Responses In Lymphoid Tissues Research Articles

Persistent human bocavirus 1 infection and tonsillar immune responses.

BackgroundPersistent human bocavirus 1 (HBoV1) infection is a common finding in patients suffering from chronic tonsillar disease. However, the associations between HBoV1 infection and specific immune reactions are not completely known. We aimed to compare in vivo expression of T‐cell cytokines, transcription factors, and type I/III interferons in human tonsils between HBoV1‐positive and ‐negative tonsillectomy patients.MethodsTonsil tissue samples, nasopharyngeal aspirate (NPA), and serum samples were obtained from 143 immunocompetent adult and child tonsillectomy patients. HBoV1 and 14 other respiratory viruses were detected in NPAs and tonsil tissues by polymerase chain reaction (PCR). Serology and semi‐quantitative PCR were used for diagnosing HBoV1 infections. Expression of 14 cytokines and transcription factors (IFN‐α, IFN‐β, IFN‐γ, IL‐10, IL‐13, IL‐17, IL‐28, IL‐29, IL‐37, TGF‐β, FOXP3, GATA3, RORC2, Tbet) was analyzed by quantitative reverse‐transcription (RT)‐PCR in tonsil tissues.ResultsHBoV1 was detected by PCR in NPA and tonsils from 25 (17%) study patients. Serology results indicated prior nonacute infections in 81% of cases. Tonsillar cytokine responses were affected by HBoV1 infection. The suppression of two transcription factors, RORC2 and FOXP3, was associated with HBoV1 infection (p < 0.05). Furthermore, intratonsillar HBoV1‐DNA loads correlated negatively with IFN‐λ family cytokines and IL‐13.ConclusionsOur study shows distinctively decreased T‐helper17 and T‐regulatory type immune responses in local lymphoid tissue in HBoV1‐positive tonsillectomy patients. HBoV1 may act as a suppressive immune modulator.

The fibroblast: An emerging key player in thymic T cell selection.

Fibroblasts have recently attracted attention as a key stromal component that controls the immune responses in lymphoid tissues. The thymus has a unique microenvironment comprised of a variety of stromal cells, including fibroblasts and thymic epithelial cells (TECs), the latter of which is known to be important for T cell development because of their ability to express self‐antigens. Thymic fibroblasts contribute to thymus organogenesis during embryogenesis and form the capsule and medullary reticular network in the adult thymus. However, the immunological significance of thymic fibroblasts has thus far only been poorly elucidated. In this review, we will summarize the current views on the development and functions of thymic fibroblasts as revealed by new technologies such as multicolor flow cytometry and single cell–based transcriptome profiling. Furthermore, the recently discovered role of medullary fibroblasts in the establishment of T cell tolerance by producing a unique set of self‐antigens will be highlighted.

Identification and expression analysis of MicroRNAs in chicken spleen in a corticosterone-induced stress model

For investigating the effects of stress on the immune response of chickens, we established a corticosterone (CORT)-induced stress model by exogenous intake of CORT. Control group was fed with a basal diet and the stress model group was fed with a 30 mg/Kg CORT-treated diet in ad libitum conditions for 7 days. Then, we used RNA-seq technology to identify the expression pattern of miRNAs, target genes, and relevant pathways in chicken spleen. Results showed that 71 differentially expressed miRNAs (DEMs) were determined, 9 of which were significantly differentially expressed miRNAs (SDEMs), and 241 target genes of DEMs were predicted. GO annotation and KEGG pathway analysis were carried out to understand the role of the DEMs. Out of 287 significantly enriched GO terms, 37 were stress- or immune-related, such as response to light stimulus, detection of oxidative stress, and immune response in mucosal-associated lymphoid tissue. Out of 85 KEGG pathways, 8 were related to stress or immunity, such as cytokine-cytokine receptor interaction, JAK-STAT signaling pathway, and RLR signaling pathway. We then constructed the interaction networks between target genes from immune-related pathways and their DEMs. The analysis results suggested that some DEMs (gga-miR-17 family, gga-miR-15/16 family, gga-miR-2954 and gga-miR-34b-5p) and target genes (SIKE1, CX3CL1, IL11Ra, PIGR, and CDKN1A) were core miRNAs and genes. This study revealed the dynamic miRNA transcriptome, target genes and related pathways in chicken spleen under CORT-induced stress model, which provided a basis for studying the molecular mechanism of stress affecting immune function.

Characterization of follicular T helper cells and donor-specific T helper cells in renal transplant patients with de novo donor-specific HLA-antibodies

T follicular helper (TFH) cells are a heterogeneous subset of immunocompetent T helper (TH) cells capable of augmenting B cell responses in lymphoid tissues. In transplantation, exposure to allogeneic tissue activates TFH cells increasing the risk of the emergence of de novo donor-specific HLA-antibodies (dnDSA). These can cause antibody-mediated rejection (AMR) and allograft loss. Follicular regulatory T (TFR) cells counteract TFH cell activity. Here, we investigated the implications of TFH and TFR cells on dnDSA formation after renal transplantation (RTX). Considering TFH cells to be CXCR5+ and IL-21+, we found by flow cytometry that patients with dnDSA produced IL-21 more abundantly compared to healthy volunteers. In in vitro alloreactivity assays, patients with dnDSA featured an enhanced alloreactive TH cell pool in response to donor-specific HLA antigens. Besides, longitudinal investigations suggested enhanced alloreactivity shortly after transplantation increasing the risk of dnDSA development. Taken together, in spite of continuous immunosuppression we report a strong IL-21 response in TFH cells and an expanded reservoir of donor-specific memory TH cells in patients with dnDSA. This warrants further investigations if aberrant TFH cell activation may precede the formation of dnDSA promoting AMR.

852 Characterizing the microbiota-reactive immune response in neonates lacking breast milk antibodies

The human gut microbiome has approximately 1000 bacterial species. To maintain intestinal health, neonates must mount a specialized immune response to allow growth of commensal microbes while limiting pathogenic tissue access. The neonatal immune system is relatively immature, thus immune cells in breast milk are important contributors to establishing appropriate host-microbiome interactions in early life. Previous work in mice shows that neonates lacking breast milk antibodies (matAbs) have increased immune response in gut lymphoid tissue, including greater presence of T-cell dependent germinal center (GC) B cells. Little is known about the function nor the persistence of these GC B cells in neonates. Here we seek to characterize the antibody isotype produced by GC B cells in matAb deficient pups and determine the relative longevity of this antibody response. This study paves the way for understanding the implications of breastfeeding on the neonate gut microbiome. An experimental group of mice were breastfed milk lacking antibodies. These mice were further divided into two groups, one of which was treated with a blocking antibody (anti-ICOSL) to prevent formation of GC B cells. Half of a control population breastfed by WT dams was treated with anti-ICOSL antibody. Feces collected from these four experimental groups from weeks 3-9 were used in ELISAs to quantify IgA abundance. Our data indicates no significant difference in amounts of IgA in mice receiving matAbs compared to those that were not. There is no difference in IgA concentrations in mice whose GC B cell formation was limited by anti-ICOSL treatment compared to control groups. A trend supporting a relative increase in IgA over weeks 3-9 of life was present. Although neonates that do not receive matAbs in breastmilk have increased GC B cells, these cells do not produce more IgA compared to WT. Further research is indicated to determine if the increased GC B cell population in matAb deprived neonates causes an increase in other antibody isotypes or has other aberrant affects on the developing microbiome.

Transcriptional Analysis of Lymphoid Tissues from Infected Nonhuman Primates Reveals the Basis for Attenuation and Immunogenicity of an Ebola Virus Encoding a Mutant VP35 Protein.

Infection with Zaire ebolavirus (EBOV), a member of the Filoviridae family, causes a disease characterized by high levels of viremia, aberrant inflammation, coagulopathy, and lymphopenia. EBOV initially replicates in lymphoid tissues and disseminates via dendritic cells (DCs) and monocytes to liver, spleen, adrenal gland, and other secondary organs. EBOV protein VP35 is a critical immune evasion factor that inhibits type I interferon signaling and DC maturation. Nonhuman primates (NHPs) immunized with a high dose (5 × 105 PFU) of recombinant EBOV containing a mutated VP35 (VP35m) are protected from challenge with wild-type EBOV (wtEBOV). This protection is accompanied by a transcriptional response in the peripheral blood reflecting a regulated innate immune response and a robust induction of adaptive immune genes. However, the host transcriptional response to VP35m in lymphoid tissues has not been evaluated. Therefore, we conducted a transcriptional analysis of axillary and inguinal lymph nodes and spleen tissues of NHPs infected with a low dose (2 × 104 PFU) of VP35m and then back-challenged with a lethal dose of wtEBOV. VP35m induced early transcriptional responses in lymphoid tissues that are distinct from those observed in wtEBOV challenge. Specifically, we detected robust antiviral innate and adaptive responses and fewer transcriptional changes in genes with roles in angiogenesis, apoptosis, and inflammation. Two of three macaques survived wtEBOV back-challenge, with only the nonsurvivor displaying a transcriptional response reflecting Ebola virus disease. These data suggest that VP35 is a key modulator of early host responses in lymphoid tissues, thereby regulating disease progression and severity following EBOV challenge.IMPORTANCE Zaire Ebola virus (EBOV) infection causes a severe and often fatal disease characterized by inflammation, coagulation defects, and organ failure driven by a defective host immune response. Lymphoid tissues are key sites of EBOV pathogenesis and the generation of an effective immune response to infection. A recent study demonstrated that infection with an EBOV encoding a mutant VP35, a viral protein that antagonizes host immunity, can protect nonhuman primates (NHPs) against lethal EBOV challenge. However, no studies have examined the response to this mutant EBOV in lymphoid tissues. Here, we characterize gene expression in lymphoid tissues from NHPs challenged with the mutant EBOV and subsequently with wild-type EBOV to identify signatures of a protective host response. Our findings are critical for elucidating viral pathogenesis, mechanisms of host antagonism, and the role of lymphoid organs in protective responses to EBOV to improve the development of antivirals and vaccines against EBOV.

Dietary Supplementation with Spray-Dried Porcine Plasma Attenuates Colon Inflammation in a Genetic Mouse Model of Inflammatory Bowel Disease

Dietary supplementation with spray-dried porcine plasma (SDP) can modulate the immune response of gut-associated lymphoid tissue. SDP supplementation reduces acute mucosal inflammation, as well as chronic inflammation associated with aging. The aim of this study was to analyze if SDP supplementation could ameliorate colitis in a genetic mouse model of inflammatory bowel disease (IBD). Wild-type mice and Mdr1a knockout (KO) mice were administered a control diet or an SDP-supplemented diet from day 21 (weaning) until day 56. The histopathological index, epithelial barrier, and intestinal immune system were analyzed in the colonic mucosa. KO mice had higher epithelial permeability, increased Muc1 and Muc4 expression, and lower abundance of E-cadherin and Muc2 (all p < 0.001). SDP prevented these effects (all p < 0.05) and decreased the colonic inflammation observed in KO mice, reducing neutrophil and monocyte infiltration and activation and the percentage of activated T helper lymphocytes in the colonic mucosa (all p < 0.05). SDP also diminished proinflammatory cytokine expression and increased the anti-inflammatory IL-10 concentration in the colonic mucosa (all p < 0.05). In conclusion, dietary supplementation with SDP enhances colon barrier function and reduces mucosal inflammation in a mouse model of IBD.

Imaging the Cancer Immune Environment and Its Response to Pharmacologic Intervention, Part 1: The Role of 18F-FDG PET/CT.

Immunotherapy agents are now entering the clinic in a wide array of malignancies and have provided a valuable addition to the therapeutic armamentarium. These agents enhance the global immune response by modulating the tumor microenvironment but can lead to unconventional patterns of response, challenging the conceptual framework that imaging is a robust surrogate for therapeutic efficacy. There is also increasing evidence that an effective antitumor response requires a systemic immune response in primary and secondary lymphoid tissues. However, an enhanced systemic immune response can lead to disruption of immunologic hemostasis in healthy tissues, causing adverse events. Better understanding of the complex interplay between tumoral and systemic immune response has been provided through tissue and liquid biopsy. However, the applicability of these methods is constrained by the biologic, spatial, and temporal heterogeneity of the processes involved. There is a growing interest in molecular imaging of cell-specific lineage markers of the immune system using biomolecules. However, the ongoing role of the more widely available 18F-FDG PET/CT for response assessment is being recognized through ongoing refinement of interpretative guidelines and emerging evidence. These noninvasive methods provide insights into the biologic basis of the global immune response to maximize potential therapeutic benefit. In this review, we aim to provide an overview of the current status of 18F-FDG PET/CT in the monitoring of tumoral and systemic immune response. In a companion review, the role of other imaging probes that might complement 18F-FDG PET/CT will be discussed.

IL-35 is critical in suppressing superantigenic Staphylococcus aureus-driven inflammatory Th17 responses in human nasopharynx-associated lymphoid tissue

The human nasopharynx is frequently exposed to microbial pathogens, including superantigen-producing Staphylococcus aureus (SAg-Sau), which activates potent pro-inflammatory T cell responses. However, cellular mechanisms that control SAg-Sau-driven T cell activation are poorly understood. Using human nasopharynx-associated lymphoid tissue (NALT), we show that SAg-Sau drove a strong Th17 activation, which was associated with an impaired CD4+ T cell-mediated immune regulation. This impairment of immune control correlated with a significant downregulation of interleukin-35 (IL-35) expression in tonsillar CD4+ T cells by SAg-Sau. Supplementing recombinant IL-35 suppressed SAg-Sau-activated Th17 responses, and this IL-35-mediated suppression positively correlated with the level of Th17 activation. Interestingly, SAg-Sau stimulation induced Foxp3+ Treg expansion and interleukin-10 (IL-10) production, which effectively suppressed the Th1 response, but failed to control the activation of Th17 cells. Overall, our results reveal an aberrant T cell regulation on SAg-Sau-driven Th17 activation and identify IL-35 as a critical cytokine to control superantigenic S.aureus-activated Th17 responses.

Патоморфологическая и иммуногистохимическая характеристика изменений лимфатических узлов у умерших от COVID-19 по данным аутопсий

Introduction. The pathogenesis of the novel coronavirus infection COVID-19 is being actively studied. Nevertheless, our up-to-date knowledge of lymphoid tissue response in the secondary immune organs dur-ing severe COVID-19 remains extremely limited. The aim of the study was to evaluate patterns of immu-nomorphological alterations in lymph nodes in patients with severe COVID-19 and to assess lymphocytes functional activity in them. Materials and methods. Lymph node tissue (autopsy material) from 17 deceased patients with severe COVID-19 was examined by histological and immunohistochemical methods using antibodies to CD4, CD8, CD20, CD30, CD123, CD138, PD-1. Results. Examined lymph nodes demonstrated lymphoid follicles reduction and paracortex expansion with reactive plasmacytosis and extrafollicular B-cell activation as well as sinus histiocytosis, variable hemophago-cytic cells, and blood vessel congestion. Red thrombi were observed in some lymph nodes. Hemorrhages in the stroma were frequent, and massive hemorrhages were found in individual nodes. Immunohistochemical study revealed CD4+ T-helpers predominance in the paracortex andcytotoxic CD8+ lymphocytes depletion together with an increase in the expression of both the PD-1 suppressor protein and the CD30 activation anti-gen on the lymphocyte surface. CD123-positive plasmacytoid dendritic cells resided in sinuses in abundance. Conclusion. Demonstrated B-associated zone reduction and cytotoxic T-lymphocytes depletion with an up-regulation of PD-1 expression in the lymph nodes in patients with severe COVID-19 indicate immune response exhaustion. At the same time, observed significant reactive plasmacytosis with the presence of numerous T-helper cells constitutes a morphological substrate of the humoral immunity. These findings might indicate the ineffec-tiveness of the humoral response at late stages of COVID-19 infection in context of cytotoxic immunity failure. Keywords: novel coronavirus infection, SARS-CoV-2, COVID-19, B- and T- immune response, lymph node

The RAG2 gene of yellow catfish (Tachysurus fulvidraco) and its immune response against Edwardsiella ictaluri infection

Recombination-activating gene 2 (rag 2) allies with recombination-activating gene 1 (rag 1) and regulates the V(D)J recombination of immunoglobulin (Ig) and T-cell receptor (TCR) genes. Being a key player in the adaptive immune response of vertebrates, functional characterization of rag 2 from yellow catfish is beneficial for understanding the biological response towards the pathogens. In this report, we have cloned and characterized the rag 2 gene of yellow catfish, and a particular pattern of expression was analysed in the major tissues of yellow catfish. The results showed that the open reading frame (ORF) of yellow catfish rag 2 was 1596 bp in length, which encodes a peptide of 531 amino acids. The multiple sequence alignment and phylogenetic analysis of rag 2 of yellow catfish with other species showed the conserved regions and the classical taxonomic evolution among the different vertebrate species. The qRT-PCR and Western blot results revealed that rag 2 transcripts and proteins were present in various tissues of yellow catfish with relatively high expression in the tissues of the thymus, head-kidney, and spleen. The systematic distribution analysis of the rag 2 expression by immunohistochemistry (IHC) using the rabbit polyclonal antibody, exposed relatively high expression in head kidney, spleen and thymus tissues after infected with Edwardsiella ictaluri. Moreover, the temporal expression of rag 2 and pro-inflammatory cytokines (IL-1β and TNF-α) were significantly upregulated at different time points in the specific lymphoid tissues of yellow catfish following E. ictaluri infection. Our findings suggest that rag 2 potentially exhibited the immunological response in primary lymphoid tissues of yellow catfish against bacterial infection. This study will provide an essential source about rag 2 gene and its relationship with the inflammatory cytokines during infection.

Do cytomegalovirus-specific memory T cells interfere with new immune responses in lymphoid tissues?

In both mice and humans, the CD8 T cell compartment is expanded with age in the presence of a cytomegalovirus (CMV) infection due to an absolute increase in the CD8+ T cell effector memory (TEM) cells. It has been hypothesized that in CMV+ subjects, such accumulated TEM cells could interfere with responses to new infection by competing for space/resources or could inhibit new responses by other, undefined, means. Here we present evidence against this hypothesis. We show that MCMV-specific CD8 T cells accumulate in blood and bone marrow, but not lymph nodes (frequent sites of immune response initiation), in either persistent lifelong CMV infection or following reactivation. Moreover, adoptive transfer of effector memory T cells from MCMV positive mice into naïve animals did not interfere with either humoral or cellular response to West Nile virus or Listeria monocytogenes infection in recipient mice. We conclude that MCMV infection is unlikely to inhibit new immune responses in old animals through direct interference of MCMV-specific CD8 T cells with the priming.

Effect of rituximab treatment on T and B cell subsets in lymph node biopsies of patients with rheumatoid arthritis.

ObjectivesThe exact underlying mechanism of rituximab treatment in patients with RA is poorly defined and knowledge about the effect of B cell depletion on immune cells in secondary lymphoid organs is lacking. We analysed lymphoid tissue responses to rituximab in RA patients.MethodsFourteen RA patients received 2 × 1000 mg rituximab intravenously, and lymph node (LN) biopsies were obtained before and 4 weeks after the first infusion. Tissues were examined by flow cytometry, immunohistochemistry and quantitative PCR. LN biopsies from five healthy individuals (HC) served as controls.ResultsLN biopsies of RA patients showed increased frequencies of CD21+CD23+IgDhighIgMvariable follicular B cells and CD3+CD25+CD69+ early activated, tissue resident T cells when compared with HCs. After treatment, there was incomplete depletion of LN B cells. There was a significant decrease in CD27−IgD+ naïve B cells, and CD27+IgD+ unswitched memory B cells including the CD27+IgD+IgM+ subset and follicular B cells. Strikingly, CD27+IgD− switched memory B cells persisted in LN biopsies after rituximab treatment. In the T cell compartment, a significant decrease was observed in the frequency of early activated, tissue resident T cells after rituximab treatment, but late activated T cells persisted. B cell proliferation inducing cytokine IL-21 was higher expressed in LN biopsies of RA patients compared with HC and expression was not affected by rituximab treatment.ConclusionRituximab does not cure RA, possibly due to persistence of switched memory B cells in lymphoid tissues suggesting that factors promoting B cell survival and differentiation need to be additionally targeted.

Abstract 5643: TCRβ repertoire analysis from a prophylactic MUC1 cancer vaccine trial

Abstract Mucin 1 (MUC1) is a large transmembrane glycoprotein that is overexpressed and hypoglycosylated in many adenocarcinomas, including colon cancer. In the hypoglycosylated state, the peptide core of the 20aa variable number of tandem repeats (VNTR) region is exposed, allowing it to function as an altered-self ligand for antibodies and T cells. VNTR peptides have been previously used as antigens in therapeutic cancer vaccines. A clinical trial was conducted to test immunogenicity and safety of a MUC1 100mer VNTR peptide+polyICLC adjuvant vaccine in a prophylactic setting in patients with premalignant colon adenomas who were at high risk for later developing colon cancer. In this premalignant setting, where patients were expected to have fewer immunosuppressive countermeasures usually present in patients with cancer, nearly half of the individuals generated IgG antibodies with no noted adverse effects. Because the antibody response was isotype-switched, it suggested simultaneous T cell activation. To determine whether changes in T cell clonal abundance following immunization could be detected, we sequenced and analyzed TCRβ repertoires from PBMCs of 21 antibody-responders collected before and after both primary and one-year booster vaccinations. Of the patients analyzed, we identified 8 TCRβ sequences from three patients that were significantly elevated after both priming and boosting immunizations, potentially identifying T cell clones that expanded in response to the vaccine. 16 TCRβ sequences from four individuals were significantly decreased at both time points, potentially identifying T cell clones that left circulation to participate in immune responses in secondary lymphoid organs or tissues. The timing of blood collection at two weeks post immunization may have been too late to allow us to detect all of the vaccine-expanded clones before they left circulation. In summary, we found 24 TCRβ CDR3 sequences from 7/21 vaccine responders that significantly changed in frequency after both primary and booster vaccinations. TCR sequencing studies from patients' T cells expanded in vitro with MUC1 peptide will be performed to determine if they overlap with those sequenced ex vivo. Citation Format: Michelle L. Miller, Jason Lohmueller, John R. McKolanis, Robert Schoen, Olivera J. Finn. TCRβ repertoire analysis from a prophylactic MUC1 cancer vaccine trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5643.

Novel MVA-based vaccine expressing influenza NP and M1 activates cross-reactive T cell responses in human nasopharynx-associated lymphoid tissue (NALT)

Abstract Influenza virus infection continues to cause widespread morbidity and mortality, resulting in major challenges for healthcare systems. Current influenza vaccines are of limited efficacy against mismatched or emerging new virus strains. Recent efforts are focused on the development of novel vaccines capable of conferring broad protection against various influenza virus subtypes. We have studied the potential of Modified Vaccinia Ankara (MVA)-vectored vaccines expressing nucleoprotein and matrix protein 1 (MVA-NP+M1) to induce cross-reactive mucosal immunity using human adenotonsillar mononuclear cell (MNC) culture system with flow cytometry and ELISPOT analysis. Influenza NP and M1 antigens were abundantly expressed following vaccine stimulation in MNC particularly in B cells and dendritic cells. M1-specific T cell response was analyzed in detail, and MVA-NP+M1 was shown to activate IFN-γ-producing CD8+ T cells against conserved M1 epitopes. M158–66-specific CD8+ T cells in HLA-matched individuals were markedly increased in an age-dependent manner. These M1-specific T cells coexpressed cytotoxic molecules such as perforin and granzyme A and B. Upon the recognition of M158–66 peptide, these cells displayed marked increase in surface CD107a expression (implicating degranulation), together with the production of pro-inflammatory cytokines including IFN-γ and TNF-α, that correlated to their ability to kill the peptide-pulsed target cells. In summary, we showed MVA-NP+M1 activates conserved influenza antigen-specific cytotoxic T cell response in human NALT. The results suggest MVA-NP+M1 vaccine has the potential to induce cross-reactive mucosal T cell immunity against different types of influenza viruses.

Intranasal Live Influenza Vaccine Priming Elicits Localized B Cell Responses in Mediastinal Lymph Nodes.

Pandemic live attenuated influenza vaccines (pLAIV) prime subjects for a robust neutralizing antibody response upon subsequent administration of a pandemic inactivated subunit vaccine (pISV). However, a difference was not detected in H5-specific memory B cells in the peripheral blood between pLAIV-primed and unprimed subjects prior to pISV boost. To investigate the mechanism underlying pLAIV priming, we vaccinated groups of 12 African green monkeys (AGMs) with H5N1 pISV or pLAIV alone or H5N1 pLAIV followed by pISV and examined immunity systemically and in local draining lymph nodes (LN). The AGM model recapitulated the serologic observations from clinical studies. Interestingly, H5N1 pLAIV induced robust germinal center B cell responses in the mediastinal LN (MLN). Subsequent boosting with H5N1 pISV drove increases in H5-specific B cells in the axillary LN, spleen, and circulation in H5N1 pLAIV-primed animals. Thus, H5N1 pLAIV primes localized B cell responses in the MLN that are recalled systemically following pISV boost. These data provide mechanistic insights for the generation of robust humoral responses via prime-boost vaccination.IMPORTANCE We have previously shown that pandemic live attenuated influenza vaccines (pLAIV) prime for a rapid and robust antibody response on subsequent administration of inactivated subunit vaccine (pISV). This is observed even in individuals who had undetectable antibody (Ab) responses following the initial vaccination. To define the mechanistic basis of pLAIV priming, we turned to a nonhuman primate model and performed a detailed analysis of B cell responses in systemic and local lymphoid tissues following prime-boost vaccination with pLAIV and pISV. We show that the nonhuman primate model recapitulates the serologic observations from clinical studies. Further, we found that pLAIVs induced robust germinal center B cell responses in the mediastinal lymph node. Subsequent boosting with pISV in pLAIV-primed animals resulted in detection of B cells in the axillary lymph nodes, spleen, and peripheral blood. We demonstrate that intranasally administered pLAIV elicits a highly localized germinal center B cell response in the mediastinal lymph node that is rapidly recalled following pISV boost into germinal center reactions at numerous distant immune sites.

CD4+ T Helper Cells Play a Key Role in Maintaining Diabetogenic CD8+ T Cell Function in the Pancreas.

Autoreactive CD8+ and CD4+ T cells have been assigned independent key roles in the destruction of insulin-producing beta cells resulting in type 1 diabetes. Although CD4 help for the generation of efficient CD8+ T cell responses in lymphoid tissue has been extensively described, whether these two cell populations cooperate in islet destruction in situ remains unclear. By using intravital 2-photon microscopy in a mouse model of diabetes, we visualized both effector T cell populations in the pancreas during disease onset. CD4+ T helper cells displayed a much higher arrest in the exocrine tissue than islet-specific CD8+ T cells. This increased arrest was major histocompatibility complex (MHC) class II-dependent and locally correlated with antigen-presenting cell recruitment. CD8+ T cells deprived of continued CD4 help specifically in the pancreas, through blocking MHC class II recognition, failed to maintain optimal effector functions, which contributed to hamper diabetes progression. Thus, we provide novel insight in the cellular mechanisms regulating effector T cell functionality in peripheral tissues with important implications for immunotherapies.

An Immunopathological Evaluation of the Porcine Cholecyst Matrix as a Muscle Repair Graft in a Male Rat Abdominal Wall Defect Model.

With the increasing use of animal-based biomaterials for regenerative medical applications, the need for their safety assessment is paramount. A porcine cholecyst-derived scaffold (CDS), intended as a muscle repair graft, prepared by a nondetergent/enzymatic method was engrafted in a rat abdominal wall defect model. Host tissue-scaffold interface samples were collected 2, 8, and 16 weeks postimplantation and evaluated by histopathology, immunohistochemistry, and electron microscopy. The nature of the tissue reaction was compared with those induced by a jejunum-derived scaffold (JDS) prepared by the same method and a commercial-grade small intestinal submucosa (CSIS) scaffold. A study of the immunopathological response in major lymphoid tissues and immunophenotyping for M1 and M2 macrophages was performed at the host tissue-scaffold interface. Further, "irritancy scores" for CDS and JDS were determined using CSIS as the reference material. Both CDS and JDS appeared to be potential biomaterials for muscle grafts, but the former stimulated a skeletal muscle tissue remodeling response predominated by M2 macrophages. The data support the notion that biomaterials with similar biocompatibility, based on local tissue response on implantation, may cause differential immunogenicity. Additionally, CDS compared to JDS and CSIS was found to be less immunotoxic.

18F]-Fluorodeoxyglucose Uptake in Lymphoid Tissue Serves as a Predictor of Disease Outcome in the Nonhuman Primate Model of Monkeypox Virus Infection.

Real-time bioimaging of infectious disease processes may aid countermeasure development and lead to an improved understanding of pathogenesis. However, few studies have identified biomarkers for monitoring infections using in vivo imaging. Previously, we demonstrated that positron emission tomography/computed tomography (PET/CT) imaging with [18F]-fluorodeoxyglucose (FDG) can monitor monkeypox disease progression in vivo in nonhuman primates (NHPs). In this study, we investigated [18F]-FDG-PET/CT imaging of immune processes in lymphoid tissues to identify patterns of inflammation in the monkepox NHP model and to determine the value of [18F]-FDG-PET/CT as a biomarker for disease and treatment outcomes. Quantitative analysis of [18F]-FDG-PET/CT images revealed differences between moribund and surviving animals at two sites vital to the immune response to viral infections, bone marrow and lymph nodes (LNs). Moribund NHPs demonstrated increased [18F]-FDG uptake in bone marrow 4 days postinfection compared to surviving NHPs. In surviving, treated NHPs, increase in LN volume correlated with [18F]-FDG uptake and peaked 10 days postinfection, while minimal lymphadenopathy and higher glycolytic activity were observed in moribund NHPs early in infection. Imaging data were supported by standard virology, pathology, and immunology findings. Even with the limited number of subjects, imaging was able to differentiate the difference between disease outcomes, warranting additional studies to demonstrate whether [18F]-FDG-PET/CT can identify other, subtler effects. Visualizing altered metabolic activity at sites involved in the immune response by [18F]-FDG-PET/CT imaging is a powerful tool for identifying key disease-specific time points and locations that are most relevant for pathogenesis and treatment.IMPORTANCE Positron emission tomography and computed tomography (PET/CT) imaging is a universal tool in oncology and neuroscience. The application of this technology to infectious diseases is far less developed. We used PET/CT imaging with [18F]-labeled fluorodeoxyglucose ([18F]-FDG) in monkeys after monkeypox virus exposure to monitor the immune response in lymphoid tissues. In lymph nodes of surviving monkeys, changes in [18F]-FDG uptake positively correlated with enlargement of the lymph nodes and peaked on day 10 postinfection. In contrast, the bone marrow and lymph nodes of nonsurvivors showed increased [18F]-FDG uptake by day 4 postinfection with minimal lymph node enlargement, indicating that elevated cell metabolic activity early after infection is predictive of disease outcome. [18F]-FDG-PET/CT imaging can provide real-time snapshots of metabolic activity changes in response to viral infections and identify key time points and locations most relevant for monitoring the development of pathogenesis and for potential treatment to be effective.

KIR3DL01 upregulation on gut natural killer cells in response to SIV infection of KIR- and MHC class I-defined rhesus macaques.

Natural killer cells provide an important early defense against viral pathogens and are regulated in part by interactions between highly polymorphic killer-cell immunoglobulin-like receptors (KIRs) on NK cells and their MHC class I ligands on target cells. We previously identified MHC class I ligands for two rhesus macaque KIRs: KIR3DL01 recognizes Mamu-Bw4 molecules and KIR3DL05 recognizes Mamu-A1*002. To determine how these interactions influence NK cell responses, we infected KIR3DL01+ and KIR3DL05+ macaques with and without defined ligands for these receptors with SIVmac239, and monitored NK cell responses in peripheral blood and lymphoid tissues. NK cell responses in blood were broadly stimulated, as indicated by rapid increases in the CD16+ population during acute infection and sustained increases in the CD16+ and CD16-CD56- populations during chronic infection. Markers of proliferation (Ki-67), activation (CD69 & HLA-DR) and antiviral activity (CD107a & TNFα) were also widely expressed, but began to diverge during chronic infection, as reflected by sustained CD107a and TNFα upregulation by KIR3DL01+, but not by KIR3DL05+ NK cells. Significant increases in the frequency of KIR3DL01+ (but not KIR3DL05+) NK cells were also observed in tissues, particularly in the gut-associated lymphoid tissues, where this receptor was preferentially upregulated on CD56+ and CD16-CD56- subsets. These results reveal broad NK cell activation and dynamic changes in the phenotypic properties of NK cells in response to SIV infection, including the enrichment of KIR3DL01+ NK cells in tissues that support high levels of virus replication.