Plasma cell immunoglobulin heavy chain repertoire dynamics before and after tetanus booster vaccination.

Antibody sequence repertoire analysis of plasma cells (PC) isolated before and 1 week after a vaccine provides time-specific snapshots of the antibody response. Comparison of the immunoglobulin (Ig) sequences pre- and post-vaccination allows analysis of maturation over time and identification of antigen specific Ig. Here we compare the Ig heavy chain (Ig-H) repertoire of circulating PCs isolated from 109 peripheral blood mononuclear cells (PBMC) collected by apheresis 1 week after a tetanus toxoid vaccine booster with the Ig-H repertoire of PCs collected 2 and 11 weeks prior to the booster. A total of 21,060 unique Ig nucleotide sequences encoding 14,307 unique heavy chain complementarity determining region 3 (CDR-H3) amino acid sequences, also called clonotypes, were identified. Only 466 clonotypes (3.3%) were present at all 3 time points. In contrast, 90% of the 30 highest frequency CDR-H3 regions at +1w were also identified at another time point and 50% were present at all time points, suggesting the rapid expansion of a memory B cell population. The tetanus toxoid specificity of the CDR-H3 region with the 7th highest frequency at +1w was confirmed using immunoprecipitation and mass spectroscopy, and two public tetanus toxoid-specific CDR-H3 regions were also overrepresented at +1w. In summary, we have used the tetanus vaccine model system to demonstrate that bulk PC Ig repertoire analysis can identify PC populations that expand and mature following antigen exposure. The application of this approach before and after clinical infections should advance our understanding of clinical protection and facilitate vaccine design.

Diagnostic differentiation of Zika and dengue virus exposure by analyzing T cell receptor sequences from peripheral blood of infected HLA-A2 transgenic mice.

Zika virus (ZIKV) is a significant global health threat due to its potential for rapid emergence and association with severe congenital malformations during infection in pregnancy. Despite the urgent need, accurate diagnosis of ZIKV infection is still a major hurdle that must be overcome. Contributing to the inaccuracy of most serologically-based diagnostic assays for ZIKV, is the substantial geographic and antigenic overlap with other flaviviruses, including the four serotypes of dengue virus (DENV). Within this study, we have utilized a novel T cell receptor (TCR) sequencing platform to distinguish between ZIKV and DENV infections. Using high-throughput TCR sequencing of lymphocytes isolated from DENV and ZIKV infected mice, we were able to develop an algorithm which could identify virus-associated TCR sequences uniquely associated with either a prior ZIKV or DENV infection in mice. Using this algorithm, we were then able to separate mice that had been exposed to ZIKV or DENV infection with 97% accuracy. Overall this study serves as a proof-of-principle that T cell receptor sequencing can be used as a diagnostic tool capable of distinguishing between closely related viruses. Our results demonstrate the potential for this innovative platform to be used to accurately diagnose Zika virus infection and potentially the next emerging pathogen(s).

Heterotypic immunity against vaccinia virus in an HLA-B*07:02 transgenic mousepox infection model.

Vaccination with vaccinia virus (VACV) elicits heterotypic immunity to smallpox, monkeypox, and mousepox, the mechanistic basis for which is poorly understood. It is generally assumed that heterotypic immunity arises from the presentation of a wide array of VACV-derived, CD8+ T cell epitopes that share homology with other poxviruses. Herein this assumption was tested using a large panel of VACV-derived peptides presented by HLA-B*07:02 (B7.2) molecules in a mousepox/ectromelia virus (ECTV)-infection, B7.2 transgenic mouse model. Most dominant epitopes recognized by ECTV- and VACV-reactive CD8+ T cells overlapped significantly without altering immunodominance hierarchy. Further, several epitopes recognized by ECTV-reactive CD8+ T cells were not recognized by VACV-reactive CD8+ T cells, and vice versa. In one instance, the lack of recognition owed to a N72K variation in the ECTV C4R70-78 variant of the dominant VACV B8R70-78 epitope. C4R70-78 does not bind to B7.2 and, hence, it was neither immunogenic nor antigenic. These findings provide a mechanistic basis for VACV vaccination-induced heterotypic immunity which can protect against Variola and Monkeypox disease. The understanding of how cross-reactive responses develop is essential for the rational design of a subunit-based vaccine that would be safe, and effectively protect against heterologous infection.

Single-cell transcriptional analyses of spasmolytic polypeptide-expressing metaplasia arising from acute drug injury and chronic inflammation in the stomach.

OBJECTIVE: Spasmolytic polypeptide-expressing metaplasia (SPEM) is a regenerative lesion in the gastric mucosa and is a potential precursor to intestinal metaplasia/gastric adenocarcinoma in a chronic inflammatory setting. The goal of these studies was to define the transcriptional changes associated with SPEM at the individual cell level in response to acute drug injury and chronic inflammatory damage in the gastric mucosa. DESIGN: Epithelial cells were isolated from the gastric corpus of healthy stomachs and stomachs with drug-induced and inflammation-induced SPEM lesions. Single cell RNA sequencing (scRNA-seq) was performed on tissue samples from each of these settings. The transcriptomes of individual epithelial cells from healthy, acutely damaged and chronically inflamed stomachs were analysed and compared. RESULTS: scRNA-seq revealed a population Mucin 6 (Muc6)+gastric intrinsic factor (Gif)+ cells in healthy tissue, but these cells did not express transcripts associated with SPEM. Furthermore, analyses of SPEM cells from drug injured and chronically inflamed corpus yielded two major findings: (1) SPEM and neck cell hyperplasia/hypertrophy are nearly identical in the expression of SPEM-associated transcripts and (2) SPEM programmes induced by drug-mediated parietal cell ablation and chronic inflammation are nearly identical, although the induction of transcripts involved in immunomodulation was unique to SPEM cells in the chronic inflammatory setting. CONCLUSIONS: These data necessitate an expansion of the definition of SPEM to include Tff2+Muc6+ cells that do not express mature chief cell transcripts such as Gif. Our data demonstrate that SPEM arises by a highly conserved cellular programme independent of aetiology and develops immunoregulatory capabilities in a setting of chronic inflammation.

CD4+T cells mediate protection against Zika associated severe disease in a mouse model of infection.

Zika virus (ZIKV) has gained worldwide attention since it emerged, and a global effort is underway to understand the correlates of protection and develop diagnostics to identify rates of infection. As new therapeutics and vaccine approaches are evaluated in clinical trials, additional effort is focused on identifying the adaptive immune correlates of protection against ZIKV disease. To aid in this endeavor we have begun to dissect the role of CD4+T cells in the protection against neuroinvasive ZIKV disease. We have identified an important role for CD4+T cells in protection, demonstrating that in the absence of CD4+T cells mice have more severe neurological sequela and significant increases in viral titers in the central nervous system (CNS). The transfer of CD4+T cells from ZIKV immune mice protect type I interferon receptor deficient animals from a lethal challenge; showing that the CD4+T cell response is necessary and sufficient for control of ZIKV disease. Using a peptide library spanning the complete ZIKV polyprotein, we identified both ZIKV-encoded CD4+T cell epitopes that initiate immune responses, and ZIKV specific CD4+T cell receptors that recognize these epitopes. Within the ZIKV antigen-specific TCRß repertoire, we uncovered a high degree of diversity both in response to a single epitope and among different mice responding to a CD4+T cell epitope. Overall this study identifies a novel role for polyfunctional and polyclonal CD4+T cells in providing protection against ZIKV infection and highlights the need for vaccines to develop robust CD4+T cell responses to prevent ZIKV neuroinvasion and limit replication within the CNS.

Conventional and Regulatory CD4+ T Cells That Share Identical TCRs Are Derived from Common Clones.

Results from studies comparing the diversity and specificity of the TCR repertoires expressed by conventional (Tconv) and regulatory (Treg) CD4+ T cell have varied depending on the experimental system employed. We developed a new model in which T cells express a single fixed TCRα chain, randomly rearranged endogenous TCRß chains, and a Foxp3-GFP reporter. We purified CD4+Foxp3- and CD4+Foxp3+ cells, then performed biased controlled multiplex PCR and high throughput sequencing of endogenous TCRß chains. We identified >7,000 different TCRß sequences in the periphery of 5 individual mice. On average, ~12% of TCR sequences were expressed by both conventional and regulatory populations within individual mice. The CD4+ T cells that expressed shared TCR sequences were present at higher frequencies compared to T cells expressing non-shared TCRs. Furthermore, nearly all (>90%) of the TCR sequences that were shared within mice were identical at the DNA sequence level, indicating that conventional and regulatory T cells that express shared TCRs are derived from common clones. Analysis of TCR repertoire overlap in the thymus reveals that a large proportion of Tconv and Treg sharing observed in the periphery is due to clonal expansion in the thymus. Together these data show that there are a limited number of TCR sequences shared between Tconv and Tregs. Also, Tconv and Tregs sharing identical TCRs are found at relatively high frequencies and are derived from common progenitors, of which a large portion are generated in the thymus.

Consumption of acidic water alters the gut microbiome and decreases the risk of diabetes in NOD mice.

Infant formula and breastfeeding are environmental factors that influence the incidence of Type 1 Diabetes (T1D) as well as the acidity of newborn diets. To determine if altering the intestinal microbiome is one mechanism through which an acidic liquid plays a role in T1D, we placed non-obese diabetic (NOD)/ShiLtJt mice on neutral (N) or acidified H2O and monitored the impact on microbial composition and diabetes incidence. NOD-N mice showed an increased development of diabetes, while exhibiting a decrease in Firmicutes and an increase in Bacteroidetes, Actinobacteria, and Proteobacteria from as early as 2 weeks of age. NOD-N mice had a decrease in the levels of Foxp3 expression in CD4(+)Foxp3(+) cells, as well as decreased CD4(+)IL17(+) cells, and a lower ratio of IL17/IFNγ CD4+ T-cells. Our data clearly indicates that a change in the acidity of liquids consumed dramatically alters the intestinal microbiome, the presence of protective Th17 and Treg cells, and the incidence of diabetes. This data suggests that early dietary manipulation of intestinal microbiota may be a novel mechanism to delay T1D onset in genetically pre-disposed individuals.

Gut Microbiota and Obesity.

The current obesity epidemic clearly has many causes, including the impact of our modern world on both our diet and our lifestyle/physical activity. Although many interventions have been recommended, the prevalence of obesity continues to rise and has forced a re-evaluation of the potential interventions that could have an impact. In recent years it has been definitively shown that microbiota in the gastrointestinal tract are altered in obese individuals. Recent data provide a potential mechanistic understanding of the interactions between microbiota and obesity and allow potential new interventions to the control of obesity to be proposed.

Microbiota downregulates dendritic cell expression of miR-10a, which targets IL-12/IL-23p40.

Commensal flora plays important roles in the regulation of the gene expression involved in many intestinal functions and the maintenance of immune homeostasis, as well as in the pathogenesis of inflammatory bowel diseases. The microRNAs (miRNAs), a class of small, noncoding RNAs, act as key regulators in many biological processes. The miRNAs are highly conserved among species and appear to play important roles in both innate and adaptive immunity, as they can control the differentiation of various immune cells, as well as their functions. However, it is still largely unknown how microbiota regulates miRNA expression, thereby contributing to intestinal homeostasis and pathogenesis of inflammatory bowel disease. In our current study, we found that microbiota negatively regulated intestinal miR-10a expression, because the intestines, as well as intestinal epithelial cells and dendritic cells of specific pathogen-free mice, expressed much lower levels of miR-10a compared with those in germ-free mice. Commensal bacteria downregulated dendritic cell miR-10a expression via TLR-TLR ligand interactions through a MyD88-dependent pathway. We identified IL-12/IL-23p40, a key molecule for innate immune responses to commensal bacteria, as a target of miR-10a. The ectopic expression of the miR-10a precursor inhibited, whereas the miR-10a inhibitor promoted, the expression of IL-12/IL-23p40 in dendritic cells. Mice with colitis expressing higher levels of IL-12/IL-23p40 exhibited lower levels of intestinal miR-10a compared with control mice. Collectively, our data demonstrated that microbiota negatively regulates host miR-10a expression, which may contribute to the maintenance of intestinal homeostasis by targeting IL-12/IL-23p40 expression.

Helicobacter felis--associated gastric disease in microbiota-restricted mice.

Human Helicobacter pylori infection leads to multiple pathological consequences, including gastritis and adenocarcinoma. Although this association has led to the classification of H. pylori as a type 1 carcinogen, it is not clear if additional nonhelicobacter gastric microbiota play a role in these diseases. In this study, we utilized either specific pathogen-free C57BL/6 mice (B6.SPF) or mice colonized with altered Schaedler flora (B6.ASF) to evaluate the role of nonhelicobacter gastric microbiota in disease development after Helicobacter felis infection. Despite similar histological changes, H. felis persisted in B6.ASF stomachs, while H. felis could no longer be detected in the majority of B6.SPF mice. The B6.SPF mice also acquired multiple Lactobacillus spp. in their stomachs after H. felis infection. Our data indicate that potential mechanisms responsible for the ineffective H. felis clearance in the B6.ASF model include the absence of new gastric microbiota to compete for the gastric niche, the lack of expression of new gastric mucins, and a reduced ratio of H. felis-specific IgG2c:IgG1 serum antibodies. These data suggest that although H. felis is sufficient to initiate gastric inflammation and atrophy, bacterial eradication and the systemic immune response to infection are significantly influenced by pre-existing and acquired gastric microbiota.