Dynamic establishment and maintenance of the human intestinal B cell population and repertoire following transplantation in a pediatric-dominated cohort.

Introduction: It is unknown how intestinal B cell populations and B cell receptor (BCR) repertoires are established and maintained over time in humans. Following intestinal transplantation (ITx), surveillance ileal mucosal biopsies provide a unique opportunity to map the dynamic establishment of recipient gut lymphocyte populations in immunosuppressed conditions. Methods: Using polychromatic flow cytometry that includes HLA allele group-specific antibodies distinguishing donor from recipient cells along with high throughput BCR sequencing, we tracked the establishment of recipient B cell populations and BCR repertoire in the allograft mucosa of ITx recipients. Results: We confirm the early presence of naïve donor B cells in the circulation (donor age range: 1-14 years, median: 3 years) and, for the first time, document the establishment of recipient B cell populations, including B resident memory cells, in the intestinal allograft mucosa (recipient age range at the time of transplant: 1-44 years, median: 3 years). Recipient B cell repopulation of the allograft was most rapid in infant (<1 year old)-derived allografts and, unlike T cell repopulation, did not correlate with rejection rates. While recipient memory B cell populations were increased in graft mucosa compared to circulation, naïve recipient B cells remained detectable in the graft mucosa for years. Comparisons of peripheral and intra-mucosal B cell repertoires in the absence of rejection (recipient age range at the time of transplant: 1-9 years, median: 2 years) revealed increased BCR mutation rates and clonal expansion in graft mucosa compared to circulating B cells, but these parameters did not increase markedly after the first year post-transplant. Furthermore, clonal mixing between the allograft mucosa and the circulation was significantly greater in ITx recipients, even years after transplantation, than in deceased adult donors. In available pan-scope biopsies from pediatric recipients, we observed higher percentages of naïve recipient B cells in colon allograft compared to small bowel allograft and increased BCR overlap between native colon vs colon allograft compared to that between native colon vs ileum allograft in most cases, suggesting differential clonal distribution in large intestine vs small intestine. Discussion: Collectively, our data demonstrate intestinal mucosal B cell repertoire establishment from a circulating pool, a process that continues for years without evidence of stabilization of the mucosal B cell repertoire in pediatric ITx patients.

Computational estimation of clonal diversity in autoimmunity.

Diversity is the cornerstone of the adaptive immune system, crucial for its effectiveness against constantly evolving pathogens that pose threats to higher vertebrates. Accurately measuring and interpreting this diversity presents challenges for immunologists, as changes in diversity and clonotype composition can tip the balance between protective immunity and autoimmunity. In this review, we present the current methods commonly used to measure diversity from single-cell T-cell receptor and B-cell receptor sequencing. We also discuss two case studies where single-cell sequencing and diversity estimations have led to breakthroughs in autoimmune disease discovery and therapeutic innovation, and reflect upon the necessity and importance of accurately defining and measuring lymphocyte diversity in these contexts.

Non B Cell-Derived Immunoglobulins in Intestinal Tract.

Intestinal epithelium constitutes a barrier to the unrestricted movement of pathogens, and other detrimental substances from the external world (gut lumen) into the interstitial environment. Intestinal epithelial cells obstruct harmful substances passing through the epithelium as a physical and chemical barrier; Moreover, the epithelial cells can express Toll-like receptors (TLRs) and cytokines to exert innate immune function. In addition, high levels of immunoglobulin A (IgA) and other antibodies exist in the intestinal mucosa, maintaining intestinal immune homeostasis in conjunction with intestinal probiotics. Traditionally, these antibodies have been deemed to be secreted by submucosal plasma cells. Nonetheless, in recent years, it has been demonstrated that intestinal epithelial cells produce a substantial amount of Igs, especially IgA or free Ig light chains, which are involved in intestinal immune homeostasis and the survival of normal epithelial cells. Furthermore, mounting evidence affirms that many human carcinoma cells, including colorectal cancer (CRC), can overexpress Igs, particularly IgG. Cancer-derived Igs exhibit a unique V(D)J rearrangement pattern distinct from B cell-derived Ig; moreover, this cancer cell-derived IgG also has a unique sialic acid modification on the 162 site of CH1 domain (SIA-IgG). The SIA-IgG plays a crucial role in promoting cancer initiation, progression, metastasis, and tumour immune escape. Simultaneously, CRC cells can also express free Ig light chains, which promote colitis, colitis-associated colon carcinogenesis, and CRC progression. Therefore, Igs expressed by CRC cells could be a potential target for diagnosing and preventing the transformation of inflammation into cancer, as well as treating CRC.

Comprehensive application of AI algorithms with TCR NGS data for glioma diagnosis.

T-cell receptor (TCR) detection can examine the extent of T-cell immune responses. Therefore, the article analyzed characteristic data of glioma obtained by DNA-based TCR high-throughput sequencing, to predict the disease with fewer biomarkers and higher accuracy. We downloaded data online and obtained six TCR-related diversity indices to establish a multidimensional classification system. By comparing actual presence of the 602 correlated sequences, we obtained two-dimensional and multidimensional datasets. Multiple classification methods were utilized for both datasets with the classification accuracy of multidimensional data slightly less to two-dimensional datasets. This study reduced the TCR ß sequences through feature selection methods like RFECV (Recursive Feature Elimination with Cross-Validation). Consequently, using only the presence of these three sequences, the classification AUC value of 96.67% can be achieved. The combination of the three correlated TCR clones obtained at a source data threshold of 0.1 is: CASSLGGNTEAFF_TRBV12_TRBJ1-1, CASSYSDTGELFF_TRBV6_TRBJ2-2, and CASSLTGNTEAFF_TRBV12_TRBJ1-1. At 0.001, the combination is: CASSLGETQYF_TRBV12_TRBJ2-5, CASSLGGNQPQHF_TRBV12_TRBJ1-5, and CASSLSGNTIYF_TRBV12_TRBJ1-3. This method can serve as a potential diagnostic and therapeutic tool, facilitating diagnosis and treatment of glioma and other cancers.

It's a balancing act: The ratio of maladaptive (vs. All) emotion regulation strategies is associated with depression.

Research on emotion regulation (ER) has increasingly recognized that people use multiple strategies simultaneously, often referred to as ER repertoire. Prior research found that ER repertoire is associated with psychopathology, but results have been mixed. Indeed, research from recent years suggests that it is the quality of ERs, more than their quantity, that needs to be considered. Based on the combination of the literatures on ER repertoire, polyregulation, and ER flexibility, we propose a novel metric: the ratio of using putatively maladaptive (vs. all) ER strategies. Using this metric, we examine (1) maladaptive ER ratio changes during the transition to adolescence, a developmental period in which the prevalence of depression sharply increases, and (2) whether changes in maladaptive ER ratio are associated with depressive symptoms. One-hundred and thirty-nine youths (baseline age: 8-15) reported ER strategies and depression daily for 21 days. One year later, 115 completed another 28-day daily-diary (Nassessments = 5631). Our results show that almost all youth use at least some maladaptive ERs. Importantly, maladaptive ER ratio decreases over a year of adolescence for most youths. Conversely, an increased maladaptive ER ratio predicted depression increases on the daily and on the yearly level. These results shed light on typical and atypical development of ER flexibility and emphasize the need to consider the balance between ERs in relation to psychopathology.

Systematic characterization of immunoglobulin loci and deep sequencing of the expressed repertoire in the Atlantic cod (Gadus morhua).

BACKGROUND: The Atlantic cod is a prolific species in the Atlantic, despite its inconsistent specific antibody response. It presents a peculiar case within vertebrate immunology due to its distinct immune system, characterized by the absence of MHCII antigen presentation pathway, required for T cell-dependent antibody responses. Thorough characterisation of immunoglobulin loci and analysis of the antibody repertoire is necessary to further our understanding of the Atlantic cod's immune response on a molecular level. RESULTS: A comprehensive search of the cod genome (gadmor3.0) identified the complete set of IgH genes organized into three sequential translocons on chromosome 2, while IgL genes were located on chromosomes 2 and 5. The Atlantic cod displayed a moderate germline V gene diversity, comprising four V gene families for both IgH and IgL, each with distinct chromosomal locations and organizational structures. 5'RACE sequencing revealed a diverse range of heavy chain CDR3 sequences and relatively limited CDR3 diversity in light chains. The analysis highlighted a differential impact of V-gene germline CDR3 length on receptor CDR3 length between heavy and light chains, underlining different recombination processes. CONCLUSIONS: This study reveals that the Atlantic cod, despite its inconsistent antibody response, maintains a level of immunoglobulin diversity comparable to other fish species. The findings suggest that the extensive recent duplications of kappa light chain genes do not result in increased repertoire diversity. This research provides a comprehensive view of the Atlantic cod's immunoglobulin gene organization and repertoire, necessary for future studies of antibody responses at the molecular level.

Corrigendum: Patients with juvenile idiopathic arthritis have decreased clonal diversity in the CD8+ T cell repertoire response to influenza vaccination.

[This corrects the article DOI: 10.3389/fimmu.2024.1306490.].

Exploring the potential of the TCR repertoire as a tumor biomarker (Review).

T cells play an important role in adaptive immunity. Mature T cells specifically recognize antigens on major histocompatibility complex molecules through T-cell receptors (TCRs). As the TCR repertoire is highly diverse, its analysis is vital in the assessment of T cells. Advances in sequencing technology have provided convenient methods for further investigation of the TCR repertoire. In the present review, the TCR structure and the mechanisms by which TCRs function in tumor recognition are described. In addition, the potential value of the TCR repertoire in tumor diagnosis is reviewed. Furthermore, the role of the TCR repertoire in tumor immunotherapy is introduced, and the relationships between the TCR repertoire and the effects of different tumor immunotherapies are discussed. Based on the reviewed literature, it may be concluded that the TCR repertoire has the potential to serve as a biomarker for tumor prognosis. However, a wider range of cancer types and more diverse subjects require evaluation in future research to establish the TCR repertoire as a biomarker of tumor immunity.

Microbiota dictate T cell clonal selection to augment graft-versus-host disease after stem cell transplantation.

Allogeneic T cell expansion is the primary determinant of graft-versus-host disease (GVHD), and current dogma dictates that this is driven by histocompatibility antigen disparities between donor and recipient. This paradigm represents a closed genetic system within which donor T cells interact with peptide-major histocompatibility complexes (MHCs), though clonal interrogation remains challenging due to the sparseness of the T cell repertoire. We developed a Bayesian model using donor and recipient T cell receptor (TCR) frequencies in murine stem cell transplant systems to define limited common expansion of T cell clones across genetically identical donor-recipient pairs. A subset of donor CD4+ T cell clonotypes differentially expanded in identical recipients and were microbiota dependent. Microbiota-specific T cells augmented GVHD lethality and could target microbial antigens presented by gastrointestinal epithelium during an alloreactive response. The microbiota serves as a source of cognate antigens that contribute to clonotypic T cell expansion and the induction of GVHD independent of donor-recipient genetics.

Improved detection of tryptic immunoglobulin variable region peptides by chromatographic and gas-phase fractionation techniques.

The polyclonal repertoire of circulating antibodies potentially holds valuable information about an individual's humoral immune state. While bottom-up proteomics is well suited for serum proteomics, the vast number of antibodies and dynamic range of serum challenge this analysis. To acquire the serum proteome more comprehensively, we incorporated high-field asymmetric waveform ion-mobility spectrometry (FAIMS) or two-dimensional chromatography into standard trypsin-based bottom-up proteomics. Thereby, the number of variable region (VR)-related spectra increased 1.7-fold with FAIMS and 10-fold with chromatography fractionation. To match antibody VRs to spectra, we combined de novo searching and BLAST alignment. Validation of this approach showed that, as peptide length increased, the de novo accuracy decreased and BLAST performance increased. Through in silico calculations on antibody repository sequences, we determined the uniqueness of tryptic VR peptides and their suitability as antibody surrogate. Approximately one-third of these peptides were unique, and about one-third of all antibodies contained at least one unique peptide.

Single cell RNA-sequencing of feline peripheral immune cells with V(D)J repertoire and cross species analysis of T lymphocytes.

Introduction: The domestic cat (Felis catus) is a valued companion animal and a model for virally induced cancers and immunodeficiencies. However, species-specific limitations such as a scarcity of immune cell markers constrain our ability to resolve immune cell subsets at sufficient detail. The goal of this study was to characterize circulating feline T cells and other leukocytes based on their transcriptomic landscape and T-cell receptor repertoire using single cell RNA-sequencing. Methods: Peripheral blood from 4 healthy cats was enriched for T cells by flow cytometry cell sorting using a mouse anti-feline CD5 monoclonal antibody. Libraries for whole transcriptome, alpha/beta T cell receptor transcripts and gamma/delta T cell receptor transcripts were constructed using the 10x Genomics Chromium Next GEM Single Cell 5' reagent kit and the Chromium Single Cell V(D)J Enrichment Kit with custom reverse primers for the feline orthologs. Results: Unsupervised clustering of whole transcriptome data revealed 7 major cell populations - T cells, neutrophils, monocytic cells, B cells, plasmacytoid dendritic cells, mast cells and platelets. Sub cluster analysis of T cells resolved naive (CD4+ and CD8+), CD4+ effector T cells, CD8+ cytotoxic T cells and gamma/delta T cells. Cross species analysis revealed a high conservation of T cell subsets along an effector gradient with equitable representation of veterinary species (horse, dog, pig) and humans with the cat. Our V(D)J repertoire analysis demonstrated a skewed T-cell receptor alpha gene usage and a restricted T-cell receptor gamma junctional length in CD8+ cytotoxic T cells compared to other alpha/beta T cell subsets. Among myeloid cells, we resolved three clusters of classical monocytes with polarization into pro- and anti-inflammatory phenotypes in addition to a cluster of conventional dendritic cells. Lastly, our neutrophil sub clustering revealed a larger mature neutrophil cluster and a smaller exhausted/activated cluster. Discussion: Our study is the first to characterize subsets of circulating T cells utilizing an integrative approach of single cell RNA-sequencing, V(D)J repertoire analysis and cross species analysis. In addition, we characterize the transcriptome of several myeloid cell subsets and demonstrate immune cell relatedness across different species.

The TCR ß chain CDR3 insights of bovine liver immune repertoire under heat stress.

Objective: The liver plays a dual role in regulating temperature and immune responses. Examining the influence of Heat stress (HS) on liver T cells contributes significantly to understanding the intricate interplay between the immune system and hepatic tissues under thermal stress. This study focused on investigating the characteristics of the T-cell receptor (TCR) ß chain CDR3 repertoire in bovine liver samples under both HS and pair-fed (PF) environmental conditions. Methods: Sequencing data from six samples sourced from the GEO database underwent annotation. Utilizing immunarch and VDJtool software, the study conducted comprehensive analyses encompassing basic evaluation, clonality assessment, immune repertoire comparison, diversity estimation, gene usage profiling, VJ gene segment pairing scrutiny, clonal tracking, and Kmers analysis. Results: All four TCR chains, namely α, ß, γ, and δ, were detected, with the α chains exhibiting the highest detection frequency, followed closely by the ß chains. The prevalence of αß TCRs in bovine liver samples underscored their crucial role in governing hepatic tissue's physiological functions. The TCR ß CDR3 repertoire showcased substantial inter-individual variability, featuring diverse clonotypes exhibiting distinct amino acid lengths. Intriguingly, HS cattle displayed heightened diversity and clonality, suggesting potential peripheral T cell migration into the liver under environmental conditions. Notably, differential VJ gene pairings were observed in HS cattle compared to the PF, despite individual variations in V and J gene utilization. Additionally, while most high-frequency amino acid 5-mers remained consistent between the HS and PF, GELHF and YDYHF were notably prevalent in the HS group. Across all samples, a prevalent trend of high-frequency 5mers skewed towards polar and hydrophobic amino acids was evident. Conclusion: This study elucidates the characteristics of liver TCR ß chain CDR3 repertoire under HS conditions, enhancing our understanding of HS implications.

Differences in Tumor-Associated T cell receptor repertoires between Early-Onset and Average-Onset colorectal cancer.

The incidence of colorectal cancer (CRC) among individuals younger than age 50 (early onset CRC; EOCRC) has substantially increased, yet the etiology and molecular mechanisms underlying this alarming rise remain unclear. We compared tumor-associated T cell repertoires between EOCRC and average-onset CRC (AOCRC) to uncover potentially unique immune microenvironment-related features by age of onset. Our discovery cohort included 242 patients who underwent surgical resection at Cleveland Clinic from 2000 to 2020. EOCRC was defined as age < 50 years at diagnosis (N = 126), and AOCRC as age ≥ 60 years (N = 116). T cell receptor (TCR) abundance and clonality were measured by immunosequencing of tumors. Logistic regression models were used to evaluate the associations between TCR repertoire features and age of onset, adjusting for sex, race, tumor location, and stage. Findings were replicated in 152 EOCRC and 1,984 AOCRC cases from the Molecular Epidemiology of Colorectal Cancer Study. EOCRC tumors had significantly higher TCR diversity compared to AOCRC tumors in the discovery cohort (Odds Ratio (OR):0.44, 95% Confidence Interval (CI):0.32-0.61, p < .0001). This association was also observed in the replication cohort (OR : 0.74, 95% CI : 0.62-0.89, p = .0013). No significant differences in TCR abundance were observed between EOCRC and AOCRC in either cohort. Higher TCR diversity, suggesting a more diverse intratumoral T cell response, is more frequently observed in EOCRC than AOCRC. Further studies are warranted to investigate the role of T cell diversity and the adaptive immune response more broadly in the etiology and outcomes of EOCRC.

Patients with juvenile idiopathic arthritis have decreased clonal diversity in the CD8+ T cell repertoire response to influenza vaccination.

Recurrent exposures to a pathogenic antigen remodel the CD8+ T cell compartment and generate a functional memory repertoire that is polyclonal and complex. At the clonotype level, the response to the conserved influenza antigen, M158-66 has been well characterized in healthy individuals, but not in patients receiving immunosuppressive therapy or with aberrant immunity, such as those with juvenile idiopathic arthritis (JIA). Here we show that patients with JIA have a reduced number of M158-66 specific RS/RA clonotypes, indicating decreased clonal richness and, as a result, have lower repertoire diversity. By using a rank-frequency approach to analyze the distribution of the repertoire, we found several characteristics of the JIA T cell repertoire to be akin to repertoires seen in healthy adults, including an amplified RS/RA-specific antigen response, representing greater clonal unevenness. Unlike mature repertoires, however, there is more fluctuation in clonotype distribution, less clonotype stability, and more variable IFNy response of the M158-66 specific RS/RA clonotypes in JIA. This indicates that functional clonal expansion is altered in patients with JIA on immunosuppressive therapies. We propose that the response to the influenza M158-66 epitope described here is a general phenomenon for JIA patients receiving immunosuppressive therapy, and that the changes in clonal richness and unevenness indicate a retarded and uneven generation of a mature immune response.

The DNA methylation landscape across the TCR loci in patients with acute myeloid leukemia.

The capacity of T cells to initiate anti-leukemia immune responses is determined by the ability of their receptors (TCRs) to recognize leukemia neoantigens. Epigenetic mechanisms including DNA methylation contribute to shaping the TCR repertoire composition and diversity. The DNA hypomethylating agents (HMAs) have been widely used in the treatment of acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). Whether DNA HMAs directly influence TCR gene loci methylation patterns remains unknown. By analyzing public datasets, we compared methylation patterns across TCR loci in AML patients and healthy controls. We also explored how HMAs influence TCR loci DNA methylation in patients with AML. While methylation patterns are largely conserved across the TCR loci, certain V genes exhibit high interindividual variability. Although overall methylation levels within the TCR loci did not show significant differences, specific sites, including 32 TRAV and 12 TRBV sites exhibited distinct methylation patterns when comparing T cells from healthy donors to those from patients with AML. In leukemic cells, decitabine treatment demethylates sites across the TRAV and TRBV genes. While not as significant, a similar pattern of demethylation is observed in T cells. Pretreatment AML samples exhibit higher methylation beta values in differentially methylated positions (DMPs) compared with non-DMPs. Methylation levels of certain TRAV and TRBV genes in leukemic cells are associated with patients' risk status. The presence of disease specific TCR loci methylated signatures that are associated with clinical outcome presents an opportunity for therapeutic intervention. HMAs can modulate the TCR loci methylation patterns, yet whether they could reprogram the TCR repertoire composition remains to be explored.

RAIN: a Machine Learning-based identification for HIV-1 bNAbs.

Broadly neutralizing antibodies (bNAbs) are promising candidates for the treatment and prevention of HIV-1 infection. Despite their critical importance, automatic detection of HIV-1 bNAbs from immune repertoire is still lacking. Here, we developed a straightforward computational method for Rapid Automatic Identification of bNAbs (RAIN) based on Machine Learning methods. In contrast to other approaches using one-hot encoding amino acid sequences or structural alignment for prediction, RAIN uses a combination of selected sequence-based features for accurate prediction of HIV-1 bNAbs. We demonstrate the performance of our approach on non-biased, experimentally obtained sequenced BCR repertoires from HIV-1 immune donors. RAIN processing leads to the successful identification of novel HIV-1 bNAbs targeting the CD4-binding site of the envelope glycoprotein. In addition, we validate the identified bNAbs using in vitro neutralization assay and we solve the structure of one of them in complex with the soluble native-like heterotrimeric envelope glycoprotein by single-particle cryo-electron microscopy (cryo-EM). Overall, we propose a method to facilitate and accelerate HIV-1 bNAbs discovery from non-selected immune repertoires.

Deciphering Abdominal Aortic Diseases Through T-Cell Clonal Repertoire of Perivascular Adipose Tissue.

BACKGROUND: Recent studies suggest that immune-mediated inflammation of perivascular adipose tissue of abdominal aortic aneurysms (AAAs) contributes to disease development and progression. Whether the perivascular adipose tissue of AAA is characterized by a specific adaptive immune signature remains unknown. METHODS AND RESULTS: To investigate this hypothesis, we sequenced the T-cell receptor ß-chain in the perivascular adipose tissue of patients with AAA and compared it with patients with aortic occlusive disease, who share the former anatomical site of the lesion and risk factors but differ in pathogenic mechanisms. Our results demonstrate that patients with AAA have a lower repertoire diversity than those with aortic occlusive disease and significant differences in variable/joining gene segment usage. Furthermore, we identified a set of 7 public T-cell receptor ß-chain clonotypes that distinguished AAA and aortic occlusive disease with very high accuracy. We also found that the T-cell receptor ß-chain repertoire differentially characterizes small and large AAAs (aortic diameter<55 mm and ≥55 mm, respectively). CONCLUSIONS: This work supports the hypothesis that T cell-mediated immunity is fundamental in AAA pathogenesis and opens up new clinical perspectives.

Defining the complexities of an occupational pattern-A concept analysis and theoretical validation.

BACKGROUND: The concept of an occupational pattern in occupational therapy and occupational science has evolved with varying definitions, ranging from activity patterns to patterns of daily occupation. AIMS: This study aimed to explore the concept of occupational pattern, develop an updated definition of the concept, and theoretically validate the concept's definition. METHOD: Walker and Avant's concept analysis method was used, where both theoretical frameworks and peer-reviewed scientific literature were searched and synthesized to clarify and define the concept. Furthermore, seven occupational therapists theoretically validated the concept. FINDINGS: The analysis included forty-nine references from various research contexts and theoretical perspectives. The synthesis yielded a conceptualization of the concept of occupational pattern, outlining it into three overarching categories: 'content in an individual's occupational pattern', 'designing an occupational pattern', and 'balancing the occupational pattern'. IMPLICATIONS: An updated operational definition of the multifaceted concept of an occupational pattern now exists, with practical implications for enhancing the education of occupational therapy students and guiding the utilization of the concept. Moreover, it holds significance for instrument development and outcome measurement in research; especially in lifestyle intervention studies within the field of occupational therapy.

Morphological and immunological characterization of primary cultured chicken caecal epithelial cells.

Cell cultures are models in biological and medical research to understand physiological and pathological processes. Cell lines are not always available depending on cell type and required species. In addition, the immortalization process often affects cell biology. Primary cells generally maintain a greater degree of similarity in short-term culture to the cells in tissue. Goal of this study was to verify the suitability of chicken primary epithelial caecal cells (PECCs) for in vitro investigations of host‒pathogen interactions. Epithelial nature of PECCs was confirmed by detection of tight and adherens junctions and cobblestone-like cell morphology. Sialic acids distribution was similar to that in caecal cyrosections. To understand the capacity of PECCs to respond to microbial challenges, the Toll-like receptors (TLRs) repertoire was determined. Exposure of PECCs to polyinosinic-polycytidylic acid (poly(I:C)) or lipopolysaccharide (LPS) led to upregulation of type I and III interferon (IFN) as well as interleukin (IL-) 1ß, IL-6 and IL-8 mRNA expression. Overall, the PECCs showed properties of polarized epithelial cells. The presence of TLRs, their differential expression, as well as pattern recognition receptor dependent immune responses enable PECCs to act as suitable in vitro model for host‒pathogen interaction studies, which are difficult to conduct under in vivo conditions.