bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterizing the mucosal-associated invariant T cell response to pulmonary infection
Authors: Melamed Jonathan , Constantinides Michael G
Published: August 2023

Mucosal-associated invariant T (MAIT) cells are an effector population of innate-like T cells that comprise up to 5% of T cells within human lungs. Rather than recognizing peptides like conventional T cells, MAIT cells utilize their semi-invariant T cell receptors to identify small molecules, such as intermediates of riboflavin synthesis, presented by MHC-class 1 related protein (MR1). Because this biosynthetic pathway is highly conserved among bacteria and fungi, MAIT cells promote immunity to a wide array of pulmonary pathogens, such as Legionella and Francisella species. However, due to the low level of MR1 expression in several major cell lineages, the populations that present microbial metabolites to MAIT cells during infection remain unknown, preventing optimization of MAIT cell vaccines. Utilizing the F. tularensis live vaccine strain to induce pneumonic tularemia, we find that MAIT cells are the predominant effector T cell population, accounting for more than 20% of IFN-γ producing T cells. Infection of MR1f/f conditional knockout mice following broad deletion of MR1 with the tamoxifen-inducible Rosa26-creERT2 allele revealed that MR1-mediated presentation is necessary for the MAIT cell response to F. tularensis. Use of MR1f/f strains that express Cre recombinase under control of cell-specific promoters has allowed us to determine the relative contributions towards MAIT cell activation from dendritic cells, macrophages, and neutrophils. This work defines the antigen presenting cells that are critical for MAIT cell activation during an intracellular bacterial infection and identifies cellular targets to enhance MAIT cell responses.

Supported by NIAID grants K22 AI146217 and R21 AI171697
Signs of Dendritic Cell Activation in the Lungs of Mice Exposed to Aerosols of Flavored Vaping Liquid
Authors: Tremblay Felix , Pineault Marie , Thériault Pierre-Olivier , Milad Nadia , Lechasseur Ariane , Routhier Joanie , Beaulieu Marie-Josée , Aubin Sophie , Côté Andréanne , Morissette Mathieu C
Published: August 2023

Rationale

Vaping products contain numerous flavor compounds that are known immunological sensitizers. Sensitization to various antigens can be responsible for the onset of serious respiratory diseases, including hypersensivity pneumonitis. However, respiratory tract sensitization and pathological consequences from exposure to aerosols of flavored vaping liquids need to be thoroughly investigated.

Methods

Female BALB/c mice were exposed to laboratory made nicotine-free vaping liquid containing a mixture of 1% citral (lemon), 1% cinnamaldehyde (cinnamon), 1% dihydrocoumarin (coconut) and 1% vanillin (vanilla) solubilized in propylene glycol and glycerol (50:50). Mice were exposed 2h/day for 4 days to vaping or room air. Bronchoalveolar lavage was performed to assess total and differential cell counts, and flow cytometry was performed on pulmonary tissue and lung-draining lymph nodes to assess immune cell populations.

Results

After 4 days of daily exposure, we found a significant increase in bronchoalveolar lavage immune cells in mice exposed to aerosols from the flavored vaping liquid. We also observed a significant increase in pulmonary conventional dendritic cells (cDCs) as well as their expression of the activation markers CD40, CD86 and MHC-II. This activation is mainly observed on type 2 cDCs. In the lung-draining lymph nodes, flavored vaping liquid lead to a significant increase in total immune cells (CD45+ cells).

Conclusions

Pulmonary conventional dendritic cells activation and the increase in total immune cells in lung-draining lymph nodes caused by exposure to aerosols from flavored vaping liquid suggest the early initiation of immunological sensitization processes in the lungs.

Fonds de recherche du Québec – Santé, Ministère de la Santé et des Services sociaux, Fondation de l’Institut de cardiologie et de pneumologie de Québec, Réseau de recherche en santé respiratoire du Québec.
The FcμR regulates IgM-BCR internalization
Authors: Blandino Rebecca A , Lou Zheng , Cook Sarah , Sciammas Roger , Baumgarth Nicole
Published: August 2023

The FcμR is a cell surface receptor that can bind to both secreted and membrane-bound IgM. In B cells it co-localizes with IgM-BCR in Golgi vesicles, where it restrains surface BCR-IgM expression. B cell-expressed FcmR is required also for maximal IgG response development after influenza A virus infection, but the mechanisms are unknown. To explore the effects of FcmR expression we generated FcmR-deficient and sufficient BCR-”SwHEL” transgenic C57BL/6 mice. The mice, containing about 20% HEL-specific B cells were immunized either directly with hen egg lysozyme (HEL) or with HEL-ovalbumin (OVA), or we created bone marrow irradiation chimeras in which about 2% of naïve B cells expressed the SwHEL-BCR. Consistent with the influenza infection studies, IgG responses following subcutaneous HEL-OVA immunization were significantly diminished in both systems when B cells lacked FcmR−/− expression compared to controls. Confocal and STED microscopy studies on B cells from these mice were done to quantify IgM-BCR internalization following in vitro exposure to HEL-bodipy. FcmR−/− SwHEL B cells did not show IgM “capping” and retained high amounts of antigen on the cell surface compared to the HEL− FcmR+/+ cells. This reduction in internalization resulted in reduced MHCII-peptide loading, as shown via flow cytometric quantification of surface pMHCII expression using the YAE-antibody. Ongoing studies are defining the subcellular compartments of antigen-processing in the presence and absence of the FcmR. We conclude that the FcmR acts as a non-redundant chaperone that optimizes IgM-BCR antigen internalization and processing.

U19AI109962 R01AI148652
HLA-DO promotes Treg differentiation by altering antigen presentation
Authors: Welsh Robin A , Song Nianbin , Antonescu Corina , Florea Liliana , Sadegh-Nasseri Scheherazade
Published: August 2023

HLA-DO in human (H2-O in mice) (DO) is a highly conserved non-classical MHC class II accessory molecule. Unlike the main Class II peptide editor HLA-DM, expression of DO is restricted to the thymic medulla, B cells and certain dendritic cell subsets. DO forms a stable complex with HLA-DM and requires DM for trafficking to MIIC. Using biochemical experiments, our lab has previously discovered that DO has differential effects on editing peptides of different sequences. DO enhances binding of DM-resistant peptides, and reduces the binding of DM-sensitive peptides to the HLA-DR1 molecules. Changes in the quantities of either DM-resistant of DM-sensitive peptides could therefore lead to alterations in both thymic negative selection, and peripheral activation of CD4 T cells. Using single-cell RNA-sequencing (scRNA-seq) of peripheral CD4 T cells from unimmunized DO knock-out (KO) mice we found an increased frequency of “activated” CD4 T cells, in support of our biochemical model. This increased frequency corresponds with a decrease in “naïve” CD4 T cells in both scRNA-seq and FACs analysis. Interestingly, scRNA-seq analysis confirmed our previous findings of elevated regulatory T cells (Treg) levels in DO-KO mice. Genetic analysis showed that unimmunized DO-KO Tregs also have a more activated phenotype. These findings suggests that absence of DO generates a more stimulatory in vivo cellular environment for CD4 T cells, likely due to altered antigen presentation.

Research supported by grants from NIH (R01 AI130210, R01 AI121174 and R37 AI060040)
Loss of HLA-DO function impairs the development of memory CD4 T cells
Authors: Song Nianbin , Welsh Robin A , Sadegh-Nasseri Scheherazade
Published: August 2023

DO is an accessory protein that binds DM for trafficking to MIIC and has peptide editing functions. DO is mainly expressed in thymic medulla and B cells. Using biochemical experiments, our lab has discovered that DO has differential effects on editing peptides of different sequences. DO increases binding of DM-resistant peptides, and reduces the binding of DM-sensitive peptides to the HLA-DR1 molecules. In a separate line of work, we have established that reduced densities of antigen presentation by B cells during the contraction phase of an infection, induces quiescence in antigen experienced CD4 T cells as they differentiate into memory T cells. This quiescent phenotype helps memory CD4 T cell survival and promotes effective memory responses to secondary Ag challenge. Based on our mechanistic understanding of DO function, it would be expected that in immunized DO-KO mice, presentation of altered densities of pMHC by B cells would lead to faulty development of memory CD4 T cells. We tracked the development of DR1 restricted H5N1 flu specific memory CD4 T cells and B cells post flu vaccine immunization in DR1+/DO-KO mice and found that immunized DR1+/DO-KO mice had fewer CD4 memory T cells and memory B cells than DR1+/DO-WT mice. By in vitro and in vivo recall experiments, we found that memory responses elicited in DR1+/DO-KO mice were compromised. Using a second model antigen, OVA, we confirmed our findings, i.e., altered memory development and secondary responses in DO-KO mice. These results demonstrate that in the absence of DO, the presentation of cognate foreign antigens in the DO-KO mice is altered and can hamper the proper development of memory cells. These findings suggest a justification for the late appearance of DO in warm blooded mammals.

R37 AI060040 R01 AI121174 R01 AI130210
Dissecting the roles of CD91 in heat shock protein-mediated cancer immunosurveillance
Authors: Harkness James F , Binder Robert J
Published: August 2023

Our lab has shown that cancer immunosurveillance is dependent on CD91, a receptor for heat shock proteins (HSPs), which is expressed on antigen-presenting cells (APCs). HSP-chaperoned tumor antigens are cross-presented following endocytosis by CD91. Binding of HSPs to CD91 also initiates an intracellular signaling pathway that is poorly understood. Two tyrosine residues on the intracellular domain (β-chain) of CD91 become phosphorylated following HSP binding, leading to downstream activation of NF-kB and STAT1. We found that the immunogenic HSPs differentially utilize these two phosphorylation sites on CD91 for downstream signaling resulting in unique cytokine profiles and co-stimulation capacity. We investigate here the CD91-interacting adaptor proteins and kinases by crosslinking and co-immunoprecipitation of CD91 and its signaling complex following stimulation with each immunogenic HSP (gp96, calreticulin or hsp70). Mass spectrometry studies with gp96 identified the adaptor protein Shc and two other receptor tyrosine kinase associated with CD91. Inhibition of these kinases leads to a significant decrease in gp96-mediated cytokine production. Further work is being done to elucidate the remainder of this signaling network for each HSP and examine any differences between APC types. This project will aid in better understanding of how CD91-mediated signaling differentially activates effector cytokine profiles in APCs. This knowledge can be used in the development of new therapeutic approaches to enhance immunosurveillance of cancers and to better eradicate established disease.
A multiscale mechanistic model of human dendritic cells for in-silico investigation of immune responses and novel therapeutics discovery
Authors: Amin Rada , Aghamiri Sara Sadat , Puniya Bhanwar Lal , Helikar Tomáš
Published: August 2023

Given the critical role of dendritic cells (DCs) in shaping immunity, they present an attractive avenue as a therapeutic target to program the immune system and reverse immune disease disorders. DC-mediated immune response grows into a complex continuum of molecular and cellular networks where each scale decides to be later translated into a seamless phenotype. Computational models open novel frontiers in research by integrating large-scale interaction to interrogate the influence of complex biological behavior across scales. The ability of modeling to digest large amounts of biological networks will likely pave the way to understanding any complex system in more approachable ways. We developed a logical and predictive model of DC function that integrates the heterogeneity of DCs population, APC function, and cell-cell interaction, spanning molecular to population levels. We provided three sample use cases to apply the model in the context of studying cell dynamics and disease environments. First, we characterized the DC molecular response to Sars-Cov-2 and influenza by comparing single to co-infection conditions. The second example presents simulations to predict the crosstalk between DCs and T cells in a cancer microenvironment. Finally, for the third example, we used the Kyoto Encyclopedia of Genes and Genomes enrichment analysis against the model’s components to identify 45 diseases and 24 molecular pathways that the DC model can address. This study presents a resource to decode the complex dynamics underlying DC-derived APC communication and provides a platform for researchers to perform in-silico experiments on human DC for vaccine design, drug discovery, and immunotherapies.

The work was supported by an NIH grant R35GM119770 to Tomáš Helikar
Decrypting pMHCI Diversity Through Noncanonical Translation Initiation
Authors: Rodriguez Jesus Contreras , Esteban Adrian Martin , Peske J David , Shastri Nilabh , Sadegh-Nasseri Scheherazade
Published: August 2023

CD8+ T-cells rely on MHCI to surveil the proteomic state of cells in the form of peptide-MHCI (pMHCI). We previously uncovered a set of ATG-like translation initiation codons that can define noncanonical open reading frames (ORFs) of cryptic peptides presented by MHCI. The model cryptic codon CTG shares key translation initiation requirements with canonical ATG initiation, yet incorporates N-terminal Methionine (nMet) or Leucine (nLeu). We aim to characterize the activity and peptide products of the remaining cryptic codons to understand how cryptic ORFs contribute to the peptidome accessible to MHCI. Therefore, we make use of the subfemtomole sensitivity of the hyperB3Z reporter T-cell hybridoma to interrogate the identity of a modified OVA peptide (SIINFEHL;[SHL8]) reporter in a fixed cryptic initiation context, while retaining information on the residue incorporated during translation initiation. Following transfection of mammalian model cell lines with reporter, purified peptides from total cell extracts are RP-HPLC fractionated to differentiate N-terminally extended and trimmed [SHL8] intermediates. We found that nMet is incorporated by CTG and ACG initiation, while nLeu was absent under CTG initiation despite coexpression of the H2-Db allotype known to protect N-terminally extended [SHL8]. Peptide reporters with a P-2 residue derived from a codon satisfying the optimal (CACCxxx G) Kozak sequence (i.e. G, D) revealed that bulky P-2 residues enhance recovery of total reporter suggesting cryptic ORF peptide stability largely depends on P-2. Thus, by understanding the products of cryptic ORFs we expand the definition of immune-self while exposing new sources of pMHCI unique to autoimmunity and viral pathogens.

Supported by grants from NIH (R01-AI130210, R01-AI121174, R37-AI060040)
Cryptic MHC-E epitope from influenza elicits a potent cytolytic T cell response
Authors: Hogan Michael J , Maheshwari Nikita , Begg Bridget E , Nicastri Annalisa , Muramatsu Hiromi , Pardi Norbert , Miller Michael A , Reilly Shanelle P , Brossay Laurent , Lynch Kristen W , Ternette Nicola , Eisenlohr Laurence C
Published: August 2023

Non-classically-restricted T cells have shown remarkable therapeutic activity in a variety of disease settings; most notably, CD8 T cells restricted to MHC-E (Qa-1 in mice; HLA-E in humans) have proven capable of eliminating a primate lentivirus infection. Here, we used mass spectrometry to identify a Qa-1-restricted 9-mer peptide, named M-SL9, that elicits a strong CD8 T cell response in mice infected with influenza A virus. The M-SL9 specificity accounted for roughly 5–10% of all lung CD8 T cells, approaching the frequency of classically restricted CD8 T cell responses to flu. A broad investigation of the M-SL9-specific T cells showed that they are generally similar in phenotype to classically restricted CD8 T cells, including strong co-expression of type 1-associated cytokines (polyfunctionality) and cytolytic markers; however, M-SL9-specific T cells were unique in that they were anatomically restricted to the lungs and were not present in blood and spleen like other specificities. Intriguingly, the M-SL9 epitope derives from a previously unknown translation product encoded by the +1 alternative reading frame of the matrix 1 (M1) protein-coding sequence. Presentation and immunogenicity of M-SL9 were completely dependent on ribosomal initiation at the second AUG codon of the matrix gene segment. Analysis of potential precursors suggests that M-SL9 is processed from a novel 16-mer peptide that is not required for viral replication in mice. These discoveries imply that unconventional epitopes may drive a large fraction of physiologically relevant T cell responses, and M-SL9 presents a highly tractable system in which to dissect the mechanisms governing MHC-E-restricted T cell responses and their protective effects.

Supported by Cancer Research Institute Irvington Fellowship, Roy and Diana Vagelos Molecular Life Sciences Program and College Alumni Society Research Grant from the Univ. of Pennsylvania, and NIH grants R01 AI146101, R01 AI153064, R01 AI046709-18S1, R01 AI046709, R01 AI125524, and R21 AI153978.
Human versus mouse Beta 2 microglobulin alters TCR antigen recognition kinetics with mouse MHCI
Authors: Rase Viva Jeanne , Kolawole Elizabeth M , Liu Baoyu , Ayres Cory M , Rogers Jhordan , Salaita Khalid , Baker Brian M , Evavold Brian D
Published: August 2023

There are three isoforms of Beta2 microglobulin (B2m) identified in mice and they differ by a single amino acid residue. To study how B2m can modify the peptide binding pocket of MHCI we turned to the comparison of mouse (mB2m) and human (hB2m). It is known that hB2m forms more stable with mouse MHCI heavy chain as compared to mb2m and hB2m is often the choice for most pMHCI tetramer production. Analysis of mB2m and hB2m tetramer (GP33 41M) following LCMV infection revealed no difference in percentage of tetramer positive cells but an increase was observed in staining intensity (MFI) of hB2m. Using steered molecular dynamics we found that there were allosteric effects with mB2m displaying more movement within the binding pocket than hB2m. Consistent with this we saw increased 2D affinity for antigen MHC Db and Kb hB2m complexes for OT1 and P14 TCRs, respectively. We also measured the force effects on bond lifetime between TCR and pMHC using biomembrane force probe (BFP) and DNA tension sensor. For OTI, we found that mB2m complexes had a longer bond lifetime measured by both methods. We observed signaling and functional effects consistent with the 2D affinity and bond lifetime measurements in mouse verses human B2m. For example, mB2m leads to increased levels of activated LCK (pY394/505) in OT1 T cells. Therefore, alterations in B2m effect the distal peptide binding domain of MHCI and suggests dynamic allostery may influence antigen recognition by TCR.

5T32NS115664-03 5R01AI147641-03
4-1BB costimulation following mRNA vaccination improves CD8 T cell responses
Authors: Sanchez Sarah , Penaloza-MacMaster Pablo
Published: August 2023

mRNA vaccines have received full FDA approval to prevent COVID-19, and are being explored for multiple infectious diseases as well as cancer. While they have shown high efficacy at preventing severe disease caused by SARS-CoV-2, waning immunity has motivated the development of improved mRNA vaccine regimens. 4-1BB (also known as CD137) is a costimulatory receptor that has been shown to be important for T cell responses following viral infections and cancer. We asked whether triggering 4-1BB costimulation with agonistic antibodies could improve immune responses elicited by mRNA vaccines in C57BL/6 mice. Here, we show that triggering 4-1BB costimulation at the time of mRNA vaccination impairs CD8 T cell responses, whereas triggering 4-1BB costimulation after day 4 of mRNA vaccination improves CD8 T cell immune responses. These data demonstrate time-dependent effects of 4-1BB costimulation on cellular responses elicited by mRNA vaccines, and suggest that delayed provision of 4-1BB costimulation could offer immunologic benefits following mRNA vaccination.

This work was supported by a DP2 New Innovator Award to P.P.M.

This work was supported by a DP2 New Innovator Award to P.P.M.
Calibrating T cell help by antigen cross-linking as a universal vaccine platform against rapidly evolving pathogenic viruses
Authors: Mallajosyula Vamsee , Chakraborty Saborni , Sola Elsa , Gao Fei , Wang Taia , Davis Mark M
Published: August 2023

The effectiveness of influenza vaccine is limited by the heterogeneous immune response it elicits. Factors such as age, sex, and pre-existing antibody titers only partially explain this heterogeneity. Using a novel deconvolution analysis we find subtype-specific bias as a major source of this inter-individual variation. Since the 1940’s it has been proposed that this phenomenon is due to prior strain exposure, referred to as “Original Antigenic Sin”. But our analysis of flu vaccine responses in a monozygotic twin cohort showed that antigenic imprinting from past exposures can only partially explain strain preference (r2=0.25, p=4.7×10−5). Thus, we hypothesized that host genetics may play a major role in subtype-bias. Consistent with this is our finding that strain-preferences are evident after vaccination in an influenza naïve infant cohort indicating that prior exposure is not essential. We determined the source of this genetic component to the wide variation in influenza hemagglutinin peptide presentation by different HLA class-II alleles resulting in an imbalanced, subtype-specific CD4+ T follicular help after vaccination. To address this, we developed a tunable, plug-and-play vaccine platform to better calibrate T cell help. In a human tonsil organoid system, we demonstrated that engineered, cross-linked HA induces a significantly higher response against multiple influenza subtypes in comparison to the inactivated influenza vaccine. In summary, we have developed an effective strategy to overcome response bias in a population with diverse genetic backgrounds and pre-existing immunity. Furthermore, we successfully adapted this platform to generate a pan-coronavirus vaccine.

Supported by grants from NIH to MMD (5U19AI090019, 5U19AI057229). Additional support was provided by ITI-YIA to VM.
Therapeutic anti-glycan antibodies against antibiotic resistant Staphylococcus aureus
Authors: Nagy Kenna K , Kain Lisa , Stanfield Robyn , Grimes Catherine , Wilson Ian A , Savage Paul B , Finn M G , Teyton Luc
Published: August 2023

Antibiotic resistance threatens clinical control of bacterial infections while the genetic adaptability of microbes continues to outpace small-molecule development. The combinatorial diversity of antibodies offers a solution to this problem. However, under normal circumstances the polymeric glycans of bacterial surfaces avoid adaptive recognition by not binding to MHC molecules. Thus, they are T cell independent antigens and targeted only by low affinity IgM responses. Glycoconjugate vaccines have been developed to provide bystander T cell help to overcome this limitation but have failed to elicit protective responses against antibiotic resistant Staphylococcus aureus in clinical trials. To address limitations in current conjugate vaccine approaches, we optimized anti-glycan B cell help through three convergent prongs: exploiting cognate T cell help, using a B cell-centric adjuvant, and using synthetic minimal glycans. This prototype next generation conjugate vaccine was used to produce nanomolar affinity anti-glycan responses in proof-of-concept studies. Focusing on antibiotic resistant Staphylococcus aureus three glycan targets of the cell wall and bacterial capsule have been selected and used to produce monoclonal antibodies. These antibodies were characterized structurally, biophysically, and by B cell sequencing to confirm high affinity, maturation, and specificity towards the intended targets. The potential therapeutic benefits are currently being tested in three preclinical mouse models: skin, lung, and systemic infection, using passive and active immunization. Preliminary studies have shown therapeutic efficacy in both a preventative and interventional model for some of these antibodies.

Kenna Nagy supported by: NIH TL1TR002551 For this project P.I. Luc Teyton supported by: NIH U01 AI160338, NIH R01 AI139748
Phenotypic analysis of HIV-1 resistance to CD4 binding site broadly-neutralizing antibodies
Authors: Shrader Hannah , Hait Sabrina Helmold , Lovelace Sarah , Kilner Meaghan , Shen Chen-Hsiang , Coates Emily , Gaudinski Martin , Rothwell Ro Shauna , Donaghy Eileen , Roa Jhoanna , Fichtenbaum Carl , Santana Jorge , Presti Rachel M , Bhatnager Anjali , Lynch Rebecca , Tebas Pablo , Ledgerwood Julie , Mascola John , Koup Richard , Pegu Amarendra
Published: August 2023

Broadly neutralizing antibodies (bNAbs) show strong immunotherapy potential by targeting conserved regions of the HIV-1 envelope. Single bNAb use in viremic individuals quickly results in the emergence of escape variants, likely originating from existing quasispecies. An ex vivo assay was developed to predict bNAb efficacy in viremic individuals. The development of a predictive bNAb efficacy model may assist with the planning of future clinical trials.

The ex vivo assay used pre-infusion CD4+ T cells from participants enrolled in the VRC 607/ACTG A5378 phase I study of the CD4 binding site (CD4bs) bNAbs’ VRC01LS (n=7) or VRC07-523LS (n=9). From these assays, the escape variant signatures of outgrowth viruses that replicated in presence of CD4bs bNAbs, were identified. We found that these escape variants contained key changes to the ß23-V5 region of the gp120 envelope, including charge changes, glycan loss, and/or glycan shifts. Outgrowth viruses from each participant had similar escape mutation patterns. Resistance mutations of the viral env sequences were confirmed in pseudovirus-based neutralization assays.

We found complementary escape profiles from 3 CD4bs bNAbs (1–18LS, N6LS, and VRC01v23) with ex vivo and neutralization assays showing suppression by 1 or 2 but not all 3 bNAbs, supporting the potential for combination bNAb therapy. Our data suggest that the simultaneous use of multiple CD4 binding site bNAbs together could aid in limiting viral escape.

Supported by intramural funds from NIH.
Modulating Innate Immune Responses for Improved Vaccine Tolerability and Durability
Authors: Esser-Kahn Aaron P , Rosenberger Matthew Glenn , Kim Jeremiah
Published: August 2023

Stimulation of the innate immune system is crucial in both effective vaccinations and immunotherapies. This is often achieved through adjuvants, molecules that usually activate pattern recognition receptors (PRRs) and stimulate two innate immune signaling pathways: the nuclear factor kappa-light-chain-enhancer of activated B-cells pathway (NF-κB) and the interferon regulatory factors pathway (IRF). Here, we demonstrate the ability to alter and improve adjuvant activity via the addition of small molecule “immunomodulators”. By modulating signaling activity instead of receptor binding, these molecules allow the customization of select innate responses. We demonstrate both inhibition and enhancement of the products of the NF-κB and IRF pathways by several orders of magnitude. Some modulators apply generally across many receptors, while others focus specifically on individual receptors. Modulators boost correlates of a protective immune responses in a commercial flu vaccine model and reduced correlates of reactogenicity in a typhoid vaccine model. These modulators have a range of applications: from adjuvanticity in prophylactics to enhancement of immunotherapy.

NIH 75N93019C00041
Innate Immune Memory for Improved Vaccine Response
Authors: Esser-Kahn Aaron P
Published: August 2023

Emerging diseases require generating new vaccines, which can often be time-consuming. An alternate method to boost host defense is by inducing nonspecific innate immune memory, called trained immunity, to develop novel prophylactics. Many molecules, most notably β-glucan, induce trained immunity, but their effects are often short-lived and uncontrolled. This lack of temporal control limits both the therapeutic ability of training and provides fundamental questions about its nature. To improve on the capabilities of β-glucan, we developed a method for temporal control of trained immunity and examined its use both as a prophylactic and as an additive for improve vaccine outcomes. We engineered controlled release nanoparticles encapsulating only 3.5% the standard dose of β-glucan to attain sustained release over a month. Nanoparticle trained mice exhibited prolonged training effects and improved resistance to a B16F10 tumor challenge compared to mice that received an equivalent amount of free β-glucan. We also showed that trained mice exhibited higher antibody responses upon vaccination. These results demonstrate that dosing and temporal control can substantially alter the trained response to unanticipated levels. As such, this approach using sustained release platforms might lead to a novel prophylactic strategy for improved disease resistance against a wide variety of diseases.

HDTRA11810052
Intranasal delivery of adenovirus-vectored vaccine confers protective mucosal immunity against SARS-CoV-2
Authors: Jung Hi Eun , Ku Keun Bon , Kang Byeong Hoon , Kim Kyun-Do , Lee Heung Kyu
Published: August 2023

The ongoing pandemic of COVID-19, caused by SARS-CoV-2 infection, has threatened the global health. Since the first case of infection was reported in December 2019, SARS-CoV-2 has rapidly spread all over the world and caused millions of deaths. As vaccination is the best way to protect the host from invading pathogens, several vaccines have been developed to prevent the infection of SARS-CoV-2 and saved huge numbers of lives so far. However, SARS-CoV-2 constantly change their antigens resulting in the escape from vaccine-induced protection, and the persistence of immunity induced by vaccine becomes an issue. Further, traditional intramuscular COVID-19 vaccines are insufficient to evoke mucosal specific immune response. Because the respiratory tract is the primary route of SARS-CoV-2 entry, the need for mucosal vaccine has been strongly suggested. Using adenoviral (Ad) vector platform, we generated recombinant COVID-19 vaccines that encode modified-spike antigen, and evaluated the vaccine efficacy in murine models. We found that intranasal delivery of Ad-vectored vaccines elicited both systemic as well as mucosal immune responses, and rescued mice from lethal SARS-CoV-2 infection. Further, we revealed that intranasal vaccination of Ad-vectored vaccines changed transcriptional landscape of lung. These results indicate that our recombinant Ad-vectored vaccine has the potential to confer protective immunity against SARS-CoV-2.

This study was supported by the National Research Foundation of Korea (NRF-2021M3A9D3026428 and NRF-2021M3A9H3015688) funded by the Ministry of Science and ICT of Korea. This work was also supported by Mobile Clinic Module Project funded by KAIST and the 2020 Joint Research Project of Institutes of Science and Technology. H.E. Jung was supported by Basic Science Research Program through the NRF funded by the Ministry of Education (2022R1I1A1A01071399)
Booster mRNA vaccination post-SARS-CoV-2 infection enhances functional qualities of T cell immunity
Authors: Cai Curtis , Gao Yu , Olsson Annika , Sette Alessandro , Wherry E John , Aleman Soo , Buggert Marcus
Published: August 2023

T cells are critical to generate protection from severe respiratory viral disease. Whether repeated SARS-CoV-2 antigen exposure through vaccination causes T cell exhaustion or gain of functions remains debated. In this study, we sampled SARS-CoV-2 convalescent donors before and after vaccination to determine the functional consequences of hybrid immunity. Donors were sampled at 6 months and 18 months convalescence and divided according to their history of mild or severe/hospitalized disease (n = 44 and 45 respectively). In between the two sampling timepoints, donors also received mRNA vaccination through the national COVID-19 vaccination program. Using activation-induced marker assays, intracellular cytokine staining, single-cell transcriptomics and single-cell T-cell receptor sequencing analyses, we show that spike-specific T cells increase in magnitude but, importantly, also gain highly functional characteristics. By contrast, T cell responses to non-spike proteins were diminished or remained unchanged. Elevated interferon-gamma expression was a common hallmark of CD4+ and CD8+ T cells post-mRNA-vaccination. These responses were founded on both pre-expanded and newly detected CD8+ T cell clones after vaccination. Collectively, these data demonstrate the cumulative benefits of booster vaccinations, leading to increased quantity and quality of cell-mediated immunity.

Supported by a grant from Karolinska Institutet Research foundation grants (2022-01708)
Trehalose-based lipid improves mRNA vaccine safety
Authors: Bae Seo-Hyeon , Bang Eun-Kyoung , Lim Byung Kwan , Lee Jisun , Yoo Soyeon , Park Hyo-Jung , Park Sang-In , Lee Yu-Sun , Roh Gahyun , Lee Seonghyun , Youn Sue Bean , Keum Gyochang , Nam Jae-Hwan
Published: August 2023

The rapid development of lipid nanoparticle (LNP)-formulated mRNA vaccines strongly contributed to overcoming the COVID-19 pandemic. However, in parallel with this success, the toxicity of ionizable cationic lipids has emerged as a potential issue in clinical safety because it might cause mRNA vaccine side effects, including myocarditis, anaphylaxis, and pericarditis. In this study, to reduce the risk posed by ionizable cationic lipids, we employed LNPs containing trehalose-based lipid (LNP-126), which increased the stability of the LNP-mRNA molecules. Trehalose has been shown to improve heart function and increase mRNA stability through hydrogen bond interactions. The LNP-126 was formulated with 50% fewer ionizable cationic lipids than a commercial LNP (LNP-128). Furthermore, the mRNA encoding the reporter gene was encapsulated by LNP-126 so that gene expression (Renilla luciferase) and organ toxicity could be examined. We also assessed immune efficiency immunizing mice that had been treated with an LNP-126-formulated mRNA vaccine expressing influenza hemagglutinin (HA). The results showed that LNP-126 reduced organ toxicity in the liver and heart compared to LNP-128, while maintaining gene expression and immune efficiency in vivo. In conclusion, the LNP-126, trehalose-based lipid will facilitate the development of safer mRNA vaccines beyond the current barriers.

A grant (22213MFDS421) from the Ministry of Food and Drug Safety in 2022 supported this research
Selection for disease resistance in Oncorhynchus mykiss increases the myeloid to lymphoid balance and is affected by rapamycin as measured by flow cytometry and scRNAseq
Authors: Reid Ross M , Osbourn Keira , Leeds Timothy , Wiens Gregory D
Published: August 2023

Heritable variation in disease resistance has been described in many species, but the effects of selection on innate immunity are unclear. Here, we compare the cellular response of two lines of rainbow trout that differ in survival due to five generations of selection for resistance to Flavobacterium psychrophilum (Fp), an important fish pathogen. We examined bacterial load, plasma biomarkers, whole blood cellular composition, and blood chemistry at days 1 and 5 after bacterial or PBS injection. We also examined the effect of 0.8 mg/kg rapamycin pretreatment, which results in immunosuppression and a ~100-fold reduction in the challenge LD50. On day 1, bacterial load did not differ between genetic lines and treatment, but by day 5, the R-line exhibited no increase in Fp load, while rapamycin increased R- and S-line loads 33- and 506-fold, respectively. Biomarkers: cathelicidin 2 and C1q-LP3 significantly differed between genetic lines and by time, corresponding to Fp load. Analysis of whole blood by flow cytometry identified a response to infection and an ~8 percentage point higher frequency of myeloid-gated cells in R-line fish that was consistent in both naïve and challenged animals. S-line fish exhibited a higher percentage of IgM+ B cells. Single-cell RNAseq was conducted on day 1 purified blood and identified 23 cell clusters including multiple B cell, neutrophil and macrophage subpopulations. In naïve animals, S-line fish harbored a cxcr4 and mych expressing B cell subpopulation absent in R-line fish. Rapamycin pretreatment caused profound loss of multiple B cell subpopulations in R- and S-line fish. In summary, selection of rainbow trout for resistance to Fp is correlated with a cellular shift in peripheral blood and is mTOR dependent.

US Department of Agriculture, Agricultural Research Service; Project number 1930-32000-007
The role of bystander activation among CD8αα T cells in controlling Mycobacterium tuberculosis infection
Authors: Winter Cassandra R , Zhou Angela , Tappen Tori , Ou Henry , Seshadri Chetan
Published: August 2023

Classical cytotoxic T cells have a CD8 coreceptor with an αβ heterodimer and recognize peptide antigens via major histocompatibility (MHC) molecules. CD8 T cells with an αα homodimer structure are nonclassical and can recognize non-MHC molecules independently of antigen. CD8αα T cells have a higher propensity to undergo T cell receptor (TCR) independent bystander activation via pro-inflammatory cytokines. Bystander activation of CD8 T cells has shown a protective effect during Mycobacterium tuberculosis (M.tb) infection, but the distinct roles of CD8αα and CD8αβ T cells have not been elucidated. To begin assessing this, we compared the levels of CD8αα and CD8αβ T cell proliferation in response to TCR dependent stimulation using anti-CD3/CD28 beads. We observed that CD8αα T cells had lower levels of proliferation compared to CD8αβ T cells after three days of stimulation. After seven days, a population of CD8αα T cells did not proliferate while nearly all CD8αβ T cells showed high proliferative capacity. We next stimulated healthy peripheral blood mononuclear cells with IL-12, IL-15, and IL-18 to induce bystander activation and found 52.8% of CD8αα T cells secreted granzyme B compared to 25.1% of CD8αβ T cells. Interestingly, CD8αα T cells also had higher levels of CD69 expression than CD8αβ T cells (62.6% and 38.2%, respectively). Finally, we present plans for defining the role of bystander activated CD8αα and CD8αβ T cells during M.tb infection using tonsil organoids. These data reveal that CD8αα T cells have a distinct bimodal response to TCR mediated activation as well as an increased cytotoxic and activated phenotype after bystander activation. We further establish a foundation for future bystander activation studies in M.tb infection.

Supported by grants from the NIH (R01 AI146072)
What did the T cell see? A deep-learning model of CD8+ T cell activation reveals sharp antigen discrimination at the single cell level
Authors: Wahlsten Madison , Akhshi Amin , Achar Sooraj , Krishnan Anagha Yogam , François Paul , Altan-Bonnet Grégoire
Published: August 2023

Immunotherapies such as checkpoint blockade antibodies to block T cell exhaustion have been successful in several cancers such as non-small cell lung cancer and melanoma, but limited in others (e.g., pancreatic or prostate carcinomas) owing to differences in tumor antigenicity. Therefore, quantifying tumor antigenicity is critical for successful immunotherapies. Our lab has shown that antigenicity can be encoded in a single parameter derived from bulk cytokine dynamics in ex vivo co-cultures between antigen presenting cells (APCs) and T cells. Here we built a model that can capture the antigenicity seen by individual cells. Using a custom robotic platform, we generated high-throughput kinetics of T cell activation in co-culture with APCs by analyzing cells at various timepoints across a large set of activation conditions. We performed spectral flow cytometry to measure the expression of up to 30 surface markers and intracellular signals per cell. To analyze our content-rich datasets, we designed a machine learning-based model that can classify the antigen seen by an individual cell using expression values from flow cytometry. The model performs well not only at classifying T cells (ROC-AUC > 0.91), but also APCs (ROC-AUC > 0.88), suggesting that each individual leukocyte may register the quality of antigen being presented. Blocking cytokine signaling disrupted this antigen classification. Our study demonstrates that every individual lymphocyte can bridge local and global response to achieve high discriminatory power of antigens.
Phenotypic and transcriptional heterogeneity of memory CXCR5+ CD8+ T cells in human blood and lymphoid tissue
Authors: Rivera Olga , Buggert Marcus
Published: August 2023

CXCR5+ CD8+ T cells are involved in antiviral and -tumor immunosurveillance. Animal models have demonstrated that CXCR5+ CD8+ T cells have multiple roles, including cytotoxic, stem-like, and regulatory functions. Many of these functions might be disease-dependent, where human studies are lacking to understand if CXCR5+ CD8+ T cells show signs of significant heterogeneity. To this end, we performed simultaneous single-cell (sc) RNA−, TCR−, and CITE-seq on CXCR5+ and CXCR5− memory CD8+ T cells from matched tonsils and blood to unravel if CXCR5+ CD8+ T cells show potential differences between human lymphoid tissue and blood. Our analysis indicates that a high proportion of CXCR5+ CD8+ T cells in tonsils show tissue residency-like (TRM) features, including CD69 and CD103 expression. Instead, blood CXCR5+ CD8+ T cells showed increased signs of cytotoxic functions in blood compared to their counterparts in tonsils. GSEA analysis identified groups of genes differentially expressed, such as the p53 pathway enriched in tonsils compared to ribosome-related pathways in blood CXCR5+ memory CD8+ T cells. Additionally, with CITE-seq markers, we identified the protein landscape of sub-populations and found increased PD-1 and CD45RO expression in tonsils compared to blood CXCR5+ memory CD8+ T cells. Overall, these findings indicate that CXCR5+ CD8+ T cells are heterogeneous with potentially distinct functional subsets that patrol blood and lymphoid tissues.
PD-1 signaling serves a dual role in suppressing T cell activation but also in protecting from activation-induced cell death
Authors: Vick Logan V , Khuat Lam Tan , Le Catherine , Dunai Cordelia , Collins Craig , Canter Robert , Monjazeb Arta , Murphy William J
Published: August 2023

PD1/PDL1 interactions exert potent inhibitory effects on T cell responses. Inhibition of this pathway results in greater T cell responses and is increasingly used in cancer immunotherapy but the long-term effects of PD1 inhibition on T cell activation responses remain unclear. We hypothesized that these gains in proliferation and activation on T cells over time may result in greater T cell loss due to apoptosis via activation induced cell death (AICD). We investigated this using both mouse and human T cell models of activation. T cells from wild-type or PD1−/− mice were activated in vitro by a variety of stimuli. We observed significantly increased total T cell numbers, as well as expression of Ki67 and CD69 in the PD-1−/− T cells compared to controls. However, we identified that this increase was accompanied by increased cell death and apoptosis at later time points. Using in vivo adoptive transfer models (allogeneic & xenogeneic) in which PD-1−/− murine cells, or human T cells given in tandem with PD-1 checkpoint blockade we assessed PD-1 inhibition in vivo on T cell activation, proliferation, and apoptosis. We observed early expansion and activation of murine PD-1−/− cells in the allogeneic environment compared to controls. Furthermore, human T cells transplanted into immunodeficient mice given anti-PD1 also had greater activation and proliferation. Yet, this was followed by a sharper contraction and increased apoptosis identified through decreased cell numbers and increased Annexin V staining. These data suggest PD-1 regulates T cell activation as a means of protection, but when this balance is manipulated, T cells experience a “short term gain” for a “long term loss” in which cells undergo greater early activation at the cost of AICD.

Supported by National Institute of Health grant numbers NIH RO1 HL140921 (WJM), National Institute of Health grant numbers NIH RO1 CA214048 (WJM).
Asymmetrical Expression of PD1 and CD25 in T-cells Post-Initial Activation
Authors: Sheng Michael K , Vick Logan , Collins Craig , Yoon Daniel J , Murphy William J
Published: August 2023

After TCR engagement, T cells induce expression of CD25, which forms the IL2 high affinity receptor complex, concurrently with PD1, which plays a down-regulatory role in activation. Cessation of TCR stimulation leads to eventual contraction of T cell responses. Chimeric antigen receptor (CAR) T cell therapy has seen increased use in the past decade. CAR T-cells are activated and transduced ex vivo and expanded with low amounts of cytokines prior to administration. The expression of CD25 and PD1 on CAR T cells at different stages of the process has been incompletely characterized. Purified T cells were activated in vitro for 3 days followed by expansion using low dose IL2. We demonstrate that both CD25 and PD-1 are strongly upregulated during initial activation, but by day 8, under expansion conditions, PD1 is downregulated to negligible levels. Despite absence of TCR signaling, CD25 expression remains moderate-to-high explaining the ability to maintain proliferation. This discrepancy was higher in transduced CAR T-cells compared to activated non-transduced (NT) T cells indicating the possibility of CAR tonic signaling. Furthermore, restimulation of both NT and CAR-T cells after this expansion phase resulted in further increased CD25 expression whereas PD-1 expression remained low. Our studies suggest that during initial activation, TCR engagement is necessary to maintain PD-1, but not CD25 expression. However, upon restimulation, TCR engagement is capable of further upregulating CD25, but not PD-1 expression. These results indicate that TCR signaling induces asymmetrical expression of PD-1 and CD25, a dynamic interplay that may regulate T-cell function and memory response and that PD1 may not be a limiting factor in the process.