Antigen Exposure Research Articles

Oral alloantigen exposure promotes donor-specific tolerance in a mouse model of minor mismatched skin transplantation

Abstract Oral tolerance is a powerful, non-invasive route to induce immune tolerance, yet it has been unsuccessful to promote graft acceptance in rodent models of transplantation. To this end, we developed a mouse model where the model antigen ovalbumin (OVA) was introduced orally, in a regimen designed to improve bioavailability, duodenal lymph node drainage and prolong duration of antigen exposure, prior to transplantation with skin grafts constitutively expressing membrane-bound OVA. We found that oral OVA treatment pre-transplantation promoted permanent graft acceptance as well as linked tolerance to skin grafts expressing OVA coupled to the additional model antigen 2W. Tolerance was donor-specific, as secondary donor-matched, but not third-party grafts were spontaneously accepted in the absence of additional immunosuppression. Treatment with this oral OVA regimen promoted an anergic phenotype in OVA-reactive CD8+ T cells, generation of iTregs, and dysfunction of OVA-reactive CD4+ T cells pre-transplantation. The tolerance induced by oral OVA was robust, as anti-CD25/anti-PD-L1 at the time of transplantation failed to prevent skin graft acceptance. Mechanistically, we revealed a critical role for the proximal gut draining lymph nodes (gLNs) in mediating this effect, as an intestinal infection that drains to the proximal gLNs prevented the induction of transplantation tolerance. Our study extends previous work applying oral antigen exposure to transplantation and serves as proof of concept that the systemic immune mechanisms supporting oral tolerance are sufficient to promote long-term graft acceptance. Regimens that improve bioavailability and duration of oral alloantigen administration may hold promise in the clinic. This work was supported by a NIAID grant to ASC and MLA (P01AI-97113). Peter Wang was supported by undergraduate research grants from the Katen Scholars Program and the Dean's Fund for Undergraduate Research.

Exploring the functionality of CD56+ MAIT cell populations across human tissues

Abstract Mucosa-associated invariant T (MAIT) cells are an evolutionarily conserved population of T cells serving a sentinel function at mucosal barrier sites to mediate immune protection. MAIT cells utilize their semi-invariant T cell receptor (TCR) to react to non-peptidic antigens derived from microbial riboflavin metabolites, presented on monomorphic MHC-class I-like MR1 molecules. In addition to TCR-dependent recognition of vitamin B2metabolites, MAIT cells sense and respond to local inflammatory cytokines in an innate-like manner. Surface expression of CD56 was previously shown to be associated with enhanced MAIT cell sensitivity to TCR-independent activation. However, the regulation, frequency, or relevance of CD56 expression on human MAIT cells is currently not well understood. Flow cytometry revealed heterogeneity of CD56 expression across donor-matched tissues obtained from human organ donors. Strikingly, the majority of liver MAIT cells expressed CD56, whereas expression was consistently lower in other tissues. We evaluated de novo CD56 expression on blood-derived sorted CD56-negative MAIT cells following IL-7 or antigen exposure which is likely to occur in the hepatic acute phase response. CD56-negative MAIT cells strongly upregulated CD56 expression after IL-7 stimulation and maintained CD56 expression for up to 19 days. Exposure to MR1-presented antigen induced CD56 expression similarly but required prior cell proliferation. In summary, we identify heterogeneity between MAIT cell populations of different tissue origin and determine the dynamic regulation of the CD56-associated innate-like characteristics to shed light on unknown aspects of MAIT cell diversity and immunobiology.

Development of a Cell-based ELISA assay to assess memory IgG produced in vitro by B cells specific for spike protein of SARS-CoV-2

Abstract Knowledge of antibody-mediated immune response to SARS-CoV-2 is crucial to understand the virus immunogenicity, establish seroprevalence and determine possible risks for infection/reinfection. An antibody presence, indicative of an humoral immune response, can be evidenced at early/intermediated stages after infection and antibodies are mainly detected by ELISA-based platforms. However, a major drawback of ELISA systems is a decrease of serum antibody titers during time. On the other hand, circulating antigen-specific memory B cells (MBC) may be present for long time after immunization/infection. The spike S1 protein has been shown to be a serological marker for Covid-19, and we describe a novel, simple and robust Cell-ELISA assay specifically designed to measure viral spike S1 protein by detecting specific IgG produced in vitro by MBC in PBMC (doi:10.3390/v12111274). By applying the Cell-ELISA assay to a cohort of 150 asympto/symptomatic individuals, age ranging from 8–100 years, we detected individuals resulted negative in ELISA but positive in Cell-ELISA, thus showing the presence of MBC that produce in vitro antibodies against SARS-CoV-2 at levels that are undetectable in the serum. These data may challenge the negative results obtained from the serological screening and indicate a previous antigen exposure in the possible absence of serum IgG. We also detected a presence of MBC specific for S1 protein for >10 months after viral infection (doi:10.3390/v13091704), suggesting a potent antigenicity, and evaluated the relative amount of in vitro secreted IgG in positive subjects. The proposed assay could be a candidate tool to monitor the presence of an effective IgG antibody memory after SARS-CoV-2 infection and vaccination.

Enhancing the antitumor response of CD8+ T cells by 4-1BB (CD137) co-stimulation with distinct inactivation of the type 2 adenosine receptors.

Abstract Activating the immune co-stimulatory receptor 4-1BB (CD137) with agonist antibody binding and crosslinking-inducing agents that elicit 4-1BB intracellular signaling potentiates the anti-tumor responses of CD8+ T cells. However, the underlying in-depth mechanisms remain to be defined. The type 2 adenosine receptors (primarily the high affinity receptor, A2AR) predominantly expressed on CD8+ T cells inhibit T cell activation and expansion by blocking TCR signaling in a cAMP-dependent manner. Here, we show that inactivation of the low affinity receptor A2BR rather than A2AR by continuous treatment of antagonists and/or genetic deletion induces superior survival advantage of effector CD8+ T cells with agonistic 4-1BB co-stimulation especially upon chronic TCR stimulation and/or long-term antigen exposure. Mechanistically, A2BR inactivation helps sustain the increased energy and biosynthetic requirements through the accumulation of intracellular glutathione (GSH) in response to agonistic 4-1BB co-stimulation. Importantly, A2BR inactivation in combination with agonistic 4-1BB co-stimulation displays a greater ability to modulate mitochondrial fitness for anti-tumor CD8+ T cell expansion while minimize T cell exhaustion. Thus, the A2BR pathway plays an unexpected role in metabolic reprogramming of GSH upon agonistic 4-1BB co-stimulation that allows the fine-tuning of the anti-tumor responses of CD8+ T cells. Supported by NCI CCSG P30 CA060553 R01CA258857

Epitope-specific T cell response to SARS-CoV-2 infection and vaccination

Abstract SARS-CoV-2 mRNA vaccines are effective for COVID-19 prevention, eliciting both robust antibody responses in naive individuals and boosting antibody levels in convalescent donors. However, the effect of repeated antigen exposures, such as vaccination following infection or breakthrough infections, on the magnitude, repertoire, and phenotype of pre-existing memory T cells, is still poorly understood. Thus, we compared epitope-specific CD8 T cells elicited after SARS-CoV-2 infection, vaccination of both naive and recovered individuals, and breakthrough infection cases. We used pools of 18 DNA-barcoded MHC-class I multimers, combined with scRNAseq and scTCRseq, to characterize T cell responses as defined by magnitude, specificity, T cell receptor (TCR) repertoire, and gene expression profile to both spike-derived and non-spike derived epitopes. In-depth analysis of over 4000 unique epitope-specific TCR sequences demonstrates that both vaccination and infection, including breakthrough cases, elicit identical repertoires as measured by dominant TCR motifs and repertoire diversity, indicating that BNT162b2 vaccination largely recapitulates spike-specific T cell repertoire generation by infection. Importantly, in COVID-19-recovered individuals receiving the vaccine, pre-existing spike-specific memory cells showed both clonal expansion and a phenotypic shift towards more differentiated CCR7-CD45RA+ effector cells, demonstrating the potency of this vaccine to recall spike-specific CD8 memory T cells. Importantly, in breakthrough infections, we observed a high proportion of T cells targeting non-spike epitopes, showing that new immune memory could be formed during SARS-CoV-2 infection after vaccination. The work was funded by 75N93019C00052, HHSN272201400006C, 3U01AI144616-02S1, R01AI136514

Hybrid immunity and vaccine breakthrough lead to robust humoral response and antibodies that effectively neutralize SARS-CoV-2 variants

Abstract Current COVID-19 vaccines significantly reduce overall morbidity and mortality and are vitally important to halting the pandemic; individuals who previously recovered from COVID-19 display enhanced immune responses after vaccination (hybrid immunity) compared to their naïve-vaccinated peers. However, the effects of vaccine breakthrough infections on humoral immune response remain to be determined. Here, we measure neutralizing antibody responses from 104 vaccinated individuals including those with breakthrough infections, hybrid immunity, and no infection history. We find that human immune sera collected after either a breakthrough infection or a vaccination post-natural infection will both broadly neutralize SARS-CoV-2 variants to a similar degree. While age negatively correlates with antibody response after vaccination alone, no such association was found in breakthrough or hybrid immune groups. Together, our data suggest that the additional antigen exposure from natural infection substantially boosts the quantity, quality, and breadth of humoral immune response regardless of whether it occurs before or after vaccination. Supported by grants from the M.J. Murdock Charitable Trust, OHSU Innovative IDEA grant (1018784), NIH (R01AI145835, T32HL083808), and an unrestricted grant from the OHSU Foundation.

Single-Cell Transcriptomics Of CD8 T Cells During CMV Viremia In Kidney Transplant Recipients Identify Transcriptional States Of Advanced Differentiation

Abstract Acute versus chronic viral infections define the dogma of strictly segregated pathways of generating memory or exhausted T cells. Paradoxically, chronic human cytomegalovirus (HCMV) infection elicits an inflationary T cell response with accumulation of progressively differentiated T cells occupying up to 30% of total CD8 T cells. The mechanisms of HCMV-driven memory T cell inflation remains poorly understood. HCMV infection is asymptomatic but immunocompromised transplant recipients often experience clinical episodes of CMV viremia correlating with graft injury and mortality. We leveraged a defined clinical cohort of renal transplant recipients and profiled the T cells from 31 CMV PCR+ patients with/without prior CMV exposure and 31 matched PCR− controls at baseline, longterm post-transplantation and at 1wk and 1month post-viremia for PCR+ patients. We observed contraction of naïve and central memory T cells and reciprocal expansion of terminal effector memory CD8 T cells post-viremia. ScRNASeq of CD8 T cells further defined early (CD28+CCR7+), transitional (CD28+GZMK+) and advanced (CD28-KLRG1+) stages of differentiation. Pseudotime analyses revealed specific transcriptomic modules and transcription factors associated with distinct CD8 T cell differentiation states. We report that HCMV induced progressively differentiated T cells with memory-progenitor features lacking inhibitory receptors but with stemness (kat6a, rnf138) and anti-apoptotic markers (bcl2, braf). Thus HCMV skewed the balance of CD8 T memory cells from pro-exhaustion to a prolonged non-classical memory phenotype. This study revealed important mechanisms underlying memory inflation over exhaustion in the setting of chronic antigen exposure. This work was funded by NIH grant U19AI128913 to EFR.

Intestinal inflammation alters the antigen-specific immune response to a skin commensal.

SUMMARYResident microbes in skin and gut predominantly impact local immune cell function during homeostasis. However, colitis-associated neutrophilic skin disorders suggest possible breakdown of this compartmentalization with disease. Using a model wherein neonatal skin colonization by Staphylococcus epidermidis facilitates generation of commensal-specific tolerance and CD4+ regulatory T cells (Tregs), we ask whether this response is perturbed by gut inflammation. Chemically induced colitis is accompanied by intestinal expansion of S. epidermidis and reduces gut-draining lymph node (dLN) commensal-specific Tregs. It also results in reduced commensal-specific Tregs in skin and skin-dLNs and increased skin neutrophils. Increased CD4+ circulation between gut and skin dLN suggests that the altered cutaneous response is initiated in the colon, and resistance to colitis-induced effects in Cd4creIl1r1fl/fl mice implicate interleukin (IL)-1 in mediating the altered commensal-specific response. These findings provide mechanistic insight into observed connections between inflammatory skin and intestinal diseases.

Sensing and alarm function of vaccine-elicited SIV-gag specific CD8 TRM in the reproductive mucosa of rhesus macaques

Abstract TRM constitute a recently identified lymphocyte lineage that occupies non-lymphoid tissues (NLT) without recirculating. Experiments in mice demonstrated that TRM reactivation triggers antiviral responses in neighboring cells by promoting local stimulation of innate and adaptive immune cells, and by recruiting immune effectors. Collectively this is referred to as ‘sensing and alarm’ function. We wished to broadly identify TRM functions in non-human primates (NHP). Here we applied a prime-boost vaccine modality in rhesus macaques to generate abundant SIVgag-specific CD8 TRM in the female reproductive tract (FRT) and 14 other NLT. To assess sensing and alarm function, macaques were challenged intravaginally with CM9 peptide from the SIV gag protein and necropsied 24 or 48 hours later. CD8 TRM reactivation increased expression of CD69 and granzyme B in SIV-gag specific CD8 T cells throughout the FRT at 24h, in situ proliferation (Ki67 expression) by 48h, and rapid antiviral and IFN response gene expression in essentially all hematopoietic and non-hematopoietic cells by 24h. Upregulation of effector genes in CD8 T cells, CD4 T cells, NK cells, and ILCs peaked at 24h and persisted at 48h. Mucosal CD4 T cells expressed various HIV restriction factors, and had reduced expression of the HIV-coreceptor CCR5. Increased numbers of vaginal T and B cells coincided with increased expression of chemokines and VCAM-1 on endothelial and stromal cells and a concomitant reduction in circulating T cells, B cells and SIV-env-specific B cells. These data demonstrate that vaccine-elicited CD8 TRM rapidly trigger local activation and the recruitment of innate, cellular, and humoral immune responses to the site of antigen exposure.

Antibody response to herpes simplex virus-1 is increased in autoimmune encephalitis.

Antibody response to herpes simplex virus-1 is increased in autoimmune encephalitis.

Efficient recall of Omicron-reactive B cell memory after a third dose of SARS-CoV-2 mRNA vaccine

We examined antibody and memory B cell responses longitudinally for ∼9–10 months after primary 2-dose SARS-CoV-2 mRNA vaccination and 3 months after a 3rd dose. Antibody decay stabilized between 6 and 9 months, and antibody quality continued to improve for at least 9 months after 2-dose vaccination. Spike- and RBD-specific memory B cells remained durable over time, and 40%–50% of RBD-specific memory B cells simultaneously bound the Alpha, Beta, Delta, and Omicron variants. Omicron-binding memory B cells were efficiently reactivated by a 3rd dose of wild-type vaccine and correlated with the corresponding increase in neutralizing antibody titers. In contrast, pre-3rd dose antibody titers inversely correlated with the fold-change of antibody boosting, suggesting that high levels of circulating antibodies may limit the added protection afforded by repeat short interval boosting. These data provide insight into the quantity and quality of mRNA-vaccine-induced immunity over time through 3 or more antigen exposures.

SARS-CoV-2 antigen exposure history shapes phenotypes and specificity of memory CD8+ T cells.

Although mRNA vaccine efficacy against severe COVID-19 remains high, variant emergence has prompted booster immunizations. However, repeated antigen exposure effects on SARS-CoV-2 memory T cells are poorly understood. Here, we utilize MHC-multimers with scRNAseq to profile SARS-CoV-2-responsive T cells ex vivo from humans with one, two, or three antigen exposures, including vaccination, primary, and breakthrough infection. Exposure order determined the distribution between spike- and non-spike-specific responses, with vaccination after infection leading to expansion of spike-specific T cells and differentiation to CCR7-CD45RA+ effectors. In contrast, individuals after breakthrough infection mount vigorous non-spike-specific responses. Analysis of over 4,000 epitope-specific T cell receptor sequences demonstrates that all exposures elicit diverse repertoires characterized by shared TCR motifs, confirmed by monoclonal TCR characterization, with no evidence for repertoire narrowing from repeated exposure. Our findings suggest that breakthrough infections diversify the T cell memory repertoire and current vaccination protocols continue to expand and differentiate spike-specific memory.

Risk Factors for the Development of Early Anti-HLA Donor Specific Antibodies After Lung Transplantation: The Role of Primary Graft Dysfunction

<h3>Purpose</h3> The development of anti-HLA donor specific antibodies (DSA) after lung transplantation has been associated poor graft survival. DSA may develop early after lung transplantation or later, usually when chronic lung allograft dysfunction (CLAD) ensues. Early DSA have recently been considered a product of a memory rather than a de-novo reaction after alloantigen exposure. Aim of this study was to analyse the risk factors for the development of early DSA after lung transplantation. <h3>Methods</h3> Records of patients transplanted at our institution between 01/2013 and 10/2021 were retrospectively reviewed. Patients were controlled for eDSA every two weeks after transplantation until hospital discharge, using the Luminex single-antigen test (mean fluorescence index threshold, 1000). Multivariable binary logistic regression analysis, including variables with univariable p-value <0.10, was performed to identify factors associated with eDSA. Goodness-of-fit of the model was tested using the Hoshmer and Lemeshow (H-L) test. <h3>Results</h3> During the study period, among the 1007 lung-transplanted patients, 275 (27%) patients showed early detectable DSA (preformed, n=56; de-novo, n=254; class I, n=71; class II, n=239). Median (IQR) time to DSA detection was 14 (11-21) days. The binary logistic regression analysis showed that pediatric (<18 years old) transplantation (OR: 2.2, 95%CI: 1.8-2.8, p=0.001), presence of pretransplant HLA antibodies (OR: 2.1, 95%CI: 1.6-2.8, p<0.01), donor lung preservation with Organ Care System (OR: 0.37, 95%CI: 0.14-0.97, p=0.04), and primary graft dysfunction (PGD) score grade 2-3 at 72 hours after transplantation (OR: 1.8, 95%CI: 1.2-1.8, p=0.007) were associated with eDSA detection (H-L statistic, p=0.92). <h3>Conclusion</h3> The graft damage and donor antigen exposure provoked by primary graft dysfunction may lead to eDSA production, that should not be considered only a product of a memory but also of a de-novo reaction (Figure 1).

Interplay of infection and vaccination in long-term protection from COVID-19

Interplay of infection and vaccination in long-term protection from COVID-19

CD4+ T-Cell Dysfunction in Severe COVID-19 Disease Is Tumor Necrosis Factor-α/Tumor Necrosis Factor Receptor 1-Dependent.

Rationale: Lymphopenia is common in severe coronavirus disease (COVID-19), yet the immune mechanisms are poorly understood. As inflammatory cytokines are increased in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, we hypothesized a role in contributing to reduced T-cell numbers. Objectives: We sought to characterize the functional SARS-CoV-2 T-cell responses in patients with severe versus recovered, mild COVID-19 to determine whether differences were detectable. Methods: Using flow cytometry and single-cell RNA sequence analyses, we assessed SARS-CoV-2-specific responses in our cohort. Measurements and Main Results: In 148 patients with severe COVID-19, we found lymphopenia was associated with worse survival. CD4+ lymphopenia predominated, with lower CD4+/CD8+ ratios in severe COVID-19 compared with patients with mild disease (P < 0.0001). In severe disease, immunodominant CD4+ T-cell responses to Spike-1 (S1) produced increased invitro TNF-α (tumor necrosis factor-α) but demonstrated impaired S1-specific proliferation and increased susceptibility to activation-induced cell death after antigen exposure. CD4+TNF-α+ T-cell responses inversely correlated with absolute CD4+ counts from patients with severe COVID-19 (n = 76; R = -0.797; P < 0.0001). In vitro TNF-α blockade, including infliximab or anti-TNF receptor 1 antibodies, strikingly rescued S1-specific CD4+ T-cell proliferation and abrogated S1-specific activation-induced cell death in peripheral blood mononuclear cells from patients with severe COVID-19 (P < 0.001). Single-cell RNA sequencing demonstrated marked downregulation of type-1 cytokines and NFκB signaling in S1-stimulated CD4+ cells with infliximab treatment. We also evaluated BAL and lung explant CD4+ T cells recovered from patients with severe COVID-19 and observed that lung T cells produced higher TNF-α compared with peripheral blood mononuclear cells. Conclusions: Together, our findings show CD4+ dysfunction in severe COVID-19 is TNF-α/TNF receptor 1-dependent through immune mechanisms that may contribute to lymphopenia. TNF-α blockade may be beneficial in severe COVID-19.

Starvation therapy enabled “switch-on” NIR-II photothermal nanoagent for synergistic in situ photothermal immunotherapy

Tumor photonic hyperthermia in the second near-infrared biowindow (NIR-II) represents the distinct therapeutic modality owing to the high tissue penetration and excellent spatiotemporal controllability. However, the non-specific “always on” nature of conventional NIR-II photothermal agents and the photothermal therapy (PTT)-associated tumor metastasis have restricted the progress of photonic hyperthermia. In this study, a cascade nanoreactor (denoted as LGT) based on clinically transformable liposomal nanoparticles is rationally engineered by encapsulating a pro-photothermal agent, 3,3′,5,5′-tetramethylbenzidine (TMB), and a tumor starvation mediator, glucose oxidase (GOD), for starvation therapy activated in-situ photothermal immunotherapy in inhibiting primary and metastasized tumors. Accompany with GOD-mediated intratumoral glucose depletion and hydrogen peroxide generation, the pro-photothermal agent is in-situ converted into the NIR-II absorbing charge transfer complex (CTC) in tumor microenvironment (TME). Collaboratively, glucose deprivation not only cuts the energy supply to tumor cells but also makes LGT sensitive to photothermal ablation. Upon NIR-II laser irradiation, LGT-mediated in-situ PTT ablates primary tumors, and further elicits the exposure of tumor-associated antigens. Importantly, the combination of starvation therapy enabled in situ PTT with anti-CTLA-4 checkpoint blockade therapy effectively inhibits distant tumor growth and tumor metastasis. Therefore, the present work provides a representative paradigm for tumor starvation therapy activated “switch-on” photothermal immunotherapy of cancer.

Imprinted SARS-CoV-2-specific memory lymphocytes define hybrid immunity

Immune memory is tailored by cues that lymphocytes perceive during priming. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic created a situation in which nascent memory could be tracked through additional antigen exposures. Both SARS-CoV-2 infection and vaccination induce multifaceted, functional immune memory, but together, they engender improved protection from disease, termed hybrid immunity. We therefore investigated how vaccine-induced memory is shaped by previous infection. We found that following vaccination, previously infected individuals generated more SARS-CoV-2 RBD-specific memory B cells and variant-neutralizing antibodies and a distinct population of IFN-γ and IL-10-expressing memory SARS-CoV-2 spike-specific CD4+ T cells than previously naive individuals. Although additional vaccination could increase humoral memory in previously naive individuals, it did not recapitulate the distinct CD4+ T cell cytokine profile observed in previously infected subjects. Thus, imprinted features of SARS-CoV-2-specific memory lymphocytes define hybrid immunity.

Antibiotic Treatment of Staphylococcus aureus Infection Inhibits the Development of Protective Immunity.

Recurrent Staphylococcus aureus infections are common, suggesting a failure to elicit protective immunity. Given the emergence of antibiotic resistance, a vaccine is urgently needed, but there is no approved vaccine for S. aureus. While antibiotics are routinely used to treat S. aureus infections, their impact on the development of protective immunity is not understood. Using an established mouse model of S. aureus skin and soft tissue infection (SSTI), we observed that antibiotic therapy effectively resolved infection but failed to elicit protection against secondary (2°) SSTI. Key contributors to protective immunity, toxin-specific antibodies and interleukin-17A (IL-17A)-producing T cells, were not strongly elicited in antibiotic-treated mice. Delaying antibiotic treatment failed to resolve skin lesions but resulted in higher antibody levels after infection and strong protection against 2° SSTI, suggesting that the development of protective immunity requires a longer period of antigen exposure. We next investigated if combining α-hemolysin (Hla) vaccination with antibiotics during primary infection would both treat infection and generate durable protective immunity. This "therapeutic vaccination" approach resulted in rapid resolution of primary infection and protection against recurrent infection, demonstrating that concurrent vaccination could circumvent the deleterious effects of antibiotic therapy on elicited immune responses. Collectively, these findings suggest that protective immunity is thwarted by the rapid elimination of antigen during antibiotic treatment. However, vaccination in conjunction with antibiotic treatment can retain the benefits of antibiotic treatment while also establishing protective immunity.

The order of immunoglobulin light chain κ and λ usage in primary and secondary lymphoid tissues of germ-free and conventional piglets

In pigs (Sus scrofa), the initial immunoglobulin rearrangement of the κ light chain is replaced by λ before the heavy chains rearrange, and the light chains may rearrange even later. This study investigates whether these developmental differences are reflected in the usage of IGK and IGL genes. We found large differences between peripheral B cells and those developing in the bone marrow, and between B cells in germ-free piglets and conventional pigs. During early B cell development in the bone marrow, more 3′ V and 5′ J gene segments for both light chains are used. However, in the peripheral naive repertoire, more 5′ IGLV and 3′ IGLJ genes are used. A similar shift toward the use of more 5′ IGKV and 3′ IGKJ genes is observed later after antigen exposure in conventional pigs. The expression profile showed that most λ+ B cells are generated earlier, while κ+ B cells develop from late precursors that already contain the λ rearrangement. The initial λ rearrangement is retained in both λ+ and κ+ B lymphocytes, and multiple λ transcripts can be found in individual cells. The overall pool of the IGLV repertoire is therefore much larger and more diversified than for IGKV. The κ repertoire is further restricted to the preferential use of only two major IGKV genes, reflecting the limitation for only two consecutive rearrangements. Tracing of silenced λ transcripts in κ+ B cells further confirmed the unconventional mechanism of differential rearrangements in pigs. Our results underline the diversity of the immune system among mammals.

Dysmotility in Eosinophilic Esophagitis.

Eosinophilic esophagitis (EoE) is an immune mediated chronic inflammatory disease resulting from antigen exposure and is characterized by mucosal inflammation with eosinophils. Diagnosis is based on the histological finding of at least 15 eosinophils per high power field in esophageal biopsy specimens from upper gastrointestinal endoscopies. These endoscopies are usually performed in the setting of esophageal dysfunction, however, EoE can occasionally be incidentally diagnosed during endoscopies performed for other indications like coeliac disease. The eosinophilia is in the absence of other causes of esophageal eosinophilia (e.g., parasitic infection, esophageal leiomyomatosis or Crohn's disease). Presentation can be wide ranging and often varies according to age. Infants and younger children can present with choking/gagging, feed refusal, failure to thrive, irritability and vomiting. Older children and adults commonly present with dysphagia, chest pain or food bolus obstruction. EoE was first described in the 1970s, but was only recognized as a distinct disease entity in the 1990s. It has been rising in incidence and prevalence, with reported prevalence ranging between 1 in 2,500 and 1 in 10,000. Although the diagnosis of EoE is dependent on clear histopathologic diagnostic criteria, there is a disconnect between the degree of esophageal eosinophilia and symptom severity especially that of reported dysphagia. Multiple anatomical changes can be seen in the spectrum of presentations of EoE which explain dysphagia, including isolated strictures, diffuse trachealisation, fixed rings, including Schatzki, as well as tissue remodeling and fibrotic changes. However, a majority of EoE patients do not have any of these findings and will still often report ongoing dysphagia. Some will report ongoing dysphagia despite histological remission. This suggests an underlying esophageal dysmotilty which cannot be assessed with endoscopy or correlated with histological changes seen in biopsies. This review will describe the types of motor disturbances seen and their prevalence, the pathophysiological basis of dysmotility seen in EoE, how best to investigate esophageal dysfunction in EoE and the role of manometry in the management of EoE.