.2% versus CDrem: 44.9%, IQR: 31.8–58.9%) in the total colonic LP immune cells (Vimentin+, lymphoid follicles/aggregates excluded) in CDrem (Fig. 1e, f). Notably, the number of Vim+ LP cells per tissue area [mm2] was not increased in colonic biopsies from CDrem patients, indicating that increased B-cell numbers are not attributable to leukocyte infiltration (Supplementary Fig. 1b). In contrast, the proportion of CD138+ PCs was not altered, nor was the percentage of non-B-cells characterized by expression of CD3, CD4, or CD8 (mostly T cells), CD11c (mostly dendritic cells (DCs)), and CD56 (mostly natural killer (NK) cells). For further characterization of the colonic B-cell compartment, LPMNCs were isolated from respective patients (Fig. 1g). Total LPMNC count as well as plasma CRP levels did not differ between CDrem and non-IBD (Supplementary Fig. 1c, d), highlighting the inactive disease state of the analyzed CD patients. In contrast, CD patients in active disease had increased colonic LPMNC counts compared to CDrem patients and elevated plasma CRP levels relative to non-IBD controls (P=0.0557) (Supplementary Fig. 1c, d). In agreement with our previous observations, colonic LPMNCs from CDrem presented increased expression levels of CD38, and to a lesser extent, CD19 and CD27 (Fig. 1h and Supplementary Fig. 1e). Strikingly, expression of TNFRSF17 and SDC1 was not increased in CDrem-derived LPMNCs. Together, these findings indicate that CD19+CD27+CD38+ B cells are overrepresented in the colonic mucosa of CDrem but do not translate into an increased amount of CD138+ PCs (Fig. 1i).

CDrem displays increased colonic IgA but reduced fecal IgA
Given the expanded colonic B-cell compartment in CDrem, we analyzed Ig classes in these patients in depth. While mass spectrometry of plasma samples revealed no quantitative differences in the presence of circulating Ig subclasses (Fig. 2a and Supplementary Fig. 1f), RNA-seq uncovered an elevated colonic expression of IGHA1, IGHA2, IGHG1 (P=0.0556), IGHG2, and IGHM in CDrem (Fig. 2b). Increased amounts of total colonic IgA and a trend towards elevated IgM, comprising intracellular, membranous as well as secreted forms, could be verified on protein level (Fig. 2c and Supplementary Fig. 1g). In line with the upregulation of TNFRSF17 (Fig. 1d), which is specifically expressed in PBs and PCs (Supplementary Fig. 1h), the amount of colonic B-cell maturation antigen (BCMA) was significantly higher in CDrem compared to non-IBD (Fig. 2c). Additionally, IHC suggested increased IgA levels in the colonic LP of CDrem (P=0.0756) (Fig. 2d).

Intriguingly, fecal IgA levels were significantly diminished in a large cohort (n=44) of CDrem patients (Fig. 2e). Since transcytosis of IgA across the intestinal epithelium and formation of SIgA depend on the basolateral pIgR (Fig. 2f), we studied its expression by RNA-seq and IHC. Levels of the colonic epithelial pIgR, however, were not different between CDrem and non-IBD (Supplementary Fig. 2a, b). Further, SSCP indicated an increased abundance of CD19+CD27+CD38+CD20-CD138- PBs in the colonic mucosa of CDrem patients (Fig. 2g and Supplementary Fig. 2c). Nevertheless, the proportion of colonic CD138+ PCs was not upregulated in CDrem, pointing to an impairment in local PC differentiation.

Shift to proliferating CD19+CD45+ PCs is associated with reduced dIgA secretion in CD
Assuming a defect in colonic PC maturation, we phenotyped colonic PCs using SSCP (Fig. 3a) to assess potential functional differences in CD remission. It has been described that human intestinal CD138+ PCs can be subdivided into three major subsets defined by selective expression of CD19 and CD454. While CD19+CD45+ PCs represent an early stage in the PC differentiation pathway and are described as immature and more proliferative with a lower antibody secretion activity and a short lifespan4,22, mature, long-lived CD19-CD45- PCs display a high capacity for antibody secretion4,22 (e.g., IgA) (Fig. 3d). Using SSCP, we validated the presence of these PC subsets in the human colonic mucosa of non-IBD controls (Fig. 3d). While CD138 expression was only slightly reduced in CD19-CD45+ and CD19-CD45- PCs, the proportion of Ki-67+ cells was significantly reduced in CD19-CD45+ PCs compared to CD19+CD45+ PCs (Fig. 3e). In line with this, NF-κB p65 as well as pan-RAS, which are involved in activating signaling pathways, were downregulated stepwise during PC differentiation in CD19-CD45+ and CD19-CD45- PCs, respectively (Fig. 3e). The cell size tended to increase slightly from CD19+CD45+ to CD19-CD45- PCs, which might reflect the expansion of organelles necessary for high-level antibody secretion, such as the rough endoplasmic reticulum (rER) and the Golgi apparatus (Supplementary Fig. 2e). Moreover, the fully mature CD19-CD45- PCs were found in closer proximity to the epithelium compared to the CD19+CD45+ and CD19-CD45+ populations, with no significant differences between CDrem patients and non-IBD controls (Supplementary Fig. 2f). Of note, SSCP identified a clear shift in these three phenotypically different colonic PC populations in CDrem (Fig. 3f, g and Supplementary Fig. 2g, h). In CD patients, the presence of colonic CD19+CD45+ PCs was significantly increased (44.6%, IQR: 29.4–57.1%) in comparison to non-IBD controls (13.2%, IQR: 9.6–33.8%), while the percentage of colonic CD19-CD45- PCs was decreased (15.5%, IQR: 5.1–24.4%) compared to non-IBD controls (32.7%, IQR: 23.0–50.5%) (Fig. 3h). In accordance with the observation that the less mature CD19+CD45+ PCs dominate over the fully differentiated CD19-CD45- PCs in CDrem, the proportion of dimeric IgA (dIgA) relative to total IgA or monomeric IgA (mIgA) was reduced in the colonic mucosa of these patients (Fig. 3i and Supplementary Fig. 2i). In contrast, the proportion of mIgA was increased, likely reflecting an expansion of IgA+ B cells. Further, the significantly reduced dIgA/BCMA ratio indicated a decreased secretion of dIgA per PB/PC in the colonic mucosa of CDrem (Fig. 3j). Next, we studied the colonic tissue microenvironment of these PCs by bulk RNA-seq regarding the expression of cytokines known to promote PC development and survival. Here, IL4 and IL6 were upregulated with respect to non-IBD, while IL10, IL21, TGFB1 (TGF-β1), and TNFSF13 (APRIL) were not significantly altered in CDrem (Fig. 3k and Supplementary Fig. 2j). Similarly, chemokines such as CCL25, CCL28, and CXCL12 that enable homing or retention of primed IgA+ PBs in the colon23,24 were not altered in these patients, suggesting that PC differentiation may rather be impaired intrinsically and independently of the mucosal microenvironment. Taken together, our analyses demonstrated a compromised PC differentiation in CDrem that is reflected by an increase in immature colonic CD19+CD45+ PCs at the expense of terminally differentiated CD19-CD45- PCs and diminished intestinal secretion of dIgA.

CD patient-derived switched memory B cells display an intrinsic defect in ex vivo IgA+ PC differentiation
Conceptualizing that the local differentiation of colonic PCs may be intrinsically affected in CDrem, we employed an ex vivo model of PC differentiation. Since the majority of intestinal PCs originate from memory B cells25 (Fig. 4a), we enriched class-switched IgA+/IgG+ memory B cells (sMBCs) from isolated colonic LPMNCs (Fig. 4b and Supplementary Fig. 3a). Subsequently, the sMBC-enriched population, mainly expressing B-cell lineage markers like MS4A1 (CD20), CD27, CD38, and TNFRSF17 (Supplementary Fig. 3b), was cultivated for 14 days and differentiated into terminal PCs according to a protocol published by ref. 26 (Fig. 4b). The presence of antibody-secreting PBs and PCs was verified by the detection of high amounts of dIgA as well as moderate levels of mIgA and IgG in the supernatant over the time of cultivation (Fig. 4c). Lower levels of mIgA and IgG detected in the medium control (IMDM+cytokines) likely result from non-specific cross-reaction with mIgA and IgG in the fetal bovine serum (FBS) or with the murine IgG1 His-Tag antibody added on Day 0 for cross-linking the histidine-tagged CD40 ligand. On Day 14, the CDrem- and non-IBD-derived PCs were compared regarding their expression of common PC markers (Fig. 4d and Supplementary Fig. 3c, d). In contrast to non-IBD-derived PCs, PCs derived from CDrem lacked detectable expression of SDC1, indicating that they were unable to develop into fully mature PCs. Additionally, the expression of TNFSRF17, X-box binding protein 1 (XBP1), CD27 (P=0.0732), and PTPRC (CD45) was reduced in patient-derived ex vivo-generated PCs (Fig. 4d and Supplementary Fig. 3c). Unlike PCs of most non-IBD controls, expression of TNFRSF13B, encoding the Transmembrane activator and CAML interactor (TACI), could not be detected in PCs of CDrem origin. While IGHM was not affected (Supplementary Fig. 3e) and IGHA1 tended to be slightly reduced (Fig. 4e), the expression of IGHA2 and the J chain (JCHAIN), which is crucial for the formation of dIgA, approached statistical significance in its reduction in CDrem-derived PCs (Fig. 4e). The similar IGHA2/IGHA1 ratio and the strong positive correlation between both isoforms (Fig. 4f) are consistent with a preserved IgA subclass composition in CDrem- compared to non-IBD-derived colonic PCs. In line with these results, CD patient-derived PCs displayed significantly decreased secretion of total and dimeric IgA into the supernatant (Fig. 4g). The comparable dIgA/total IgA ratio (Supplementary Fig. 3f) excludes a compensatory increase in mIgA secretion by CDrem- versus non-IBD-derived PCs, which is further supported by the positive correlation between dIgA and total IgA (Fig. 4g). The expression of IGHG1, IGHG2, IGHG3, and IGHG4 tended to be slightly lower in patient-derived PCs with no significant differences (Supplementary Fig. 3g). However, IgG secretion did not differ between PCs originating from non-IBD or CDrem (Fig. 4h). Concisely, the data indicate an intrinsic defect in the differentiation of IgA+ colonic PCs from sMBCs of CDrem patients, leading to an impaired capacity for dIgA secretion, particularly dIgA2.

PCs in CD patients show increased levels of the NADase CD38 and mitochondrial dysfunction
To unravel underlying molecular mechanisms of impaired colonic PC differentiation in CD patients, we further phenotyped distinct PC subtypes (CD19+CD45+, CD19-CD45+, and CD19-CD45-) by SSCP using FFPE tissue slides of six CDrem patients and six non-IBD controls. Three proteins were specifically regulated during PC maturation and differed between CD patients and controls. A slight increase in CD27 and CD45 was observed, while CD38 displayed the strongest upregulation in all three colonic PC subtypes in CDrem (Fig. 5a, b). In line with this, the CD38high PCs were found primarily in the CDrem PC cluster identified previously (Fig. 5c and Supplementary Fig. 4a). Although nearly all PCs were CD38+ (Fig. 5a) and CD38 expression decreased during PC differentiation ranging from CD19+CD45+ to CD19-CD45- PCs, median CD38 expression per cell was increased in CDrem versus non-IBD for each PC subpopulation (Fig. 5d). Moreover, CD38 levels positively correlated with levels of the activating signaling proteins NF-κB and pan-RAS across all PC subtypes (Supplementary Fig. 4b). CD38 is known to act as a cell surface NADase/ADP-ribosylcyclase as well as a cyclic ADPR (cADPR) hydrolase that catalyzes the synthesis of nicotinamide (NAM) and (c)ADPR using nicotinamide adenine dinucleotide (NAD+) as a substrate27 (Fig. 5e). NAD+, which can be synthesized from tryptophan or various vitamin B3 derivatives, is crucial for mitochondrial energy metabolism. Accordingly, several studies linked CD38 overexpression to the depletion of intracellular NAD+ levels and subsequent mitochondrial dysfunction28,29. Moreover, the formation of cADPR can induce a Ca2+ release-mediated activation of the NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome, further exacerbating mitochondrial dysfunction30. In support of this, colonic CD38 expression correlated significantly with the expression of the inflammasome components NLRP3 and CASP1 (Fig. 5f).

As mitochondrial respiration—supported by high levels of mitochondrially localized, OXPHOS-promoting P32 (Fig. 6a)—is essential for proper differentiation and function of PCs12, we investigated the systemic as well as the local energy metabolism in the colonic mucosa of CDrem patients. Proton nuclear magnetic resonance (1H NMR)-based metabolomics detected no difference in the global metabolic profile of plasma samples from CDrem and non-IBD (Fig. 6b). However, elevated levels of ketone bodies and the significantly increased L-lactate/D-glucose ratio (Fig. 6c and Supplementary Fig. 4c) may indicate an upregulation of ketogenesis and glycolytic activity in CDrem, respectively, both of which are known compensatory mechanisms in response to OXPHOS deficiency31. Higher succinic acid and citric acid levels might reflect a blockage of the citric acid cycle (CAC). Moreover, CD patients displayed a dyslipidemia with several upregulated lipoprotein fractions (Fig. 6c), which has often been linked to mitochondrial dysfunction32,33. In accordance with these systemic metabolic observations, GSEA of the “HALLMARK_OXIDATIVE_PHOSPHORYLATION” gene set using colonic tissue RNA-seq data demonstrated a significant depletion of OXPHOS-related genes in CDrem (Fig. 6d). In detail, GSEA identified enrichment of various REACTOME pathways involved in mitochondrial ATP synthesis, β-oxidation, CAC, and electron transport chain (ETC) among the downregulated gene transcripts (Fig. 6e). Differential gene expression analysis uncovered multiple transcripts related to mitochondrial function that were significantly reduced in the colonic mucosa of CDrem (Fig. 6f) and correlated negatively with CD38 expression (Supplementary Fig. 4d). The majority of these targets are mitochondrially encoded subunits of the complexes I, III, IV, and V of the ETC (Fig. 6g). Further transcriptional analyses revealed ex vivo differentiated PCs to display low expression of the glucose transporters SLC2A1 and SLC2A3, but higher levels of the amino acid transporters SLC3A2 and SLC38A2 (Supplementary Fig. 4e, f), which facilitate the uptake of neutral amino acids, including branched-chain amino acids and glutamine—both of which serve as critical substrates for PC metabolism34. These findings were validated in functional metabolic analyses, where CDrem patient-derived PCs exhibited a very low consumption of D-glucose and a significantly reduced L-lactate secretion compared to non-IBD-derived ex vivo differentiated colonic PCs (Fig. 6h). Consistent with a significantly decreased expression of the mitochondrially encoded genes MT-ATP6, MT-ATP8, and MT-ND3 (Fig. 6i and Supplementary Fig. 4g), these findings highlight a metabolically quiescent state of CDrem patient-derived PCs. Together, our data indicate a dysfunction in mitochondrial energy metabolism in the colonic mucosa of CDrem patients that potentially plays a causative role in defective colonic terminal PC differentiation and dIgA production in these patients.

Mitochondrial dysfunction is associated with impaired IgA+ PC maturation in mice
To examine whether mitochondrial dysfunction may causally impede PC differentiation in vivo, we investigated the maturation of PBs and PCs in conplastic respiratory chain complex V mutant mice. These mice carry a mutation in the mitochondrially encoded ATP synthase membrane subunit 8 (ATP8), resulting in diminished respiratory capacity and ATP production with a compensatory increase in glycolysis in various cell entities, including lymphocytes35 (Fig. 7a), and display an impaired intestinal barrier homeostasis36,37. Splenic resting B cells isolated from adult Atp8-mutant versus B6-WT (wild type) mice (n=3 per group) were ex vivo differentiated into IgG1+ PBs under stimulation with lipopolysaccharide (LPS) and IL4 for four days (Fig. 7b and Supplementary Fig. 5a, b). Using the mitochondrial membrane potential-sensitive dye tetramethylrhodamine ethyl ester (TMRE), flow cytometry revealed a significant increase in mitochondrial activity in LPS+IL4-stimulated PBs (mean: 54.2% TMREhigh) compared to resting B cells (mean: 17.1% TMREhigh) obtained from B6-WT mice. As expected, the percentage of TMREhigh PBs under LPS+IL4 stimulation was significantly lower in Atp8-mutant-derived B cells (Fig. 7c and Supplementary Fig. 5a). However, Atp8-mutant B cells displayed a significant boost in proliferation upon activation and PB induction in contrast to B6-WT B cells (Fig. 7d). While murine PBs exhibit high levels of surface B220, B220 expression is downregulated in short-lived PCs (SLPCs) and lost completely in most fully mature PCs22. Hence, the trend towards elevated B220 expression in Atp8-mutant PBs may indicate a less differentiated phenotype compared to B6-WT PBs (Fig. 7e). In line with this, splenic IgG levels were increased in Atp8-mutant mice under chronic DSS-induced colitis (Supplementary Fig. 5c), whereas expression of Sdc1 (CD138) was significantly diminished compared to B6-WT mice (Supplementary Fig. 5d).

Additionally, we isolated cells from mesenteric lymph nodes (MLNs) and PPs for flow cytometry analysis from three animals per group (Supplementary Fig. 5e). While the percentages of B220+ B cells and CD138+ PCs in MLNs and PPs did not differ between Atp8-mutant and B6-WT mice (Supplementary Fig. 5f), the proportion of B220- PCs was decreased in MLNs and PPs of Atp8-mutant mice (MLNs: 21.6%, IQR: 20.3–22.3% and PPs: 24.3%, IQR: 18.0–31.3%) compared to B6-WT mice (MLNs: 30.8%, IQR: 21.4–52.3% and PPs: 34.2%, IQR: 28.6–42.1%) (Fig. 7f and Supplementary Fig. 5g). Consistent with our previous observations, colonic expression of B-cell and PB/PC differentiation markers, including Cd19, Igha, Tnfrsf17, Prdm1, and Sdc1, was significantly reduced in Atp8-mutant mice (Fig. 7g and Supplementary Fig. 5h). Notably, expression of complement component 1q binding protein (C1qbp), encoding p32, which is essential for maintaining mitochondrial OXPHOS38, was decreased in Atp8-mutant mice (Supplementary Fig. 5d) and significantly correlated with colonic expression of Igha, Prdm1, and Sdc1 (Fig. 7h, i). In line with this, the percentage of colonic IgA+ cells tended to be decreased in Atp8-mutant (0.5%, IQR: 0.5–0.7%, n=3) compared to B6-WT mice (3.0%, IQR: 2.2–4.5%, n=3) (Fig. 7j), accompanied by significantly diminished levels of mIgA and dIgA in the colonic mucosa (Supplementary Fig. 5i).

In a previous study36, we demonstrated that removal of glucose from the diet and isocaloric replacement by the protein casein promotes colonic p32 levels and mitochondrial OXPHOS in mice (Fig. 7k). Indeed, adult C57BL/6 mice that were fed a glucose-free, high-protein (GFHP) diet for an average of 70 days displayed significantly upregulated colonic expression of Cd19, Cd79a, Ighm, and Tnfrsf17 (Supplementary Fig. 5j), higher percentages of colonic IgA+ cells (2.7%, IQR: 2.1–3.3%) than isocaloric chow diet-fed mice (1.9%, IQR: 0.7–2.3%) (Fig. 7l), and increased fecal IgA levels (Supplementary Fig. 5k). In summary, these findings demonstrate that PC maturation and colonic humoral immunity are highly dependent on mitochondrial function and could potentially be promoted by nutritional intervention boosting OXPHOS efficiency.

Methods
Study cohort and sample collection
Protocols were approved by the ethics committees of the University of Lübeck (AZ12-138; AZ19-233; AZ21-415), the University of Münster (AZ 2016-305-b-S), the University Hospital Regensburg (No. 21-2390-101) and the Hannover Medical School (No. 8738_B0_S_2019 (DRKS00019082); No. 10847_BO_S_2023 (DRKS00032771)) and written informed consent was obtained from participants before sample collection.

Blood samples were collected at the University Hospital Schleswig-Holstein, Campus Lübeck, Germany. Blood was drawn from fasting patients into a hirudin blood collection tube, centrifuged at 4122×g for 15 min, and plasma was stored at −80°C. Stool samples were obtained from the University Hospital Schleswig-Holstein, Campus Lübeck, Germany; the University Hospital Münster, North Rhine-Westphalia, Germany; the University Hospital Regensburg, Bavaria, Germany, and the Hannover Medical School Hospital, Lower Saxony, Germany and stored at −80°C. Stepwise colon biopsies were taken during endoscopy as part of regular patient management in the Department of Medicine I, University Hospital Schleswig-Holstein, Campus Lübeck, Germany. Biopsies were immediately fixed in 4.5% formaldehyde, snap-frozen in liquid nitrogen, and stored at −80°C or placed in ice-cold RPMI for cell isolation. Clinical characteristics of CD patients and non-IBD controls included in this study are listed in Supplementary Tables 1–4. Due to the limited number of patients, analyses were not stratified by sex.

Patients with a confirmed diagnosis of CD and colonic involvement were classified as being in remission or active disease based on clinical and endoscopic parameters. CD patients for stool sample collection were defined as being in remission if the available parameters met the following criteria: Harvey–Bradshaw index (HBI) ≤3, CRP ≤5 mg/l, and fecal calprotectin <100 µg/g ±1 month around stool sampling. CD patients for blood draw and endoscopic removal of colon biopsies were classified as being in remission if they were in clinical remission, the colon presented without macroscopic evidence of mucosal inflammation, and the ileum showed no or at most minor inflammatory signs during endoscopy. The age- and sex-matched control group included patients without a diagnosis of IBD and who presented without macroscopic or histological evidence of mucosal inflammation if endoscopy had been performed ±1 month around sample collection. Non-IBD controls with autoimmune diseases such as primary sclerosing cholangitis (PSC) or multiple sclerosis (MS), prior organ transplantation, immunosuppressive medication, diverticulitis, or non-specific intestinal inflammation were excluded from data analysis.

Due to limitations in available patient sample material, experimental methods were applied to different, overlapping patient subgroups (Supplementary Fig. 6).

Animal models
All animal experiments were approved by the Ministry of Energy, Agriculture, the Environment, Nature and Digitalization Schleswig-Holstein, Germany (mitochondrial mutation: V 242-63560/2017 [5-1/18] and V 312-72241.122-4 [93-10/10]; dietary intervention: V 242-27664/2018 [64-5/17]) and performed in accordance with the ARRIVE guidelines. Mice were housed under specific pathogen-free conditions at 21±2°C and 55±10% humidity on a regular 12-h light-dark cycle with ad libitum access to food (Altromin #1324, unless otherwise specified) and water.

C57BL/6J mice were purchased from the Jackson Laboratory and bred in the animal facility of the University of Lübeck. The conplastic strain C57BL/6J-mtFVB/NJ, which carries a mutation in the mitochondrial gene encoding the ATP synthase membrane subunit 8 (Atp8), was generated as described previously51 and maintained by repeatedly backcrossing female conplastic offspring with male C57BL/6J mice. Here, 3- to 6-month-old male and female Atp8-mutant mice (median age 14.9 weeks, IQR 13.3–16.0 weeks) and corresponding C57BL/6J control animals (B6-WT; median age 17.5 weeks, IQR 15.0–18.0 weeks) were euthanized for organ collection in independent experimental rounds. Due to variations in baseline mRNA expression levels of targets of interest, data were normalized to the median B6-WT target expression for each of three experiments. In a separate experiment, chronic colitis was induced in 3- to 4-month-old male and female Atp8-mutant and B6-WT mice (median age at organ harvest – Atp8-mutant: 13.3 weeks, IQR 13.3–13.3 weeks/B6-WT: 18.0 weeks, IQR 18.0–18.0 weeks) using three cycles of 5 days of 2% dextran sodium sulfate (DSS) in the drinking water, each followed by 5 days of regular drinking water for recovery. Due to the limited number of mice available from in-house breeding, sex-specific stratification was not performed.

Glucose-free high-protein (GFHP) and isocaloric control diets were obtained from Ssniff (Soest, Germany), with the respective compositions specified in Supplementary Table 5. Female C57BL/6 mice, aged 7 to 8 weeks, were purchased from Charles River Laboratories (Wilmington, MA) and were allowed to acclimate on a standard chow diet. At 20 weeks, mice were randomly assigned to groups receiving the GFHP diet or the isocaloric control diet. Mice were maintained on the respective diet for an average of 10 weeks before organ sampling. The dietary intervention was conducted in two independent experimental runs.

Isolation of colonic lamina propria mononuclear cells (LPMNCs)
Seven stepwise biopsies were collected from the cecum to rectum during colonoscopy and immediately placed in ice-cold RPMI 1640 supplemented with 10% (v/v) fetal bovine serum (FBS), 100 U/ml penicillin, and 100 µg/ml streptomycin. Biopsies were centrifuged at 300×g and 4°C for 5 min and treated with 1 mM dithiothreitol (DTT) at room temperature for 10 min to disintegrate the mucus layer and the extracellular matrix. Then, biopsies were centrifuged again (300×g, 4°C, 5 min), digested with 2.5 U/ml Dispase (STEMCELL Technologies #07913) and 1% (w/v) BSA in PBS at 37°C for 1 h, and isolated cells were collected by passage through a 70-µm cell strainer and placed on ice.

For further digestion, the remaining tissue was incubated in pre-warmed 200 CDU/ml collagenase type IV (Sigma-Aldrich #C5138) in supplemented RPMI 1640 at 37°C for 30 min, and isolated cells were separated using a 100 µm cell strainer. Both isolated cell fractions were pooled, centrifuged (300×g, 4°C, 5 min), and resuspended in 5 ml of supplemented RPMI 1640. To purify lamina propria mononuclear cells (LPMNCs) by density gradient centrifugation, the cell suspension was layered onto 6 ml of 63% (v/v) Percoll solution and centrifuged at 2,454×g and 4°C for 20 min without brake. The turbid layer of MNCs was removed, centrifuged (300×g, 4°C, 5 min), resuspended in 1 ml supplemented RPMI 1640 and the number of living cells was determined in a counting chamber using trypan blue staining.

Enrichment of colonic IgA+ and IgG+ switched memory B cells
IgA+ and IgG+ switched memory B cells (sMBCs) were enriched from colonic LPMNCs using negative selection magnetic-activated cell sorting (MACS; a cocktail of biotin-conjugated monoclonal antibodies against CD2, CD14, CD16, CD36, CD43, CD235a, IgM, and IgD) following the manufacturer’s instructions of the Switched Memory B Cell Isolation Kit (Miltenyi #130-093-617).

Ex vivo differentiation of colonic switched MBCs into PCs
The sMBC-enriched cell population in the flow-through was centrifuged at 300×g and 4°C for 5 min, resuspended in 120 µl Iscove’s Modified Dulbecco’s Medium (IMDM) supplemented with 10% (v/v) FBS, 100 U/ml penicillin, 100 µg/ml streptomycin, 5 mg/ml human insulin, and 50 mg/ml human transferrin26 and seeded in one round-bottom well of a 96-well suspension cell culture plate. Cells were cultivated at 37°C and 5% CO2 in a humidified incubator for 14 days, and differentiation into plasmablasts (PBs) and PCs was induced by sequentially adding different combinations of cytokines as established by ref. 26. SMBCs were initially cultured from Day 0 to 4 with IL-2 (20 U/ml, R&D Systems #202-IL), IL-10 (50 ng/ml, R&D Systems #217-IL), IL-15 (10 ng/ml, Thermo Fisher Scientific #200-15), CpG oligodeoxynucleotide (ODN) 2006 (10 µg/ml, InvivoGen #tlrl-2006), histidine-tagged soluble recombinant human CD40L (50 ng/ml, R&D Systems #2706-CL), and anti-polyhistidine mAb (5 µg/ml, R&D Systems #MAB050) to induce B-cell activation. Between Day 4 and 7, PBs were generated in the presence of IL-2 (20 U/ml), IL-6 (50 ng/ml, Thermo Fisher Scientific, #200-06), and IL-15 (10 ng/ml), followed by PC differentiation from Day 7 to 10 induced by IL-6 (50 ng/ml), IL-15 (10 ng/ml), and IFNα (500 U/ml, Sigma-Aldrich #IF007). Stimulation with IL-6 (50 ng/ml) and APRIL (200 ng/ml, R&D Systems #5860-AP) between Day 10 and 14 promoted PC survival and terminal differentiation. Supernatants were collected on Day 4, 7, 10, and 14 after centrifugation at 300×g and 4°C for 5 min and cells were washed with 100 µl of IMDM before fresh supplemented IMDM and cytokines were added.

D-glucose assay
D-glucose levels were measured in supernatants of Day 10–14 (1:10 v/v) from patient-derived ex vivo differentiated colonic PCs using the D-glucose assay kit (Megazyme #K-GLUC) according to the manufacturer’s instructions.

L-lactate assay
L-lactate levels were quantified in supernatants of Day 10–14 (1:4 v/v) from patient-derived ex vivo differentiated colonic PCs following the manufacturer’s instructions of the L-lactate assay kit (Megazyme #K-LATE).

RNA extraction and RT-qPCR
Total RNA was extracted from isolated colonic LPMNCs, sMBCs, or ex vivo differentiated PCs (Day 14) following the manufacturer’s instructions using the Single Cell RNA Purification Kit (Norgen Biotek #51800). Native frozen murine colon tissue was homogenized, and total RNA was isolated according to the innuPREP RNA Mini Kit 2.0 (IST Innuscreen #845-KS-2040010). The RNA was on-column treated with DNase I (Sigma-Aldrich #AMPD1-1KT), and its concentration was determined using the NanoDropTM OneC spectrophotometer. CDNA synthesis was performed using Oligo(dT) primers and the RevertAid H Minus reverse transcriptase (Thermo Fisher Scientific #EP0451). Real-time quantitative polymerase chain reaction (RT-qPCR) was performed on the StepOnePlusTM or the QuantStudioTM 1 system (Thermo Fisher Scientific) using the PerfeCTaTM SYBR® Green SuperMix (Quantabio #95055-02K) and target-specific oligonucleotides (Supplementary Table 6). Target expression levels were normalized to UBC or ACTB/Actb and displayed as 2−ΔCt or 2−ΔΔCt (relative expression to the control group).

Bulk RNA sequencing of patient-derived colonic tissue
Native frozen colon biopsies were homogenized and total RNA was isolated following the manufacturer’s instructions of the innuPREP RNA Mini Kit 2.0 (IST Innuscreen #845-KS-2040010) including on-column DNA digestion using DNase I. RNA was quantified using the NanoDropTM OneC spectrophotometer (Thermo Fisher Scientific) and verified to have an RNA integrity number (RIN) >5 using the RNA 6000 Nano Kit and the 2100 Bioanalyzer instrument (Agilent Technologies #5067-1511). Bulk mRNA sequencing was performed at Novogene Corporation Inc. in Cambridge, United Kingdom. Following poly(A) enrichment and non-directional mRNA library preparation, paired-end 150 bp sequencing was performed to a depth of 30 million reads per sample on the NovaSeq X Plus platform (Illumina).

Demultiplexed FastQ files were mapped onto the human reference genome (GRCh38) using kallisto (v0.46.1) with sequence bias correction and 30 bootstraps per sample. Differentially expressed genes were identified by applying the R package sleuth (v0.30.1) on kallisto quantifications using a likelihood ratio test to calculate P values. Effect sizes, which approximate log fold changes, were calculated using the Wald test implemented in sleuth. Genes with P≤0.05 and absolute effect sizes >1 were considered significantly different between conditions. Gene set enrichment analysis (GSEA) against REACTOME pathways (MSigDB v2023.2, Oct 2023) was performed on effect sizes using the R package mitch (v1.16.0). Pathways with q values <0.001 and absolute enrichment scores >0.4 were considered significant. GSEA for the gene set HALLMARK_OXIDATIVE_PHOSPHORYLATION (MSigDB v2023.2) was conducted using the R package fgsea (v1.24.0).

Protein isolation from stool samples and colon biopsies
For fecal protein extraction, stool samples were homogenized in a tenfold volume of native protein buffer (20 mM Na2HPO4, 650 mM NaCl, 1 mM EDTA, 1 mM PMSF; pH 7.4) and cleared by centrifugation at 20,000×g and 4°C for 10 min.

To isolate proteins from colon tissue, native frozen biopsies were homogenized in denaturing lysis buffer containing 1% (w/v) sodium dodecyl sulfate (SDS), 10 mM Tris (pH 7.4), 2% (v/v) phosphatase inhibitor 2 and 3 (Sigma-Aldrich #P5726 and #P0044), and 1% (v/v) protease inhibitor (Sigma-Aldrich #P8340). The homogenate was heated at 100°C for 5 min, treated with ultrasonication, and cleared by centrifugation at 12,000×g and 4°C for 15 min. Whole protein concentration was determined using the Roti® Quant universal assay (Carl Roth #0120.1).

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting
For SDS-PAGE, whole protein extracts were separated on a CriterionTM TGXTM 4–15% polyacrylamide gel (Bio-Rad #5671085). To differentiate between monomeric and dimeric IgA, SDS-PAGE was performed under non-reducing conditions using a 3–8% CriterionTM XT Tris-Acetate polyacrylamide gel (Bio-Rad #3450131) and XT Tricine Running Buffer (Bio-Rad #1610790). The amount of IgG secreted into the SN by ex vivo PCs was quantified by SDS-PAGE under non-reducing conditions on a 4–15% precast polyacrylamide gel (Bio-Rad #5671085). Separated proteins were blotted to a PVDF membrane by semi-dry transfer.

After blocking in 5% (w/v) non-fat milk in Tween 20-tris-buffered saline (T-TBS), membranes were probed with specific primary antibodies, followed by respective horseradish peroxidase (HRP)-conjugated secondary antibodies (Supplementary Table 7). Specific proteins were detected using chemiluminescent HRP substrate on the ChemiDocTM XRS+ (Bio-Rad) or the iBright 1500 imaging system (Thermo Fisher Scientific) and quantified in ImageJ. To ensure similar transfer and equal loading of proteins, the expression of target proteins was normalized to α-Tubulin or β-Actin. The amount of secreted IgG by ex vivo-generated PCs was normalized to the RNA concentration, a measure of viable cell mass, isolated from respective cells.

IgA-specific enzyme-linked immunosorbent assay (ELISA)
For total IgA quantification, fecal protein samples or SNs from ex vivo-generated PCs were coated onto NuncTM MaxiSorp 96-well plates (Thermo Fisher Scientific #442404) at 4°C overnight, followed by three washing steps and blocking. A serial dilution of purified human IgA kappa (Southern Biotech #0155K-01) served as a standard curve for quantification.

For the detection of dimeric IgA secreted by ex vivo differentiated PCs, we established a dIgA-specific ELISA, which was validated to exclusively detect binding of dIgA to the polymeric immunoglobulin receptor (pIgR) using human monomeric IgA from serum (Sigma-Aldrich #I4036), secretory IgA from colostrum (Sigma-Aldrich #I2636), IgG1 from myeloma (Sigma-Aldrich #I5154) and recombinant anti-CD38 mIgA2 as negative controls (Supplementary Fig. 7). For coating, 150 ng/well of pIgR (R&D Systems #2717-PG) was incubated on a Half-Area High-Bind microplate (Corning #3690) at 4°C overnight. Following three washing steps and blocking, the supernatant was incubated for 1.5 h at room temperature.

After an additional three washes, wells were incubated with a human IgA-specific antibody conjugated to HRP (Supplementary Table 7). After six washing steps, 3,3’,5,5’-tetramethylbenzidine (TMB) substrate was added, and the enzymatic reaction was stopped using 3 M HCl. Optical density (OD) was measured at 450 nm on the SpectraMax® iD3 microplate reader (Molecular Devices) and normalized to a reference wavelength of 540 nm. OD detected in control samples (only supplemented IMDM from Day 10–14) was subtracted from signals of ex vivo-generated PC SNs for background correction. Quantified amount of secreted IgA was normalized to the RNA concentration, a measure for viable cell mass, of respective ex vivo generated PCs isolated on Day 14.

Immunohistochemical (IHC) and immunofluorescence (IF) tissue staining
Immunohistochemical staining of 2 µm tissue sections of formalin-fixed and paraffin-embedded (FFPE) colon samples was performed according to standard protocols. After deparaffinization, rehydration, antigen retrieval, endogenous peroxidase inhibition, and blocking, the tissue was probed with specific primary antibodies against IgA or pIgR, followed by respective HRP-labeled secondary antibodies (Supplementary Table 7). Subsequently, tissue sections were incubated with 3,3’-diaminobenzidine (DAB) substrate solution, counterstained with Mayer’s hemalum solution, dehydrated, and mounted in EntellanTM. In the case of immunofluorescence, tissue was permeabilized using 0.1% Triton X-100 and incubated with primary antibodies against human IgA and P32, followed by respective fluorochrome-labeled IgG secondary antibodies (Supplementary Table 7). Afterwards, nuclei were counterstained with 4,6’-diamidino-2-phenylindole (DAPI).

Images were obtained on an Axio Scope.A1 microscope (Zeiss) using the ZEN lite imaging software (v2.3; Zeiss). The area fraction of the epithelium or the lamina propria stained for human pIgR or IgA, respectively, was quantified by applying the Color Deconvolution 2 plugin in ImageJ (v1.53t; National Institutes of Health). To account for differences in cell density, IgA staining was normalized by hematoxylin nuclear staining. For murine colonic tissue, the percentage of IgA+ cells was determined by normalization to the total number of nuclei.

Spatial single-cell proteomics (SSCP)
Spatial single-cell proteomics of 5 µm FFPE colonic biopsy sections was performed on the CosMxTM Spatial Molecular Imager (SMI, NanoString/Bruker) using the 64-plex CosMxTM Human Immuno-Oncology Protein Panel (NanoString/Bruker #121500010) according to the manufacturer’s protocol. Two independent experiments were performed, each including n=3 CD patients in remission and n=3 non-IBD controls. Cell segmentation was performed based on the markers B2M/CD298, PanCK, CD45, and CD3, as well as nuclear staining with DAPI.

An expression matrix with raw fluorescence intensities was exported from the AtoMxTM platform. The positivity cut-off was set to 30 or 100, using mouse IgG1 and rabbit IgG isotype controls, respectively. All fields of view (FOVs, 0.5×0.5 mm) containing (part of) a lymphoid follicle/aggregate were excluded from the data analysis of total immune cells. To account for differences in area and cell density of the LP, immune cell counts were normalized to the number of total Vimentin+ LP cells. Following isotype fluorescence subtraction from each target per cell (negative values set to zero), Uniform Manifold Approximation and Projection (UMAP) of total Vim+ LP cells or CD138+ Vim+ PCs was performed using the Qlucore Omics Explorer (v3.9; Qlucore, Lund, Sweden). Thirteen targets unrelated to the B-cell compartment (β-catenin, CD16, CD3, CD4, CD56, CD68, CD8, EGFR, EpCAM, fibronectin, FOXP3, Her2, and SMA) were excluded from the phenotyping of PCs (UMAPs and dot plot). This approach allowed for the analysis of a median of 16,444 (IQR: 11,947–30,849) cells per patient, of which 1,943 (IQR: 920–2,722) were PCs. The median distance of each PC subtype to the nearest EpCAM+ epithelial cell was calculated using R (v4.2.3).

Plasma proteomics using liquid chromatography-coupled mass spectrometry (LC-MS)
Untargeted label-free proteome profiling was performed by liquid chromatography-coupled mass spectrometry (LC-MS). Plasma proteins were precipitated by incubation in ice-cold acetone (100%) followed by centrifugation. The protein pellet was dried and reconstituted in DOC buffer (1% (w/v) sodium deoxycholate (DOC) and 100 mM ammonium bicarbonate) to determine the protein concentration according to the Roti® Quant universal assay. Fifty micrograms of protein were incubated with dithiothreitol (1 M) and iodoacetamide (0.5 M). Proteins were digested using trypsin (Promega #V5280) overnight. After treatment with formic acid and centrifugation, peptides were lyophilized, and pellets were reconstituted in 0.1% (v/v) formic acid in water for mass spectrometric analysis.

Extracted peptides were loaded onto C18 EvoTip disposable trap columns and separated by chromatography on the EvoSep One (EvoSep) using a C18 Performance column (EV1137, 15 cm × 150 µm, 1.5 µm) paired with the extended 15 SPD method (88 min gradient, 220 nl/min). Peptides were analyzed on the TIMS (trapped ion mobility spectrometry) quadrupole TOF (time-of-flight) mass spectrometer (timsTOF Flex HT, Bruker), including a CaptiveSpray nano-electrospray ion source and operating in the diaPASEF mode52. The acquired diaPASEF mass spectra were processed using the DIA-NN software tool (v1.8.1)53, employing an optimized window design by using py_diAID54.

A unique protein matrix containing only proteins that were identified and quantified by at least two proteotypic peptides and that passed the FDR cut-off of 0.01 was created using the DIA-NN package in R. Protein abundances resulting from the MaxLFQ algorithm55 were preprocessed using the R package DEP56. Following the exclusion of proteins identified in less than 70% of the samples, a variance stabilizing normalization57, including a log2 transformation, was applied to the data. Missing values were imputed by employing the k-nearest neighbor method, and the observed batch effect (two batches) was corrected using the ComBat function58 of the Surrogate Variable Analysis (sva) R package59.

Analysis of plasma metabolites using 1H NMR spectroscopy
Plasma metabolomics was performed using proton nuclear magnetic resonance (1H NMR) spectroscopy, applying Bruker’s standardized in vitro Diagnostic Research (IVDr) procedure (Bruker BioSpin). Plasma samples were mixed 1:1 (v/v) with buffer consisting of 75 mM sodium phosphate (pH 7.4), 0.04% (w/v) sodium azide (NaN3), 4.6 mM 3-trimethyl-silyl-[2,2,3,3-2H4]propionic acid (TSP-d4), and 20% (v/v) deuterium oxide (D2O) by manual panning for one minute and 600 µl of sample were transferred to a 5 mm NMR tube. NMR spectrometry was carried out on a Bruker Avance III HD 600 MHz NMR spectrometer with a TXI probe at 310 K. The spectrometer was equipped with a SampleJetTM (Bruker) for automation, in which samples were kept at 279 K until measurement. Prior to analysis, a standard operating procedure was performed to check temperature calibration, quantification, and water suppression performance. A one-dimensional (1D) nuclear Overhauser effect spectroscopy (NOESY) experiment (pulse program: noesygppr1d) and a 1D Carr–Purcell–Meiboom–Gill (CPMG) spin-echo experiment (pulse program: cpmgpr1d) for the suppression of protein and other macromolecular signals were recorded per sample. Quantification in plasma/serum (B.I.QUANT-PSTM) and lipoprotein subclass analysis (B.I.LISATM) of the Bruker IVDr platform were used to automatically quantify 39 metabolites (+2 technical additives) and 112 lipoprotein parameters, including several subfractions of apolipoproteins (Apo), cholesterol (Chol), free cholesterol (FC), phospholipids (PL), and triglycerides (TG). Concentrations below the limit of detection were assigned a value of zero. For principal component analysis (PCA), metabolites identified in fewer than 40% of the samples were excluded, and data were transformed using the Yeo–Johnson method.

Isolation of primary murine cells and culture of splenic B cells
Murine MLNs, PPs, and splenic tissue were processed through a 70-µm cell strainer to obtain single-cell suspensions.

Splenic resting B cells were enriched using negative selection MACS (a cocktail of biotin-conjugated monoclonal antibodies against CD43, CD4, and Ter-119) following the manufacturer’s instructions of the B Cell Isolation Kit (Miltenyi #130-090-862). Subsequently, the isolated splenic B cells were cultivated in RPMI medium with 10% (v/v) FBS, 100 µM 2-mercaptoethanol, 1 mM HEPES, 100 U/ml penicillin, and 100 µg/ml streptomycin in the presence or absence of 3 µg/ml LPS-EB ultrapure (InvivoGen #tlrl-3pelps), and 10 ng/ml IL4 (Thermo Fisher Scientific #214-14) for 4 days. After cultivation, live cells were counted in the Attune NxT flow cytometer (Thermo Fisher Scientific) using trypan blue staining and prepared for flow cytometry as described below.

Flow cytometry
For mitochondrial staining, cultivated splenic B cells were incubated with 150 nM tetramethylrhodamine ethyl ester (TMRE, Thermo Fisher Scientific #T669) and 100 nM MitoTrackerTM Green (Thermo Fisher Scientific #M7514) at 37°C for 15 min.

Following Fc receptor blocking with an anti-CD16/32 antibody (Supplementary Table 7) at 4°C for 10 min, splenic B cells, as well as cells from MLNs and PPs, were stained with fluorochrome-labeled primary antibodies against B220, CD138, or IgG1 (Supplementary Table 7) on ice for 30 min. Subsequently, cells were washed, resuspended in PBS containing 0.5% (w/v) BSA, and measured immediately using the Attune NxT flow cytometer. Data analysis was performed using the FlowJo software (v10.10; FlowJo, Ashland, OR) and cells were selected using a lymphocyte gate, with debris and doublets excluded. Gating strategies for splenic B cells and cells isolated from MLNs/PPs are specified in Supplementary Fig. 5a and e, respectively.

Statistical analysis
Statistical analyses were performed using GraphPad Prism (v10.0.2; GraphPad Software, San Diego, CA). No statistical method was used to predetermine sample sizes. Outliers were identified and removed by the ROUT test (Q=1%, for stool IgA ELISA Q=2%). Normality was assessed using either the D’Agostino–Pearson or the Kolmogorov–Smirnov test, followed by an unpaired two-tailed t-test or a two-tailed Mann–Whitney U-test for the comparison of two groups. Statistical differences between more than two groups were analyzed by multiple Mann–Whitney U-test or Kruskal–Wallis test with Dunn’s post hoc test. Two-way analysis of variance (ANOVA) with Bonferroni’s, Dunnett’s, Šídák’s, Tukey’s, or Fisher’s least significant difference (LSD) post hoc test was applied for datasets with two variables. The correlation between variables was assessed by determining the Spearman correlation coefficient. P values were calculated, and null hypotheses were rejected when P≤0.05. Data were shown as median with range. For data visualization, GraphPad Prism, Qlucore Omics Explorer (v3.9; UMAPs), and R (v4.2.3) in RStudio (v2024.12.0+467; metabolomics PCA and enrichment plot) were employed. All schemes were created using BioRender (Toronto, Canada).

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The article by Raschdorf and colleagues examines the phenotype and functionality of plasma cells and plasmablasts in the colon of Chron’s disease patients in remission. The authors find that CDrem patients had a higher proportion of B cells and plasmablasts in the colonic mucosa, but an overall reduction in secretory IgA compared to healthy controls. They hypothesise that the lack of IgA may be due to incomplete or exacerbated differentiation of B cells/PBs into fully mature plasma cells (PCs), which they confirm by ex vivo differentiation. Moreover, they propose a mechanisms by which the B cell lineage in CD shows a reduced capacity for oxidative phosphorylation, correlating to increased CD38 expression, leading to a reduction in mature PC development. The authors further link OxPhos and PC development by showing that Atp8 mutant mice, which have impaired respiratory capacity, demonstrate a lack of intestinal PC development.\nOverall, the manuscript is clear and well-presented and contains exhaustive and interesting data from human samples that suggest a molecular mechanism which may explain changes in the B cell lineage in the colonic mucosa. The analysis of human material is very thorough and I only have some minor concerns about the experimental methods and some of the interpretation of the data. The last section of the paper, using a mouse model, is very interesting but here I have some more pressing concerns regarding the reproducibility of the data and some missing readouts which would be essential in assessing the importance and relevance of the mouse experiments. However, if the authors address these concerns I would be happy to support the publication of this manuscript in Nature Communications.\n- The authors note an increase in CD19+ CD138- B cell in the colonic mucosa of CDrem patients, but the localisation of these cells is not clear. Are the B cells the authors observe in colonic biopsies localised in the lamina propria or in lymphoid aggregates (lymphoid follicles, inflammation-induced tertiary lymphoid tissue etc.)? Is the increased proportion of B cells in the colon of CDrem due to a higher number of lymphoid follicles and/or lymphoid tissues? This is especially important for the interpretation of the data since an increased number of follicles would disproportionally affect B cells counts.\n- The authors demonstrate an increased proportion of B cells and plasmablasts in the colons of CDrem patients but no overall change in the proportion of plasma cells. However, it is unclear if there is an overall increase in infiltrate in the colons of these patients, i.e. whether there are more leukocytes overall. If yes, this would actually indicate an increase in the overall number of PCs, albeit disproportionally when compared to PBs. Could the authors provide any indication whether the numbers of leukocytes is increased in CD patients (e.g. quantified per unit area)?\n- When discussing figure 3E, the authors state “As expected, CD138 expression was similar across the 2 populations, while the proportion of Ki-67+ cells was reduced in CD19-CD45+ and CD19-CD45- PCs compared to CD19+CD45+ PCs”. However the data in 3E show no significant difference between the latter two subpopulations. Please amend the text accordingly. Moreover, it would be helpful to show whether the Ki67 pattern seen in CD patients is also present in healthy colonic PC subpopulations.\n- In 3I the authors quantify dIgA/BCMA ratios by western blot and show a reduced ratio in CD compared to healthy controls and state that “… the significantly reduced dIgA/BCMA ratio indicated a decreased secretion of dIgA per PB/PC in the colonic mucosa of CDrem” However, it is unclear if the reduced ratio is primarily due to more BCMA or less dIgA? The authors already show an increase in the number of colonic B cells, which would presumably also increase the BCMA signal and lead to a skewed ratio (Less dIgA per B/PB/PC would then be tautological). A more informative quantification might be a ratio of dIgA/CD138 to get a measure of dIgA production per PC instead.\n- Related to this, could a reduction in dIgA production be due to an increase in monomeric IgA production? Can the method used accurately quantify mIgA?\n- The data in figure 4 is very informative and convincingly shows an intrinsic defect in CD B cell differentiation into IgA-secreting PCs. Could the authors specify whether the ELISA used for quantification of IgA secretion in 4G detects only dIgA or whether it would also measure mIgA? If not, could the western blot quantification (from 4C) be utilised instead? I think this could be an important point in understanding the defect of CD PCs, as a reduction of J chain production could indicate more mIgA secretion which would also account for total reduced SIgA. Similarly, does the ELISA used discriminate between IgA1 and IgA2, as the latter seems to be disproportionately affected in CD?\n- Could the authors provide a more detailed explanation of what the images in Figure 6A are supposed to illustrate?\n- In figure 7, the overall number of mice used seems very low with only two mice per group in 7E and three mice in 7F and J. The authors should increase the number of biological replicates and perform all experiments on at least two occasions to demonstrate reproducibility. In addition, could the authors indicate why some experiments were performed only on male mice (qPCR) and some only on female mice (GFHP diet).\n- The authors use in vitro differentiation of splenic B cells to show a defect in the capacity of Atp8 mutant cells to develop into IgG-producing PCs, however the relevance of these data is unclear. Since these experiments are supposed to provide a mechanistic explanation for the human data, which shows a selective defect in IgA-PC induction, the authors should utilise conditions that favour induction of IgA producing PCs (i.e. IL-6, IL-21, TGFb) for their in vitro assay.\n- Similarly, the relevance of Figure 7F is uncertain. Since the main phenotype observed in human concerns colonic PC in the lamina propria, it is unclear why the authors chose to analyse lymphoid tissues associated with the small intestine (MLN and PP). This analysis should be complemented by FACS (or multi-coloured immunohistochemistry) of PCs in the colonic LP. This has the added benefit that it would allow for a much more detailed phenotypic description of these cells than the rather uninformative “IgA+ cells” obtained from the histology in 7J and 7I.", "reviewer_2": "In this manuscript Raschdorf and colleagues study the B cell compartment in Crohn’s disease patients in remission. Several recent studies evaluated the composition and activity of B cells in inflammatory bowel disease. As stated by the authors, the main difference with the current study is the fact that in the earlier studies, only patients with active disease were included, or disease activity was not addressed specifically. I agree with the authors that disease in remission is an interesting subject in its own right, but I do have several concerns regarding this study.\nMajor general concerns:\n- The authors emphasize the fact that they want to study patients in remission. However, when assessing their patient characteristics, a considerable proportion of the ‘remission’ patients had macroscopic inflammation in the ileum at the time of sampling. Although I realize the samples were obtained from the colon, this does stress these patients were in fact not in remission. For any systemic measurements (stool/serum) this is a problem in itself, and for the specific colonic analyses, the study is actually not different from the ‘non-inflamed’ samples which have in fact been included in earlier studies (e.g doi.org/10.1038/s41591-022-01680-y). As this concerns up to 33% of patients in many analysis, this is the difference between showing activity in actual remission, or just repeating earlier studies.\n- On a analytical levels, this study lacks rigor. The biggest problem is an unacceptable level of cherry picking in the statistics. In several panels, effects which they deem relevant are described as ‘increased’ or ‘decreased’ without acknowledgement of the fact that the difference lacks statistical significance. At the same time, effects apparently deemed unwanted are described as ‘not different’ and mentioned without any p-values, just as ‘NS’. An example is figure 2c, where the change in IgM is described as ‘increased’ (p value 0.1), while in figure 2g the proportion of PC is ‘not upregulated’, even though there is a clear trend as well, and no p value is provided. This is just one example, but occurs throughout the manuscript. The authors need to be be consistent in their description (it is fine to describe non-significant effects as well, but then for all) and provide p values for all comparisons.\nOther general concerns:\n1) the use of low sample numbers, which for sure has underpowered several analyses. Already in the first paragraph, the authors mention there was no clustering of samples in the PCA plot based on medication. However, most medications only included a single patient, and therefore can not possibly cluster. RNASeq analysis with primary material using 12 vs 7 samples is very limited etc. This is also evident from the volcano plot (fig 1b) which depicts p values rather than p values adjusted for high level multiple testing (Bonferroni or False Discovery Rate) as should be done for RNASeq. Using the appropriate metric would show no significant DEG.\n2) Use of less than conventional methods. Determination of immune cell subsets is usually performed by surface marker evaluation rather than transcription, in particular bulk expression. For example in figure 2g/h, immune cells were actually isolated from the colon, but rather than perform flow cytometry, were assessed by bulk expression. Production and secretion of antibodies is consistently measured by Western Blot, while this is mostly done by more specific ELISA assay (which can be done on the sample sample type). This make quantification much more reliable, as the western blots are not all convincing (eg Suppl figure 2g really does not show reliable bands, and thus quantifications is not very meaningful).\n3) The role of metabolic changes seems to be an overreach. Metabolomics are performed on plasma, and can therefore not be correlated to local B cells. Bulk RNASeq similarly does not indicate the involvement of B cells. The mechanism is mainly suggested based on RNA transcription (at best suggestive, not definitive for metabolic activity) and correlations. If they authors really want to state that decreased OXPHOS is involved in the impaired differentiation of PC in IBD, they need to perform the actual experiment, which would be Seahorse assays on the B cells themselves.\n4) I don’t understand the use of Vimentin as a positive marker in this study. I understand the need to correct in some way for the amount of lamina propria included in the respective samples. However, this is commonly done using DAPI or another nuclear/DNA stain. Correcting against total vimentin as a LP marker is unusual but I can understand to some degree. However, the authors refer to the plasmablasts/cells (as well as T cells) as Vim+, which seems odd, as this is not a marker for these cells at all?\nOther specific concerns\n- Several graphs contain delta-Ct values as a measure of absolute expression. Delta-CT is a perfectly fine methods for assing relative expression of a particular genes, but can not be compared between different primers, and thus different genes. These values should not be in a single graph (eg Suppl fig 2e), and definitely not in a stacked bar such as figure 4e. The statement ‘cells mainly expressed CD20, CD27 etc’ is incorrect from this figure (I do believe they do, but the conclusion can not be drawn from this figure).\n- Several figures are presented in a way which does not allow any statistical analyses, even though the data itself would. For example figure 2h shows a compounds alteration in expression, with no information regarding distribution and thus no insight into the robustness of the data. Fig 3j is used to suggest increased expression of IL6, which is highly doubtful based on the image (seems mainly driven by 1 donor).\n- Since identification of cells in the spatial analysis is crucial, some images of the separation and analysis should be provided as supplementary information. Showing the staining of a single double/triple stained cells does not prove anything else than the fact one exists. For example, the number of T cells (or at least CD3+ cells) appears extremely low, and should be addressed.", "reviewer_3": "The manuscript by Raschdorf et al. provides a deep dive into the role of IgA+ plasma cells and blasts in Crohn’s disease. Unconventionally, they target patients in remission (i.e., without active inflammation). They apply bulk RNA sequencing, fluorescent microscopy, CosMX, mass-spec, ex vivo profiling and in vitro mouse modelling to propose a cell-intrinsic defect in the terminal differentiation of CD19-CD45-CD138+ long-lived plasma cells. Increased CD38 expression and the employment of ketogenesis and glycolytic activity suggest OXPHOS deficiency in these cells. Together, the manuscript sheds light on altered IgA plasma cell profiles driving dysregulated barrier control in CD, rather than being simply in response to inflammation. The manuscript is well written and logical. The numbers of participants included in the study are not large (i.e., 7 vs 12, 6 vs 5 and 6 vs 6), but the chosen methods are appropriate and executed effectively. I have only minor comments.\n1. To further support defective regulation of the microbiota in CD rem, the authors could consider B cell receptor repertoire analysis or analysis of IgA binding to faecal microbiota (e.g., by flow cytometry).\n2. Methods page 17 line 18: Patients were classified as being in remission if the colon presented without macroscopic evidence of mucosal inflammation. Was this confirmed histologically?\n3. Methods page 19 line 19: The ‘different combinations of cytokines’ used in the differentiation of PBs and PCs should be explicitly stated.\n4. Please include catalogue numbers for kits included throughout the methods section."}, "author_response": ""}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have made significant changes to the manuscript and have addressed or provided reasonable explanations for most of the concerns I raised in the initial review. Indeed, the authors clearly went to great lengths to seriously engage with the reviewers’ comments, which should be commended. The development of a dIgA ELISA in such a short timeframe is especially impressive.\nThe one potential difficulty still remaining is the extremely limited number of biological replicates in the mouse experiments, which the authors were able to only partially address. My understanding is that this is due to unfortunate and unpredictable circumstances and certainly not due to any lack of willingness by the authors to engage with the problem. At this point, the possible solutions would be to either remove the mouse data or to proceed with the small number of replicates to publication. On the whole, I very much favour the latter option, providing that the limitation inherent in the small sample sizes is clearly indicated in the text. The mouse data is not the primary focus of the manuscript but is still informative and provides a potential mechanistic explanation for the observed phenotype that is novel and interesting.\nUltimately, the final decision lies with the editors and adherence to the editorial policy of Nature Communications. However, from the point of view of scientific peer review I am happy to endorse the publication of the manuscript.", "reviewer_2": "The authors have resolved most of my concerns in their rather lenghty rebuttal. Some concerns remina, which can probably be handled textually.\nConcern1\n- The use of samples including those with inflammtion in the small bowel\nThe authors extensively describe their samples in their response, which largely satisfies my concerns regarding their patient selection. The only thing I would like to confirm before assigning these patients as ‘in remission’ is whether these patients previously showed colonic disease. I agree that someone with previous colonic involvement and currently only aftous lesions on the ileocecal anastomosis is in remission. However, to differentiate their study from earlier work, it is crucial these patients did in fact have colonic involvement at some point in their disease history. I am willing to assume that is the case, but it would be important to confirm this in the patient characteristics.\nConcern 2\n- The use of low sample numbers, which for sure has underpowered several analyses\nI appreciate the fact that the study was focused on techniques not suitable for high throughput analysis (ie spatial transcriptomics) and would not require to increase sample size for these expeirments. However, I would be very careful in stating anything regarding the RNASeq, which clearly is underpowered. Representation of the data is now improved, and I can agree to that. However, still any mention regarding the effect of medication here should be removed to the small group sizes.\nThe additional analysis of IgA based on medication does unfortunately not resolve matters. These data show no significant differences, but include many different treatments grouped together or still in too small numbers to provide meaningful information. Although not significant, for example anti-TNF vs other biologicals does appear to show a trend to higher IgA in the ‘other’ group. This may very well be explained by differences in initial disease activity (patients receiving other biologicals almost always have failed anti-TNF and thus suffer more refractory disease), but still confounds the data.\nAdditionally, no patient in this sub-cohort appears to be using no biologic at all, making this an unusual patient cohort.\nSince the authors do not focus on the effect of medication at all, I would remove most comments regarding this data, and at best state that although numbers were too small for statistical analysis, they did not find data suggesting an effect of the medication (if they want to mention medication at all cost).\nConcern3\n- The data regarding metabolomics was mainly associative and lacked functional assays\nI appreciate the initial focus of the study did not allow for the inclusion of for example Seahorse experiments and the added metabolomic analysis did add support for the claims of the authors. I would appreciate some statements that this requires further study etc in the discussion.", "reviewer_3": "The authors have satisfactorily addressed each of my comments to their original manuscript and I have no additional comments. The revised manuscript has been improved through the revision process."}, "author_response": "The authors thank the reviewer for raising this comment and fully agree that the localization of, for example, B cells is very important for the interpretation of the data.\nThe main focus of our study was on investigating PC differentiation in the colonic mucosa of CDrem patients, for instance using spatial single-cell proteomics on FFPE tissue slices (CosMxTM SMI). Since CD138+ PCs are not located in lymphoid follicles/aggregates but mainly in the LP (Figure 1), no regions were excluded from the PC-specific analyses in Figure 3c-h and Figure 5a-d of the original manuscript.\nFig. 1 | Representative image of a lymphoid aggregate in the colonic biopsy section of a CDrem patient showing that CD138+ PCs (green) are almost absent in the lymphoid aggregate and are exclusively located in the LP.\nIn the first version of the manuscript, the exclusion of lymphoid follicles/aggregates from the analysis of total immune cells was only described in the methods section on page 22, lines 28-29 and page 23, line 1. Additionally, we edited a sentence in the results section of the revised manuscript file on page 7, line 21 (highlighted in yellow) to clarify that the analysis focuses on immune cell frequencies in the LP.\nFig. 2 | Representative image of a lymphoid aggregate in the colonic biopsy section of a CDrem patient with strong B (CD19, CD20) and T cell signals (CD3, CD4, CD8) that was excluded from analysis of immune cell proportions.\n(2) The authors demonstrate an increased proportion of B cells and plasmablasts in the colons of CDrem patients but no overall change in the proportion of plasma cells. However, it is unclear if there is an overall increase in infiltrate in the colons of these patients, i.e. whether there are more leukocytes overall. If yes, this would actually indicate an increase in the overall number of PCs, albeit disproportionally when compared to PBs. Could the authors provide any indication whether the numbers of leukocytes is increased in CD patients (e.g. quantified per unit area)?\n- We are grateful to the reviewer for pointing out this aspect and recognize the critical importance of distinguishing between relative and absolute immune cell numbers to interpret the data properly.\nTherefore, it is essential to determine the density of total leukocytes/LP cells in the colonic tissue. For this purpose, we determined cell numbers of lamina propria mononuclear cells (LPMNCs) isolated from seven sequential colonic biopsies per patient, which is presented in Supplementary Figure 1b of the original manuscript. In this context, we identified a small error in the graph, where one active CD patient without colitis was inadvertently included, while another CD patient with endoscopically confirmed active colitis had been excluded. This has now been corrected in Supplementary Figure 1c and Supplementary Table 4 of the revised manuscript. Although absolute LPMNC counts show considerable inter-patient variability (mainly due to differences in biopsy size), it is evident that the CDrem patients do not exhibit an increased LPMNC count when compared to non-IBD controls. In contrast, the number of colonic LPMNCs isolated from CD patients with active colitis was significantly increased compared to CDrem patients.\nMoreover, the reviewer’s suggestion prompted us to also consider the LP cell density within the colonic biopsy sections analyzed using spatial single-cell proteomics. Therefore, we quantified the total number of Vimentin+ cells, effectively excluding all epithelial cells (Figure 3a), and normalized the counts to the tissue area [mm2]. We preferred the quantification of Vim+ cells over CD45+ cells for two main reasons.\nFirst, vimentin is also expressed by non-immune cells such as fibroblasts, which increase during inflammation. This is supported by single-cell transcriptomics data of the Human Gut Cell Atlas, published by Elmentaite et al. in Nature (2021)1. While all LP cells, such as immune cells, endothelial cells, neuronal cells, and mesenchymal cells, express VIM, it is absent in colonic epithelial cells (Figure 3b). Second, not all immune cells express CD45. For example, mature PCs, which are the focus of our study, typically lose surface CD45 during differentiation.\nFig. 3 | Vimentin serves as a marker for colonic lamina propria (LP) cells. (a) Representative image of Vimentin staining (yellowish green) of the colonic LP cells of a non-IBD control in the CosMxTM SMI. (b) VIM (Vimentin) expression in colonic immune, endothelial, neuronal, mesenchymal, and epithelial cells. Single-cell transcriptomics data originate from the publicly available Human Gut Cell Atlas1.\nOmitting FOVs comprising lymphoid follicles/aggregates, there is no significant difference in the number of Vim+ cells/mm2 between CDrem patients and non-IBD controls (Figure 4). This observation rules out leukocyte/cell infiltration in these patients, as it would result in a higher density of LP cells.\nFig. 4 | Absolute number of Vim+ cells per tissue area [mm2] quantified in colonic biopsy sections of CDrem patients (n=6) and non-IBD controls (n=6). Mann-Whitney U test.\nReferring to the reviewer’s comment, we found that the absolute number of B cells per mm2 is significantly increased in CDrem patients, PBs show a strong upward trend, while the absolute number of PCs per mm2 is not significantly different from non-IBD controls (Figure 5).\nFig. 5 | Absolute numbers of B cells (a; CD19+), PBs (b; CD19+CD27+CD38+CD20-CD138-), and PCs (c; CD138+) per tissue area [mm2] in CDrem patients (n=6) compared to non-IBD controls (n=6).\n(3) When discussing figure 3e, the authors state “As expected, CD138 expression was similar across the 2 populations, while the proportion of Ki-67+ cells was reduced in CD19-CD45+ and CD19-CD45- PCs compared to CD19+CD45+ PCs”. However the data in 3E show no significant difference between the latter two subpopulations. Please amend the text accordingly. Moreover, it would be helpful to show whether the Ki67 pattern seen in CD patients is also present in healthy colonic PC subpopulations.\n- The authors thank the reviewer for pointing out this mistake. We have amended the sentence on page 9 lines 10-12 to clarify that only CD19-CD45+ PCs, but not CD19-CD45- PCs, displayed a significantly decreased percentage of Ki-67+ cells compared to CD19+CD45+ PCs.\nSince the focus of Figure 3e is on the general characterization of the three PC subtypes, the data presented include only the six non-IBD controls. The authors have now added this information to the results section on page 9, line 10 (highlighted in yellow), in addition to the legend of Figure 3e.\nMoreover, the dotplot in Figure 5a of the original manuscript demonstrates that there is no significant difference in the percentage of Ki-67+ cells between CDrem patients and non-IBD controls in any of the PC subpopulations.\nTo further improve the general characterization of the three PC subpopulations, we have now added a spatial analysis to the revised manuscript that allowed determination of the distance between the three PC types and the colonic epithelial cells. While no differences were observed between CDrem patients and non-IBD controls, the fully mature CD19-CD45- PCs were found in closer proximity to the epithelium compared to the CD19+CD45+ and CD19-CD45+ PCs (Figure 6). The data were included as Supplementary Figure 2f in the revised Supplementary Material file and respective changes in the text have been highlighted in yellow and added to the results section on page 9, lines 17-19 and the methods section on page 25, lines 14-16.\n(4) In 3I the authors quantify dIgA/BCMA ratios by western blot and show a reduced ratio in CD compared to healthy controls and state that “… the significantly reduced dIgA/BCMA ratio indicated a decreased secretion of dIgA per PB/PC in the colonic mucosa of CDrem”. However, it is unclear if the reduced ratio is primarily due to more BCMA or less dIgA? The authors already show an increase in the number of colonic B cells, which would presumably also increase the BCMA signal and lead to a skewed ratio (Less dIgA per B/PB/PC would then be tautological). A more informative quantification might be a ratio of dIgA/CD138 to get a measure of dIgA production per PC instead.\nIndeed, the reviewer raises an important point regarding the amount of colonic dIgA in relation to the number of mucosal B cells/PBs/PCs. The significantly decreased dIgA/BCMA ratio in CDrem patients from Western blot experiments reflects a combined effect of increased BCMA, shown in Figure 2c of the original manuscript, and a strong trend towards reduced dIgA levels, depicted in Figure 7a. Additionally, decreased dIgA/total IgA and dIgA/mIgA ratios in CDrem patients compared to non-IBD controls are displayed in Supplementary Figure 2g of the original manuscript. Increased levels of BCMA could be validated by upregulated expression of TNFRSF17, the gene encoding BCMA, in the colonic tissue of CDrem patients, as shown in the RNA-seq data in Figure 1d of the original manuscript.\nHowever, we respectfully disagree with the reviewer’s assumption that the increased BCMA levels are caused by the expansion of colonic B cells in these patients. According to published data, BCMA/TNFRSF17 is marginally expressed on B cells but is highly expressed in later differentiation stages like PBs and PCs. This is supported, among others, by single-cell transcriptomics data of the Human Gut Cell Atlas (Figure 7b), published by Elmentaite et al. in Nature (2021)1. Given that CosMx spatial single-cell proteomics revealed no increase in PCs but elevated PBs (Figure 2g of the original manuscript file), we conclude that the increased BCMA levels in these patients are primarily driven by mucosal PB expansion. We understand the reviewer’s suggestion that normalizing dIgA levels to CD138 would be more informative in this context. However, while CD138 is a well-established marker for identifying PCs within immune cell populations, its interpretation in colonic tissue is confounded by its lower mRNA expression in epithelial cells (Figure 7c). In the CosMx analyses, selection of Vim+ cells allowed us to focus on CD138+ LP cells. However, in Western blot experiments, CD138+ PCs cannot be distinguished from CD138+ epithelial cells by the commercially available antibodies we have utilized. Considering the specific expression of BCMA on PBs and PCs, in contrast to B cells, and the fact that CD138 is also expressed by epithelial cells in colonic tissue, we consider the dIgA/BCMA ratio as a valid indicator of the amount of dIgA secreted per PB/PC. We hope that the reviewer agrees with it.\nTo illustrate specific expression of TNFRSF17 (BCMA) in PBs and PCs, but not B cells, we have included the respective data from the Human Gut Cell Atlas as Supplementary Figure 1i in the revised manuscript and included this information on page 8 lines 15-16 (highlighted in yellow).\nFig. 7 | Colonic dIgA levels per PB/PC. (a) Dimeric IgA levels were quantified in colonic biopsies from CDrem patients and non-IBD controls using Western blot experiments. (b) and (c) TNFRSF17 (BCMA; b) and SDC1 (CD138; c) expression in colonic B cells, PBs/PCs and epithelial cells. Single-cell transcriptomics data originate from the publicly available Human Gut Cell Atlas1. (a) Mann-Whitney U test. One outlier was identified and removed by the Grubb’s test (α = 0.05).\n(5) Related to this, could a reduction in dIgA production be due to an increase in monomeric IgA production? Can the method used accurately quantify mIgA?\nThe authors appreciate the reviewer’s comment and can follow their line of thought. To our knowledge, there is no commercial ELISA available for the specific detection of monomeric or dimeric IgA2.\nConsidering this, along with the limited sample volume, we believe that non-reducing Western blotting represents the most suitable method to differentiate between the two IgA forms in this context.\nHowever, in response to a comment raised by reviewer 2, we optimized the Western blotting conditions, now using XT Tricine Running Buffer (Bio-Rad #1610790) in combination with a 3-8% CriterionTM XT Tris-Acetate polyacrylamide gel (Bio-Rad #3450131) to improve visualization of mIgA and dIgA (Figure 17a below). While the amount of dIgA relative to total IgA or mIgA was reduced in the colonic mucosa of CDrem patients, the proportion of mIgA was increased (Figure 17b below). At this stage, it remains uncertain whether this reflects an increase of mIgA-secreting PCs or an expansion of IgA+ precursor cells. However, considering the broader context of our data, the latter appears more likely. Since the mIgA signal detected by Western blotting cannot differentiate between intracellular, membrane-bound, or secreted forms, the IgA levels measured in the supernatant of the ex vivo differentiated PCs provide a more accurate indication of secreted forms.\nWe substituted the original Western blot and corresponding quantifications in Supplementary Figure 2g with the optimized Western blot and quantification, which is now provided in Figure 3i and j and Supplementary Figure 2i in the revised manuscript. Additionally, we have addressed the increased proportion of mIgA relative to total IgA in the results section on page 9, lines 27-28.\n6\n(6) The data in figure 4 is very informative and convincingly shows an intrinsic defect in CD B cell differentiation into IgA-secreting PCs. Could the authors specify whether the ELISA used for quantification of IgA secretion in 4G detects only dIgA or whether it would also measure mIgA? If not, could the western blot quantification (from 4C) be utilised instead? I think this could be an important point in understanding the defect of CD PCs, as a reduction of J chain production could indicate more mIgA secretion which would also account for total reduced SIgA. Similarly, does the ELISA used discriminate between IgA1 and IgA2, as the latter seems to be disproportionately affected in CD?\nThe authors appreciate the reviewer’s insightful comment regarding the experimental distinction of IgA forms in the supernatant of the ex vivo differentiated colonic PCs. The ELISA used for quantification of IgA secretion in Figure 4g of the original manuscript detects total IgA (monomeric and dimeric). To our knowledge, there is no commercial ELISA available that can differentiate between total IgA and dIgA2.\nIn response, we have established an ELISA for the specific detection of dIgA during the course of the revision process. First, we characterized the IgA variants in different (purified) IgA samples via Western blotting (Figure 8a). Accordingly, we used monomeric IgA from human serum (lane 1), human IgA from colostrum (lane 2, mainly sIgA), human IgG1 from myeloma (lane 3), and recombinantly expressed human anti-CD38 mIgA2 (lane 6) as negative controls. Native human colon protein lysate (lane 5) contained all three IgA forms (mIgA, dIgA, and sIgA) and served as a positive control. As human IgA κ-UNLB (#0155K-01, SouthernBiotech) is mainly comprised of dIgA (lane 4), it was used as a standard in the ELISA. For specific detection of J-chain-bound dIgA, we coated the plate with increasing amounts of human recombinant pIgR protein or bovine serum albumin (BSA) and validated specific binding of human IgA κ-UNLB to pIgR (Figure 8b). Based on these results, we coated 150 ng pIgR/well and observed a concentration-dependent binding of human IgA κ-UNLB to pIgR, with saturation at higher concentrations (Figure 8c). The specificity of dIgA detection was validated by the absence of signal from mIgA and sIgA, whereas the native colon protein produced a strong signal (Figure 8d). Measuring the supernatant of ex vivo differentiated colonic PCs between Day 10-14 revealed significantly decreased dIgA levels for CDrem- versus non-IBD-derived PCs (Figure 8e), comparable to the total IgA ELISA depicted in Figure 4g of the original manuscript.\nAs we cannot directly quantify mIgA in the supernatant using ELISA, we calculated the ratio of dIgA levels measured by the newly established dIgA-specific ELISA to total IgA levels measured by ELISA in the original manuscript. The similar dIgA/total IgA ratio excludes a compensatory increase in mIgA secretion in CDrem- versus non-IBD-derived colonic PCs (Figure 8f). This is further supported by the observed positive correlation between dIgA and total IgA in the supernatant samples (Figure 8g).\nAs we cannot directly quantify mIgA in the supernatant using ELISA, we calculated the ratio of dIgA levels measured by the newly established dIgA-specific ELISA to total IgA levels measured by ELISA in the original manuscript. The similar dIgA/total IgA ratio excludes a compensatory increase in mIgA secretion in CDrem- versus non-IBD-derived colonic PCs (Figure 8f). This is further supported by the observed positive correlation between dIgA and total IgA in the supernatant samples (Figure 8g).\nAs already depicted in Supplementary Figure 3e and Figure 4f of the original manuscript, we have quantified IGHA1 and IGHA2 mRNA expression in the ex vivo differentiated colonic PCs using qPCR. Although the effect was slightly less prominent compared to IGHA2 (P=0.0619), IGHA1 expression also tended to be decreased in CDrem- compared to non-IBD-derived colonic PCs (P=0.1469). We here provide an additional analysis of the IGHA2/IGHA1 ratio, which was found to be comparable between CDrem and non-IBD (Figure 9a). Based on these findings, we do not assume a shift in IgA subclass composition.\nTogether, colonic PC-specific IGHA1 and IGHA2 expression levels display a strong positive correlation, with most CDrem-derived colonic PCs expressing lower amounts of both, IGHA1 and IGHA2 (Figure 9b).\nAs we are confident that it adds value to the manuscript, we included the quantified dIgA levels in the supernatant of the ex vivo differentiated PCs in Figure 4g of the revised version, together with the correlation between dimeric and total IgA secretion. The interpretation of the results is provided in the results section on page 11, lines 3-6 (highlighted in yellow). In the interest of reproducibility, we briefly described the procedure and validation of this newly established ELISA in the results section on page 23, lines 22-29 and added the respective data as Supplementary Figure 7 in the revised manuscript.\nFurther, we decided to include the IGHA2/IGHA1 ratio and the correlation of both isoforms as Figure 4f in the revised manuscript and added the corresponding interpretation in the results section on page 10, line 30 and page 11, line 1-3 (highlighted in yellow).\n(7) Could the authors provide a more detailed explanation of what the images in Figure 6A are supposed to illustrate?\n- The authors thank the reviewer for identifying the missing explanation of the immunofluorescence staining presented in Figure 6a of the manuscript. The images depict a co-staining of IgA and P32, a pivotal protein targeted to the mitochondria to facilitate mitoribosome assembly and enable the translation of mitochondrially encoded subunits of the electron transport chain3,4. In previous studies, the authors could verify that mitochondrial translocation of P32 is essential for respiration in different cell models5,6. Given the specificity of the antibody to mitochondrial P32, the strong signal observed in IgA+ LP cells underscores their high OXPHOS activity.\nThe authors added information on the function of P32 to the results section on page 12 lines 4-5 and to the legend of Figure 6a in the revised manuscript (highlighted in yellow) to provide a more comprehensive explanation.\n(8) In figure 7, the overall number of mice used seems very low with only two mice per group in 7E and three mice in 7F and J. The authors should increase the number of biological replicates and perform all experiments on at least two occasions to demonstrate reproducibility. In addition, could the authors indicate why some experiments were performed only on male mice (qPCR) and some only on female mice (GFHP diet).\nWe appreciate the reviewer’s careful reading and fully understand the concern regarding the limited sample size in Figure 7c-e (n=2 mice per group) and Figure 7f and j (n=3 mice per group).\nUnfortunately, we are unable to further increase the number of biological replicates, as the breeding of the conplastic mouse strain C57BL/6J-mtFVB/NJ has been unexpectedly discontinued and no living animals are currently available. Further details can be found in the separate file ‘Official statement_mice availability’ by PD Dr. Misa Hirose (breeding coordinator).\nIn addition to the four male mice (n=2 Atp8-mutant, n=2 B6-WT) in the splenic B-cell stimulation experiment (Figure 7c-e of the original manuscript), we also analyzed two female mice (n=1 Atp8-mutant, n=1 B6-WT). However, it is well established that mice exhibit sex-specific differences in immune system characteristics, with females generally displaying stronger adaptive immune responses7,8. For this reason, we initially decided against including the data from the female mice in the analysis.\nHowever, several studies indicate that sex-related disparities in adaptive immunity become more evident only in middle-aged or older mice and are partially driven by hormonal factors9. Additionally, in an ongoing study using C57BL/6J WT mice, we compared the colonic proteome of males and females using liquid chromatography-coupled mass spectrometry (LC-MS) and observed that several adaptive immunity-associated proteins were upregulated in 17-month-old female mice compared to age-matched males (Figure 10a). Notably, such differences were absent in younger, 4-month-old animals (Figure 10a), which corresponds to the age of the Atp8-mutant and B6-WT mice used in our experiments. Data from the colonic compartment were validated by qPCR analyses performed on splenic samples from 4-month-old mice, which similarly showed no significant differences in Cd19, Sdc1 (CD138), or immunoglobulin heavy chain expression between females and males (Figure 10b).\nFig. 10 | Sex-specific differences in adaptive immunity in mice depend on age. (a) Colonic levels of humoral immunity-related proteins were quantified via liquid chromatography-coupled mass spectrometry (LC-MS) in 4-month-old (left) and 17-month-old (right) B6-WT mice and compared between sexes. (b) Comparison of expression levels of humoral immunity-associated transcripts in splenic tissue of 4-month-old male and female B6-WT mice using RT-qPCR. (a) and (b) Two-way ANOVA with Šídák’s multiple comparisons test. ***P.001.\nSince the experiments presented in the manuscript were conducted using 3- to 4-month-old mice (median: 15.14 weeks, IQR: 14.86-18.00), we decided to include the data from the two female animals (n=1 Atp8-mutant, n=1 B6-WT) in Figure 7c-e and Supplementary Figure 5b of the revised manuscript in order to increase the statistical power (Figure 11a-d). Respective changes in the text have been highlighted in yellow and added to the methods section on page 19, line 14 and the results section on page 13, lines 13, 14, and 16.\nAs the percentages of CD138+ PCs in Atp8-mutant and B6-WT mice are very similar between MLNs and PPs (Supplementary Figure 5c of the original manuscript), we decided to combine the data of both organs into one graph to improve statistical power (Figure 11e) and hope that the reviewer agrees with it. PCs from MLNs and PPs are displayed as circles or squares, respectively, and the legend of Supplementary Figure 5g of the revised manuscript file has been updated accordingly (highlighted in yellow). However, in Figure 7f of the revised manuscript, the data for MLNs and PPs are still presented separately.\nFig. 11 | Ex vivo IgG1+ PB differentiation of splenic B cells with mitochondrial dysfunction. (a) Generation of IgG1+ cells upon LPS+IL4 stimulation is shown for splenic B cells from three B6-WT mice. (b) Percentage of TMREhigh cells in LPS+IL4 stimulated or unstimulated splenic B220+ cells from Atp8-mutant (n=3) versus B6-WT mice (n=3). (c) Expansion of Atp8-mutant and B6-WT B cells under LPS+IL4 stimulation relative to the unstimulated controls. (d) B220 level of LPS+IL4 stimulated or unstimulated splenic B220+ cells from Atp8-mutant compared to B6-WT mice. (e) Percentages of B220+ and B220- CD138+ PCs isolated from the MLNs (circles) and PPs (squares) of Atp8-mutant (n=3) and B6-WT mice (n=3). (a) Mann-Whitney U test, (b)-(e) Two-way ANOVA with Fisher’s least significant difference (LSD) test. *P.05, **P.01.\nTo validate the dysregulated B-cell compartment in a larger number of independent mice, we retrospectively analyzed spleen tissue samples from one of our previous studies involving n=8 Atp8-mutant and n=4 B6-WT mice, in which chronic colitis was induced by administering 2% DSS via the drinking water for three cycles of 5 days each, with recovery periods in between. Western blotting revealed significantly increased IgG levels in the spleen of Atp8-mutant compared to B6-WT mice (Figure 12a). Elevated IgG levels in the spleen of Atp8-mutant mice could reflect enhanced B-cell proliferation, as we also observed in isolated splenic B cells stimulated with LPS+IL4, shown in Figure 7d of the original manuscript. Consistent with the mitochondrial dysfunction confirmed by reduced mt-Nd3 expression, PC numbers seem diminished, as indicated by significantly lower Sdc1 mRNA levels in the spleen of Atp8-mutant mice (Figure 12b).\nFig. 12 | Dysregulated splenic B-cell compartment in Atp8-mutant mice under chronic DSS-induced colitis. Quantification of (a) splenic IgG levels by Western blot and (b) splenic expression of mt-Nd3 and Sdc1 mRNA via RT-qPCR for Atp8-mutant (n=8) versus B6-WT (n=4) mice under chronic DSS-induced colitis. Data are displayed as relative values to B6-WT mice. (a) Mann-Whitney U test, (b) Two-way ANOVA with Holm- Šídák’s multiple comparisons test. *P.05. AU, arbitrary units.\nHowever, the stimulation experiment of splenic B cells (Figure 7c-e of the original manuscript) was performed using six mice on two separate days. The six mice used for the analysis of MLNs and PPs (Figure 7f of the original manuscript) were also sampled on two different days, and the corresponding flow cytometry experiments were performed independently on each day to demonstrate reproducibility. The six mice used for colonic IgA IHC staining were sacrificed on two consecutive days, and the reduction of colonic IgA in Atp8-mutant mice was additionally confirmed by qPCR analysis in a substantially larger number of animals (Figure 7g of the original manuscript, n=12 per group).\nMoreover, it is important to note that different animals were used for the individual experiments (spleen, MLNs/PPs, colon), yet all consistently showed a compromised B-cell compartment in Atp8-mutant mice, supporting the reproducibility of this phenotype.\nThe predominant use of male or female mice in the Atp8-mutant or glucose-free high-protein diet (GFHPD) experiment, respectively, is attributable to technical reasons.\nThe predominant use of male Atp8-mutant and matching WT mice was not intentional, but resulted from the fact that almost exclusively male mice were available from the breeding at that time.\nHowever, for the analysis of mesenteric lymph nodes (MLNs) and Peyer’s patches (PPs), female Atp8-mutant and B6-WT mice were used. To improve clarity for the readership, we now explicitly state the sex of the animals in the corresponding legends of Figure 7b, g, j, and l and Supplementary Figure 5i in the revised manuscript file (highlighted in yellow).\nThe female GFHPD- and isoenergetic chow diet-fed mice originate from the control arm of a large-scale mouse study10. In this study, colonic tumors were induced by AOM/DSS treatment in half of the mice. After 10 weeks, the mice were assigned to different intervention groups (GFHPD/chow diet; anti-EGFR mAb/isotype ctrl. mAb/PBS) in a way that ensured approximately equal body weight distribution across groups. Reorganization of cage groups, however, is only feasible with female mice, as male mice that were not raised together tend to fight when housed in the same cage.\n(9) The authors use in vitro differentiation of splenic B cells to show a defect in the capacity of Atp8-mutant cells to develop into IgG-producing PCs, however the relevance of these data is unclear. Since these experiments are supposed to provide a mechanistic explanation for the human data, which shows a selective defect in IgA-PC induction, the authors should utilise conditions that favour induction of IgA producing PCs (i.e. IL-6, IL-21, TGFb) for their in vitro assay.\nWe understand the reviewer’s perspective and would like to clarify that the purpose of the Atp8-mutant versus B6-WT mouse experiments was to demonstrate a general impairment in PC differentiation, which is evidently not restricted to mucosal IgA+ PCs. The defect in the colonic PC compartment is subsequently characterized in Figure 7g-j of the original manuscript. We also attempted to differentiate isolated splenic resting B cells into IgA+ PBs/PCs by stimulation with 10 ng/ml IL-6 and 0.1 µM retinoic acid (RA). However, this approach was not successful, as the cells did not proliferate, comparable to the unstimulated controls (Figure 13).\nUnder physiological conditions, IgA class switch recombination (CSR) does not typically occur in the spleen, but primarily takes place in mucosa-associated lymphoid tissues, such as PPs, MLNs, or LP. As a systemic lymphoid organ, the spleen predominantly supports CSR to IgG isotypes, especially IgG1 and IgG2a. Therefore, we focused here on the ex vivo differentiation of LPS-activated B cells into IgG1+ PBs/PCs, which is specifically induced by IL-4 stimulation11,12.\nFig. 13 | Ex vivo differentiation of splenic B cells from Atp8-mutant and B6-WT mice into PBs. (a) Expansion of Atp8-mutant and B6-WT splenic B cells under LPS+IL-4 or IL-6+retinoic acid (RA) stimulation for four days. (b) Representative contour plots of B220 and IgG1 (top) or IgA (bottom) on splenic B cells of a B6-WT mouse upon stimulation with LPS + IL-4 (top right) or IL-6+RA (bottom right). (a) Two-way ANOVA with Tukey’s multiple comparisons test. **P..01, ***P.001.\n(10) Similarly, the relevance of Figure 7F is uncertain. Since the main phenotype observed in human concerns colonic PC in the lamina propria, it is unclear why the authors chose to analyse lymphoid tissues associated with the small intestine (MLN and PP). This analysis should be complemented by FACS (or multi-coloured immunohistochemistry) of PCs in the colonic LP. This has the added benefit that it would allow for a much more detailed phenotypic description of these cells than the rather uninformative “IgA+ cells” obtained from the histology in 7J and 7I.\nHere, the authors would like to point out that the aim was to demonstrate a systemic impairment of B-cell/PC differentiation in Atp8-mutant compared to B6-WT mice. Accordingly, we included different organs and tissues for analysis, such as spleen, MLNs, PPs, and colonic mucosa.\nSince we no longer have access to additional conplastic C57BL/6J-mtFVB/NJ mice (see comment 8 above), we unfortunately are not in a position to carry out the experiments suggested by the reviewer at this time. However, we would like to draw the reviewer’s attention to the qPCR data presented in Figure 7g and Supplementary Figure 5d of the original manuscript, which characterize the PC compartment in the colonic LP in more detail. Notably, the reduced expression of colonic B-cell and PC markers such as Cd19, Igha, Tnfrsf17, Sdc1, and Prdm1 in Atp8-mutant animals is reflected in the decreased number of IgA+ cells observed by IHC (Figure 7j of the original manuscript).\nRegarding the glucose-free high-protein diet (GFHPD)- versus isocaloric chow diet-fed mice, we fully agree with the reviewer that quantifying IgA+ cells in the colonic mucosa alone (Figure 7l of the original manuscript) is not sufficiently informative. Therefore, we performed the following additional experiments to further characterize the effect of OXPHOS-promoting dietary intervention on colonic humoral immunity in mice. First, we quantified the expression of various markers along the B cell to PC lineage in colonic tissue from the same animals analyzed by IHC using qPCR. We observed that both, B-cell markers, such as Cd19, Cd79A, and Ighm, and the PB/PC-expressed Tnfrsf17, which encodes BCMA, were significantly upregulated in GFHPD-fed compared to chow diet-fed mice (Figure 14a). These findings suggest that the dietary intervention not only promotes PC differentiation but also affects the entire mucosal B-cell compartment. Furthermore, we performed non-reducing Western blotting using fecal protein samples collected after 10 weeks on the respective diets to quantify IgA. In line with our qPCR and IHC data, we detected significantly increased total IgA and dIgA levels in fecal samples from GFHPD-fed mice compared to controls (Figure 14b), indicating a promoting effect on IgA+ PC maturation. We decided to include these data in the revised manuscript file as Supplementary Figures 5j and k. Respective changes in the results section and discussion have been highlighted in yellow and can be found on page 14, lines 11-14 and on page 17, lines 13-14, respectively.\nFig. 14 | Effect of glucose-free high-protein diet (GFHPD) on colonic humoral immunity in mice. (a) Colonic expression of different B-cell and PC markers was quantified for GFHPD-fed (n=6) and chow diet-fed mice (n=5) via RT-qPCR. Data are displayed as relative values to chow diet-fed mice for two independent experiments. (b) Quantification of fecal IgA levels in mice after 10 weeks of nutritional intervention (chow diet n=3, GFHPD n=3) using non-reducing Western blotting. (a) Mixed-effects model with Fisher’s least significant difference (LSD) test, (b) Two-way ANOVA with Fisher’s LSD test. *P.05, **P.01."}, {"round": 3, "reviewer_comments": {"reviewer_2": "The authors have now satisfied my remaining concerns."}, "author_response": ""}]

Decision letter