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bioRxiv2021

Expansion of tissue-resident CD8+ T cells and CD4+ Th17 cells in the nasal mucosa following mRNA COVID-19 vaccination

Vaccines against SARS-CoV-2 have shown high efficacy in clinical trials, yet a full immunologic characterization of these vaccines, particularly within the upper respiratory tract, remains lacking. We enumerated and phenotyped T cells in nasal mucosa and blood before and after vaccination with the Pfizer-BioNTech COVID-19 vaccine (n =21). Tissue-resident memory (Trm) CD8+ T cells expressing CD69+CD103+ expanded [~]12 days following the first and second doses, by 0.31 and 0.43 log10 cells per swab respectively (p=0.058 and p=0.009 in adjusted linear mixed models). CD69+CD103+CD8+ T cells in the blood decreased post-vaccination. Similar increases in nasal CD8+CD69+CD103-T cells were observed, particularly following the second dose. CD4+ Th17 cells were also increased in abundance following both doses. Following stimulation with SARS-CoV-2 spike peptides, CD8+ T cells increased expression of CD107a and CD154. These data suggest that nasal T cells may be induced and contribute to the protective immunity afforded by this vaccine.

immunology↗

bioRxiv2021

Evidence for Deleterious Original Antigenic Sin in SARS-CoV-2 Immune Response

A previous report demonstrated the strong association between the presence of antibodies binding to an epitope region from SARS-CoV-2 nucleocapsid, termed Ep9, and COVID-19 disease severity. Patients with anti-Ep9 antibodies (Abs) had hallmarks of antigenic imprinting (AIM), including early IgG upregulation and cytokine-associated injury. Thus, the immunological memory of a previous infection was hypothesized to drive formation of suboptimal anti-Ep9 Abs in severe COVID-19 infections. This study identifies a putative primary antigen capable of stimulating production of cross-reactive, anti-Ep9 Abs. Binding assays with patient blood samples directly show cross-reactivity between Abs binding to Ep9 and only one bioinformatics-derived, homologous potential antigen, a sequence derived from the neuraminidase protein of H3N2 Influenza A virus. This cross-reactive binding is highly influenza strain specific and sensitive to even single amino acid changes in epitope sequence. The neuraminidase protein is not present in the influenza vaccine, and the anti-Ep9 Abs likely resulted from the widespread influenza infection in 2014. Therefore, AIM from a previous infection could underlie some cases of COVID-19 disease severity. ImportanceInfections with SARS-COV-2 result in diverse disease outcomes, ranging from asymptomatic to fatal. The mechanisms underlying different disease outcomes remain largely unexplained. Previously, our laboratory identified a strong association between the presence of an antibody and increased disease severity in a subset of COVID-19 patients. Here, we report that this severity-associated antibody cross-reacts with viral proteins from an influenza A viral strain from 2014. Therefore, we speculate that antibodies generated against previous infections, like the 2014 influenza A, play a significant role in directing some peoples immune responses against SARS-COV-2. Such understanding of the sources and drivers of COVID-19 disease severity can help early identification and pre-emptive treatment.

immunology↗

bioRxiv2021

Disc Large Homolog 1 is critical for early microcluster formation and activation in human T cells

T cell activation by antigen involves multiple sequential steps, including TCR-microcluster (MC) formation, immunological synapse formation and phosphorylation of mediators downstream of the TCR. The adaptor protein Disc Large Homolog 1 (DLG1) is known to regulate proximal TCR signaling and, in turn, T cell activation, acting as a molecular chaperone that organizes specific kinases downstream of antigen recognition. Here, we report using knockdown and knockout studies in human T cells that DLG1 functions even earlier to regulate T cell activation by promoting TCR-MC formation. Moreover, we found that DLG1 can act as a bridge between the TCR-{zeta} chain and ZAP70 while inhibiting binding of the phosphatase SHP1 to TCR-{zeta}. Together, these effects drive dysregulation of T cell activation in DLG1-deficient T cells.

immunology↗

bioRxiv2021

Early Th2 cytokine production in Heligmosomoides polygyrus and Toxoplasma gondii co-infected mice is associated with reduced IFNγ production, increased parasite loads and increased mortality

Co-infections are a common reality but understanding how the immune system responds in this context is complex and can be unpredictable. Heligmosomoides bakeri (parasitic roundworm, previously Heligmosomoides polygyrus) and Toxoplasma gondii (protozoan parasite) are well studied organisms that stimulate a characteristic Th2 and Th1 response, respectively. Several studies have demonstrated reduced inflammatory cytokine responses in animals co-infected with such organisms. However, while general cytokine signatures have been examined, the impact of the different cytokine producing lymphocytes on parasite control/clearance is not fully understood. We investigated five different lymphocyte populations (NK, NKT, {gamma}{delta} T, CD4 + T and CD8+ T cells), five organs (small intestine, Peyers patches, mesenteric lymph nodes, spleen and liver), and 4 cytokines (IFN {gamma}, IL-4, IL-10 and IL-13) at two different time points (days 5 and 10 post T. gondii infection). We found that co-infected animals had significantly higher mortality than either single infection. This was accompanied by transient and local changes in parasite loads and cytokine profiles. Despite the early changes in lymphocyte and cytokine profiles, severe intestinal pathology in co-infected mice likely contributed to early mortality due to significant damage by both parasites in the small intestine. Our work demonstrates the importance of taking a broad view during infection research, studying multiple cell types, organs/tissues and time points to link and/or uncouple immunological from pathological findings. Our results provide insights into how co-infection with parasites stimulating different arms of the immune system can lead to drastic changes in infection dynamics.

immunology↗

bioRxiv2021

Topological and Structural Plasticity of the single Ig fold and the double Ig fold present in CD19

The Ig-fold has had a remarkable success in vertebrate evolution, with a presence in over 2% of human genes. The Ig-fold is not just the elementary structural domain of antibodies and TCRs, it is also at the heart of a staggering 30% of immunologic cell surface receptors, making it a major orchestrator of cell-cell-interactions. While BCRs, TCRs, and numerous Ig-based cell surface receptors form homo or heterodimers on the same cell surface (in cis), many of them interface as ligand-receptors (checkpoints) on interacting cells (in trans) through their Ig domains. New Ig-Ig interfaces are still being discovered between Ig-based cell surface receptors, even in well known families such as B7. What is largely ignored however is that the Ig-fold itself is pseudo-symmetric, a property that makes the Ig-domain a versatile self-associative 3D structure and may in part explain its success in evolution, especially through its ability to bind in cis or in trans in the context of cell surface receptor-ligand interactions. In this paper we review the Ig domains tertiary and quaternary pseudo symmetries, with a particular attention to the newly identified double Ig fold in the solved CD19 molecular structure to highlight the underlying fundamental folding elements of Ig domains, i.e. Ig protodomains. This pseudosymmetric property of Ig domains gives us a decoding frame of reference to understand the fold, relate all Ig-domain forms, single or double, and suggest new protein engineering avenues.

immunology↗

bioRxiv2021

Persistence of SARS CoV-2 S1 Protein in CD16+ Monocytes in Post-Acute Sequelae of COVID-19 (PASC) Up to 15 Months Post-Infection

The recent COVID-19 pandemic is a treatment challenge in the acute infection stage but the recognition of chronic COVID-19 symptoms termed post-acute sequelae SARS-CoV-2 infection (PASC) may affect up to 30% of all infected individuals. The underlying mechanism and source of this distinct immunologic condition three months or more after initial infection remains elusive. Here, we investigated the presence of SARS-CoV-2 S1 protein in 46 individuals. We analyzed T-cell, B-cell, and monocytic subsets in both severe COVID-19 patients and in patients with post-acute sequelae of COVID-19 (PASC). The levels of both intermediate (CD14+, CD16+) and non-classical monocyte (CD14Lo, CD16+) were significantly elevated in PASC patients up to 15 months post-acute infection compared to healthy controls (P=0.002 and P=0.01, respectively). A statistically significant number of non-classical monocytes contained SARS-CoV-2 S1 protein in both severe (P=0.004) and PASC patients (P=0.02) out to 15 months post-infection. Non-classical monocytes were sorted from PASC patients using flow cytometric sorting and the SARS-CoV-2 S1 protein was confirmed by mass spectrometry. Cells from 4 out of 11 severe COVID-19 patients and 1 out of 26 PASC patients contained ddPCR+ peripheral blood mononuclear cells, however, only fragmented SARS-CoV-2 RNA was found in PASC patients. No full length sequences were identified, and no sequences that could account for the observed S1 protein were identified in any patient. Non-classical monocytes are capable of causing inflammation throughout the body in response to fractalkine/CX3CL1 and RANTES/CCR5.

immunology↗

bioRxiv2021

Clonally expanded virus-specific CD8 T cells acquire diverse transcriptional phenotypes during acute, chronic, and latent infections

CD8+ T cells play a crucial role in the control and resolution of viral infections and can adopt a wide range of phenotypes and effector functions depending on the inflammatory context and the duration and extent of antigen exposure. Similarly, viral infections can exert diverse selective pressures on populations of clonally related T cells. Technical limitations have nevertheless made it challenging to investigate the relationship between clonal selection and transcriptional phenotypes of virus-specific T cells. We therefore performed single-cell T cell receptor (TCR) repertoire and transcriptome sequencing of virus-specific CD8 T cells in murine models of acute, chronic and latent infection. We observed clear infection-specific populations corresponding to memory, effector, exhausted, and inflationary phenotypes. We further uncovered a mouse-specific and polyclonal T cell response, despite all T cells sharing specificity to a single viral epitope, which was accompanied by stereotypic TCR germline gene usage in all three infection types. Persistent antigen exposure during chronic and latent viral infections resulted in a higher proportion of clonally expanded T cells relative to acute infection. We furthermore observed a relationship between transcriptional heterogeneity and clonal expansion for all three infections, with highly expanded clones having distinct transcriptional phenotypes relative to lowly expanded clones. Finally, we developed and utilized a bioinformatic pipeline integrating pseudotime and clonality, termed Clonotyme, to further support a model in which expanded virus-specific CD8+ T cells adopt heterogenic, yet preferentially, effector-like phenotypes. Together our work relates clonal selection to gene expression in the context of viral infection and further provides a dataset and accompanying software for the immunological community.

immunology↗

bioRxiv2021

Comparative computational modeling of the bat and human immune response to viral infection with the Comparative Biology Immune Agent Based Model

Given the impact of pandemics due to viruses of bat origin there is increasing interest in comparative investigation into the differences between bat and human immune responses. The practice of comparative biology can be enhanced by computational methods used for dynamic knowledge representation to visualize and interrogate the putative differences between the two systems. We present an agent-based model that encompasses and bridges the differences between bat and human responses to viral infection: the Comparative Biology Immune Agent-based Model, or CBIABM. The CBIABM examines differences in innate immune mechanisms between bats and humans, specifically regarding inflammasome activity and Type 1 Interferon dynamics, in terms of tolerance to viral infection. Simulation experiments with the CBIABM demonstrate the efficacy of bat-related features in conferring viral tolerance and also suggest a crucial role for endothelial inflammasome activity as a mechanism for bat systemic viral tolerance and affecting the severity of disease in human viral infections. We hope that this initial study will inspire additional comparative modeling projects to link, compare, and contrast immunological functions shared across different species, and in so doing, provide insight and aid in the preparation for future viral pandemics of zoonotic origin.

immunology↗

bioRxiv2021

TAILORED DC INDUCE PROTECTIVE HIV-1 SPECIFIC POLYFUNCTIONAL CD8+ T CELLS IN THE LYMPHOID TISSUE FROM HUMANIZED BLT MICE

Effective function of CD8+ T cells and enhanced innate activation of dendritic cells (DC) in response to HIV-1 is linked to protective antiviral immunity in controllers. Manipulation of DC targeting the master regulator TANK-binding Kinase 1 (TBK1) might be useful to acquire controller-like properties. Here, we evaluated the impact of TBK1-primed DC inducing protective CD8+ T cell responses in lymphoid tissue and peripheral blood and their association with reduced HIV-1 disease progression in vivo in the humanized bone marrow, liver and thymus (hBLT) mouse model. A higher proportion of hBLT-mice vaccinated with TBK1-primed DC exhibited less severe CD4+ T cell depletion following HIV-1 infection compared to control groups. This was associated with infiltration of CD8+ T cells in the white pulp from the spleen, reduced spread of infected p24+ cells to secondary lymphoid organs and with preserved abilities of CD8+ T cells from the spleen and blood of vaccinated animals to induce specific polyfunctional responses upon antigen stimulation. Therefore, TBK1-primed DC might be an useful tool for subsequent vaccine studies. Author summaryEmulating protective immunological characteristics from individuals capable of spontaneously controlling HIV-1 infection might be useful for the development of a protective vaccine. Enhanced function of dendritic cells (DC) in these HIV-1 controllers depends on the activation of TANK-binding Kinase 1 (TBK1) and might associate with protective T cells. Our study evaluated the ability of DCs trained through TBK1 activation inducing protective adaptive immune responses against HIV-1 and reducing disease progression in vivo, using a humanized mouse model. Our data indicate that mice vaccinated with tailored DC exhibit delayed disease progression, increased induction of protective CD8+ T lymphocyte subsets in the lymphoid tissue and blood upon antigen recognition. Therefore, trained-DC might be an useful tool for future HIV-1 vaccine designs.

immunology↗

bioRxiv2021

Single-domain antibodies for targeting, detection and in vivo imaging of human CD4+ cells

The advancement of new immunotherapies necessitates appropriate probes to monitor the presence and distribution of distinct immune cell populations. Considering the key role of CD4+ T cells in regulating immunological processes, we generated novel single-domain antibodies (nanobodies, Nbs) that specifically recognize human CD4. After in depth analysis of their binding properties, recognized epitopes, and effects on T cell proliferation, activation and cytokine release, we selected CD4 Nbs that did not interfere with crucial T cell processes in vitro and converted them into immune tracers for non-invasive molecular imaging. By optical imaging, we demonstrate the ability of a high-affinity CD4-Nb to specifically visualize CD4+ cells in vivo using a xenograft model. Furthermore, time-resolved immune positron emission tomography (immunoPET) of a human CD4 knock-in mouse model showed rapid accumulation of 64Cu-radiolabeled CD4-Nb in CD4+ T cell-rich tissues. We propose that the CD4 Nbs presented here could serve as versatile probes for stratifying patients and monitoring individual immune responses during personalized immunotherapy in both cancer and inflammatory diseases.

immunology↗

bioRxiv2021

The PD-1 checkpoint receptor maintains tolerance of self-reactive CD8 T cell in skin

Peripheral tolerance is thought to result from anergy or deletion of self-reactive T cells shortly after antigen encounter. However, the frequent occurrences of immune-related Adverse Events (irAEs) following checkpoint inhibitor (CPI) treatment suggest a hypothesis that immunologically healthy individuals have self-reactive effector T cells that are kept in a non-pathogenic state through checkpoint receptor-mediated suppression, instead of anergy or deletion. We expressed self-antigens in healthy skin and found that antigen-specific CD8 T cells infiltrated the tissue, but remained tolerant, despite having a transcriptional program that resembled effector T cells found after CPIs. These self-reactive PD-1+ CD8 T cells drove IFN{gamma}-dependent increases in PD-L1 on skin myeloid cells. Blockade of PD-1 or PD-1/CTLA-4 led to post-transcriptional upregulation of effector proteins by antigen-specific CD8 T cells and elimination of antigen-expressing epithelial cells, resulting in localized tissue pathology with features of human cutaneous irAEs. This data supports the hypothesis that myeloid cells in healthy skin prevent pathology from self-reactive effector CD8 T cells through the PD-1/PD-L1 pathway.

immunology↗

bioRxiv2021

IgG surface mobility promotes antibody-dependent cellular phagocytosis by Syk and Arp2/3 mediated reorganization of Fcγ receptors in macrophages.

By visualizing the movements of Rituximab during Antibody dependent cellular phagocytosis (ADCP) of B lymphoma cells by macrophages, we found that Fc{gamma} receptors (Fc{gamma}R) on the macrophage surface microcluster, recruit Syk and undergro large-scale reorganization at the phagocytic synapse prior to and during engulfment of the target cell. Given these dramatic rearrangements, we analyzed how the surface mobility of Rituximab contributes to Fc{gamma}R signal amplification and ADCP efficiency. Depolymerization of the target cell actin cytoskeleton resulted in free diffusion of Rituximab docked to CD20, enhanced microcluster reorganization, Syk recruitment and ADCP. Conversely, immobilization of Rituximab by chemical fixation impaired microcluster formation and diminished Syk recruitment and ADCP. In macrophages lacking Syk, Rituximab accumulated at the base of the phagosome and were trogocytosed, consistent with Syk kinase activity being necessary to trigger redistribution of Rituximab-Fc{gamma}R during engulfment and to prevent antigenic modulation of the target. Total internal reflection fluorescence analysis of Fc{gamma}R-IgG on fluid supported lipid bilayers revealed a membrane topography displaying inward reaching leading edges and protruding contact sites reminiscent of podosomes. This topography was distinct from the closely apposed macrophage/target membranes observed during engagement of IgG displayed on immobile supported lipid bilayers. The organization of this contact, pseudopod extension and the rearrangement of microclusters depended critically on Arp 2/3. Thus, Syk and Arp2/3 coordinate actin rearrangements and Fc{gamma}R-IgG complexes that were of previously unrecognized complexity for the clearance of cells displaying surface-mobile antigens. Significance StatementADCP is an important effector mechanism for the removal of malignant, immunologically aberrant, and infected cells during treatment with therapeutic antibodies or adaptive immune responses. Most transmembrane protein antigens are mobile with transient confinement from the actin of the target cell. This work demonstrates that macrophage forces overcome these confinements to rearrange Fc{gamma}R-IgG-antigen complexes before and during ADCP. Thus, new paradigms are needed as ADCP has largely been studied using model target particles that display immobile antigens. Moreover, we found that the mobility of the therapeutic antibody, Rituximab, on the surface of B lymphoma cells foretells ADCP efficacy, with lower densities of IgG mediating ADCP on increasingly mobile antigens.

immunology↗

bioRxiv2021

Regulation of MHC-I and MHC-II by CIITA in transmissible cancers

MHC-I and MHC-II molecules are critical components of antigen presentation and T cell immunity to pathogens and cancer. The two monoclonal transmissible devil facial tumours (DFT1, DFT2) exploit MHC-I pathways to overcome immunological anti-tumour and allogeneic barriers. This exploitation underpins the ongoing transmission of DFT cells across the wild Tasmanian devil population. We have previously shown that constitutive expression of NLRC5 can induce stable upregulation of MHC-I on DFT1 and DFT2 cells, but unlike IFNG-treated cells, NLRC5 does not upregulate PDL1. MHC-II expression is crucial for CD4+ T cell activation and is primarily confined to haematopoietic antigen presenting cells. Transcriptomic analysis of DFT1 and DFT2 cell lines showed that several genes of the MHC-I and MHC-II pathways were upregulated in response to constitutive overexpression of the class II transactivator (CIITA) gene. This was further supported by upregulation of MHC-I protein on DFT1 and DFT2 cells, but interestingly MHC-II protein was upregulated only on DFT1 cells. The functional significance of the MHC upregulation on DFT cells was shown using serum from devils with natural or immunotherapy-induced DFT1 regressions; binding of serum IgG was stronger in CIITA-transfected cells than wild type cells, but was less than binding to NLRC5 transfected cells. This new insight into regulation of MHC-I and MHC-II in cells that naturally overcome allogeneic barriers can inform vaccine, immunotherapy, and tissue transplant strategies for human and veterinary medicine.

immunology↗

bioRxiv2021

Echidna: integrated simulations of single-cell immune receptor repertoires and transcriptomes

Single-cell sequencing now enables the recovery of full-length immune repertoires [B cell receptor (BCR) and T cell receptor (TCR) repertoires], in addition to gene expression information. The feature-rich datasets produced from such experiments require extensive and diverse computational analyses, each of which can significantly influence the downstream immunological interpretations, such as clonal selection and expansion. Simulations produce validated standard datasets, where the underlying generative model can be precisely defined and furthermore perturbed to investigate specific questions of interest. Currently, there is no tool that can be used to simulate a comprehensive ground truth single-cell dataset that incorporates both immune receptor repertoires and gene expression. Therefore, we developed Echidna, an R package that simulates immune receptors and transcriptomes at single-cell resolution. Our simulation tool generates annotated single-cell sequencing data with user-tunable parameters controlling a wide range of features such as clonal expansion, germline gene usage, somatic hypermutation, and transcriptional phenotypes. Echidna can additionally simulate time-resolved B cell evolution, producing mutational networks with complex selection histories incorporating class-switching and B cell subtype information. Finally, we demonstrate the benchmarking potential of Echidna by simulating clonal lineages and comparing the known simulated networks with those inferred from only the BCR sequences as input. Together, Echidna provides a framework that can incorporate experimental data to simulate single-cell immune repertoires to aid software development and bioinformatic benchmarking of clonotyping, phylogenetics, transcriptomics and machine learning strategies. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=60 SRC="FIGDIR/small/452792v1_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@1c9930eorg.highwire.dtl.DTLVardef@d5a50eorg.highwire.dtl.DTLVardef@47584forg.highwire.dtl.DTLVardef@1f6bb17_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2021

Previously unrecognized non-reproducible antibody-antigen interactions and their implications for diagnosis of viral infections including COVID-19

Antibody-antigen (Ab-Ag) interactions are canonically described by a model which exclusively accommodates non-interaction (0) or reproducible-interaction (RI) states, yet this model is inadequate to explain often-encountered non-reproducible signals. Here, by monitoring diverse experimental systems and confirmed COVID-19 clinical sera using a peptide microarray, we observed that non-specific interactions (NSI) comprise a substantial proportion of non-reproducible antibody-based results. This enabled our discovery and capacity to reliably identify non-reproducible Ab-Ag interactions (NRI), as well as our development of a powerful explanatory model ("0-RI-NRI-Hook four-state model") that is [mAb]-dependent, regardless of specificity, which ultimately shows that both NSI and NRI are not predictable yet certain-to-happen. In experiments using seven FDA-approved mAb drugs, we demonstrated the use of NSI counts in predicting epitope type. Beyond challenging the centrality of Ab-Ag interaction specificity data in serology and immunology, our discoveries also facilitated the rapid development of a serological test with uniquely informative COVID-19 diagnosis performance.

immunology↗

bioRxiv2021

CD8+ T cell signature in acute SARS-CoV-2 infection identifies memory precursors

Immunological memory is a hallmark of adaptive immunity and facilitates an accelerated and enhanced immune response upon re-infection with the same pathogen1,2. Since the outbreak of the ongoing coronavirus disease 19 (COVID-19) pandemic, a key question has focused on whether severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific T cells stimulated during acute infection give rise to long-lived memory T cells3. Using spectral flow cytometry combined with cellular indexing of transcriptomes and T cell receptor (TCR) sequencing we longitudinally characterize individual SARS-CoV-2-specific CD8+ T cells of COVID-19 patients from acute infection to one year into recovery and find a distinct signature identifying long-lived memory CD8+ T cells. SARS-CoV-2-specific memory CD8+ T cells persisting one year after acute infection re-express CD45RA and interleukin-7 receptor (CD127), upregulate T cell factor-1 (TCF1), and maintain low CCR7, thus resembling CD45RA+ effector-memory T (TEMRA) cells. Tracking individual clones of SARS-CoV-2-specific CD8+ T cells, we reveal that an interferon signature marks clones giving rise to long-lived cells, whereas prolonged proliferation and mammalian target of rapamycin (mTOR) signaling are associated with clone contraction and disappearance. Collectively, we identify a transcriptional signature differentiating short-from long-lived memory CD8+ T cells following an acute virus infection in humans.

immunology↗

bioRxiv2021

Multi-tier mechanics control stromal adaptations in swelling lymph nodes

Lymph nodes (LNs) comprise two main structural elements: Fibroblastic reticular cells (FRCs) that form dedicated niches for immune cell interaction and capsular fibroblasts that build a shell around the organ. While LNs are fairly stable in size during homeostatic conditions, immunological challenge causes more than 10-fold increase in size within only a few days. How a solid organ can accommodate such extreme volumetric changes is poorly understood. Here, we characterize the biomechanics of LN swelling on the cellular and organ scale. We identify lymphocyte trapping by influx and proliferation as drivers of an outward pressure force, causing FRCs and their associated conduits to stretch. After an initial phase of relaxation, FRCs sense the resulting strain via cell matrix adhesions, which coordinates local growth and remodeling of the stromal network. While the expanded FRC network adopts its typical configuration, a massive fibrotic reaction of the organ capsule sets in and counters further organ expansion. Thus, different fibroblast populations mechanically control LN swelling in a multi-tier fashion.

immunology↗

bioRxiv2021

Epithelial Immunomodulation by Aerosolized Toll-like Receptor Agonists Attenuates Allergic Responsiveness in Mice

Allergic asthma is a chronic inflammatory respiratory disease associated with eosinophilic infiltration, increased mucus production, airway hyperresponsiveness (AHR), and airway remodeling. Epidemiologic data has revealed that the prevalence of allergic sensitization and associated diseases has increased in the twentieth century. This has been hypothesized to be partly due to reduced contact with microbial organisms (the hygiene hypothesis) in industrialized society. Airway epithelial cells, once considered a static physical barrier between the body and the external world, are now widely recognized as immunologically active cells that can initiate, maintain, and restrain inflammatory responses, such as those that mediate allergic disease. Airway epithelial cells can sense allergens via myriad expression of Toll-like receptors (TLRs) and other pattern-recognition receptors (PRRs). We sought to determine whether the innate immune response stimulated by a combination of Pam2CSK4 ("Pam2", TLR2/6 ligand) and a class C oligodeoxynucleotide ODN362 ("ODN", TLR9 ligand) when delivered together by aerosol ("Pam2ODN"), can modulate the allergic immune response to allergens. Treatment with Pam2ODN 7 days before sensitization to House Dust Mite (HDM) extract resulted in a strong reduction in eosinophilic and lymphocytic inflammation. This Pam2ODN immunomodulatory effect was also seen using Ovalbumin (OVA) and A. oryzae (Ao) mouse models. The immunomodulatory effect was observed as much as 30 days before sensitization to HDM, but ineffective just 2 days after sensitization, suggesting that Pam2ODN immunomodulation lowers the allergic responsiveness of airway epithelial cells. Furthermore, Pam2 and ODN cooperated synergistically suggesting that this treatment is superior to any single agonist in the setting of allergen immunotherapy. One Sentence SummaryA synergistic combination of Toll-like Receptor agonists, delivered directly into the lung mucosa, can attenuate allergic responsiveness of airway epithelial cells and prevent host sensitization to aeroallergens. What is already knownO_LIAllergic sensitization has increased in the 20th century due to reduced contact with microbial organisms in industrialized society (ie. hygiene hypothesis) C_LIO_LIWe have previously identified a pharmacological means to stimulate innate immunity of lung epithelial cells. C_LI What this study addsO_LIActivation of innate immunity in lung epithelial cells attenuates the allergic responsiveness of mice. C_LIO_LISynergistic cooperation of pattern recognition receptors induces stronger immunomodulatory responses C_LI What is the clinical significanceO_LIAerosolized Toll-like Receptor agonists have been demonstrated as safe in human clinical trials C_LIO_LIThis study provides proof-of-principle that aerosolized toll-like receptor agonists could have clinical efficacy in the setting of the allergen immunotherapy C_LI

immunology↗