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The ideal vaccine against viruses such as influenza and SARS-CoV-2 must provide a robust, durable and broad immune protection against multiple viral variants. However, antibody responses to current vaccines often lack robust cross-reactivity. Here we describe a polymeric Toll-like receptor 7 agonist nanoparticle (TLR7-NP) adjuvant, which enhances lymph node targeting, and leads to persistent activation of immune cells and broad immune responses. When mixed with alum-adsorbed antigens, this TLR7-NP adjuvant elicits cross-reactive antibodies for both dominant and subdominant epitopes and antigen-specific CD8+ T-cell responses in mice. This TLR7-NP-adjuvanted influenza subunit vaccine successfully protects mice against viral challenge of a different strain. This strategy also enhances the antibody response to a SARS-CoV-2 subunit vaccine against multiple viral variants that have emerged. Moreover, this TLR7-NP augments antigen-specific responses in human tonsil organoids. Overall, we describe a nanoparticle adjuvant to improve immune responses to viral antigens, with promising implications for developing broadly protective vaccines.

Fully effective vaccines for viruses such as Influenza and SARS-CoV-2 must elicit a diverse repertoire of antibodies against multiple drifted virus strains. However, how to achieve a diverse response has no general solution except to combine multiple strains, which risks diluting the response for all strains included. Here, we describe the synthesis of a universal, toll-like receptor 7 agonist (TLR7)-nanoparticle adjuvant, TLR7-NP, constructed from TLR7 agonist-initiated ring-opening polymerization of lactide and self-assembly with poly(ethylene glycol)-b-poly(lactic-co-glycolic acid). When mixed with Alum-adsorbed antigens, this TLR7-NP adjuvant elicited cross-reactive antibodies for both dominant and subdominant epitopes, as well as antigen-specific CD8+ T cell responses. TLR7-NPs adjuvanted influenza subunit vaccine successfully protected mice from heterologous viral challenge. TLR7-NPs also enhanced the antibody response to a SARS-CoV-2 subunit vaccine against multiple variants and revealed the mobilization of a virus-like response. We further demonstrate enhanced antigen-specific responses in human tonsil organoids with this novel adjuvant.

The germinal center (GC) response is critical for both effective adaptive immunity and establishing peripheral tolerance by limiting autoreactive B cells. Dysfunction in these processes can lead to defective immune responses to infection or contribute to autoimmune disease. To understand the gene regulatory principles underlying the GC response, we generated a single-cell transcriptomic and epigenomic atlas of the human tonsil, a widely studied and representative lymphoid tissue. We characterize diverse immune cell subsets and build a trajectory of dynamic gene expression and transcription factor activity during B cell activation, GC formation, and plasma cell differentiation. We subsequently leverage cell type-specific transcriptomic and epigenomic maps to interpret potential regulatory impact of genetic variants implicated in autoimmunity, revealing that many exhibit their greatest regulatory potential in GC-associated cellular populations. These included gene loci linked with known roles in GC biology (IL21, IL21R, IL4R, BCL6) and transcription factors regulating B cell differentiation (POU2AF1, HHEX). Together, these analyses provide a powerful new cell type-resolved resource for the interpretation of cellular and genetic causes underpinning autoimmune disease.

Accurate prediction of antigen presentation by human leukocyte antigen (HLA) class II molecules would be valuable for vaccine development and cancer immunotherapies. Current computational methods trained on in vitro binding data are limited by insufficient training data and algorithmic constraints. Here we describe MARIA (major histocompatibility complex analysis with recurrent integrated architecture; https://maria.stanford.edu/ ), a multimodal recurrent neural network for predicting the likelihood of antigen presentation from a gene of interest in the context of specific HLA class II alleles. In addition to in vitro binding measurements, MARIA is trained on peptide HLA ligand sequences identified by mass spectrometry, expression levels of antigen genes and protease cleavage signatures. Because it leverages these diverse training data and our improved machine learning framework, MARIA (area under the curve = 0.89–0.92) outperformed existing methods in validation datasets. Across independent cancer neoantigen studies, peptides with high MARIA scores are more likely to elicit strong CD4+ T cell responses. MARIA allows identification of immunogenic epitopes in diverse cancers and autoimmune disease. A neural network trained on diverse datasets improves prediction of HLA class II epitope presentation.

The germinal center (GC) response is critical for both effective adaptive immunity and establishing peripheral tolerance by limiting auto-reactive B cells. Dysfunction in these processes can lead to defects in immune response to pathogens or contribute to autoimmune disease. To understand the gene regulatory principles underlying the GC response, we generated a single-cell transcriptomic and epigenomic atlas of the human tonsil, a widely studied and representative lymphoid tissue. We characterize diverse immune cell subsets and build a trajectory of dynamic gene expression and transcription factor activity during B cell activation, GC formation, and plasma cell differentiation. We subsequently leverage cell type-specific transcriptomic and epigenomic maps to interpret potential regulatory impact of genetic variants implicated in autoimmunity, revealing that many exhibit their greatest regulatory potential in GC cell populations. Together, these analyses provide a powerful new cell type-resolved resource for the interpretation of cellular and genetic causes underpinning autoimmune disease.One sentence summarySingle-cell chromatin accessibility landscapes of immune cell subsets reveal regulatory potential of autoimmune-associated genetic variants during the germinal center response.

Most of what we know about adaptive immunity has come from inbred mouse studies, using methods that are often difficult or impossible to confirm in humans. In addition, vaccine responses in mice are often poorly predictive of responses to those same vaccines in humans. Here we use human tonsils, readily available lymphoid organs, to develop a functional organotypic system that recapitulates key germinal center features in vitro, including the production of antigen-specific antibodies, somatic hypermutation and affinity maturation, plasmablast differentiation and class-switch recombination. We use this system to define the essential cellular components necessary to produce an influenza vaccine response. We also show that it can be used to evaluate humoral immune responses to two priming antigens, rabies vaccine and an adenovirus-based severe acute respiratory syndrome coronavirus 2 vaccine, and to assess the effects of different adjuvants. This system should prove useful for studying critical mechanisms underlying adaptive immunity in much greater depth than previously possible and to rapidly test vaccine candidates and adjuvants in an entirely human system.

The distal lung contains terminal bronchioles and alveoli that facilitate gas exchange and is affected by disorders including interstitial lung disease, cancer, and SARS-CoV-2-associated COVID-19 pneumonia. Investigations of these localized pathologies have been hindered by a lack of 3D in vitro human distal lung culture systems. Further, human distal lung stem cell identification has been impaired by quiescence, anatomic divergence from mouse and lack of lineage tracing and clonogenic culture. Here, we developed robust feeder-free, chemically-defined culture of distal human lung progenitors as organoids derived clonally from single adult human alveolar epithelial type II (AT2) or KRT5+ basal cells. AT2 organoids exhibited AT1 transdifferentiation potential, while basal cell organoids progressively developed lumens lined by differentiated club and ciliated cells. Organoids consisting solely of club cells were not observed. Upon single cell RNA-sequencing (scRNA-seq), alveolar organoids were composed of proliferative AT2 cells; however, basal organoid KRT5+ cells contained a distinct ITGA6+ITGB4+ mitotic population whose proliferation segregated to a TNFRSF12Ahi subfraction. Clonogenic organoid growth was markedly enriched within the TNFRSF12Ahi subset of FACS-purified ITGA6+ITGB4+ basal cells from human lung or derivative organoids. In vivo, TNFRSF12A+ cells comprised ~10% of KRT5+ basal cells and resided in clusters within terminal bronchioles. To model COVID-19 distal lung disease, we everted the polarity of basal and alveolar organoids to rapidly relocate differentiated club and ciliated cells from the organoid lumen to the exterior surface, thus displaying the SARS-CoV-2 receptor ACE2 on the outwardly-facing apical aspect. Accordingly, basal and AT2 “apical-out” organoids were infected by SARS-CoV-2, identifying club cells as a novel target population. This long-term, feeder-free organoid culture of human distal lung alveolar and basal stem cells, coupled with single cell analysis, identifies unsuspected basal cell functional heterogeneity and exemplifies progenitor identification within a slowly proliferating human tissue. Further, our studies establish a facile in vitro organoid model for human distal lung infectious diseases including COVID-19-associated pneumonia.

There are fundamental differences between humans and the animals we typically use to study the immune system. We have learned much from genetically manipulated and inbred animal models, but instances in which these findings have been successfully translated to human immunity have been rare. Embracing the genetic and environmental diversity of humans can tell us about the fundamental biology of immune cell types and the elasticity of the immune system. Although people are much more immunologically diverse than conventionally housed animal models, tools and technologies are now available that permit high-throughput analysis of human samples, including both blood and tissues, which will give us deep insights into human immunity in health and disease. As we gain a more detailed picture of the human immune system, we can build more sophisticated models to better reflect this complexity, both enabling the discovery of new immunological mechanisms and facilitating translation into the clinic.

The induction of long-lived heterotypic T-cell protection against influenza virus remains elusive, despite the conservation of T-cell epitopes. T-cell protection against influenza is critically dependent on lung-resident memory T cells (Trm). Here we show that intranasal administration of 4-1BBL along with influenza nucleoprotein in a replication-defective adenovirus vector to influenza pre-immune mice induces a remarkably stable circulating effector memory CD8 T-cell population characterized by higher IL-7Rα expression than control-boosted T cells, as well as a substantial lung parenchymal CD69+ CD8 Trm population, including both CD103+ and CD103- cells. These T-cell responses persist to greater than 200 days post-boost and protect against lethal influenza challenge in aged (year old) mice. The expansion of the nucleoprotein-specific CD8 Trm population during boosting involves recruitment of circulating antigen-specific cells and is critically dependent on local rather than systemic administration of 4-1BBL as well as on 4-1BB on the CD8 T cells. Moreover, during primary influenza infection of mixed bone marrow chimeras, 4-1BB-deficient T cells fail to contribute to the lung-resident Trm population. These findings establish both endogenous and supraphysiological 4-1BBL as a critical regulator of lung-resident memory CD8 T cells during influenza infection.

Cancer somatic mutations can generate neoantigens that distinguish malignant from normal cells. However, the personalized identification and validation of neoantigens remains a major challenge. Here we discover neoantigens in human mantle-cell lymphomas by using an integrated genomic and proteomic strategy that interrogates tumour antigen peptides presented by major histocompatibility complex (MHC) class I and class II molecules. We applied this approach to systematically characterize MHC ligands from 17 patients. Remarkably, all discovered neoantigenic peptides were exclusively derived from the lymphoma immunoglobulin heavy- or light-chain variable regions. Although we identified MHC presentation of private polymorphic germline alleles, no mutated peptides were recovered from non-immunoglobulin somatically mutated genes. Somatic mutations within the immunoglobulin variable region were almost exclusively presented by MHC class II. We isolated circulating CD4+ T cells specific for immunoglobulin-derived neoantigens and found these cells could mediate killing of autologous lymphoma cells. These results demonstrate that an integrative approach combining MHC isolation, peptide identification, and exome sequencing is an effective platform to uncover tumour neoantigens. Application of this strategy to human lymphoma implicates immunoglobulin neoantigens as targets for lymphoma immunotherapy.

Antibodies are typically thought to mediate antiviral immunity by blocking host-cell infection. However, immunoglobulin G (IgG) can also stimulate antiviral responses through its constant region. A recent study in Nature (Bournazos et al., 2020) shows that modifying Fc binding affinities to better target FcɣRIIa enhances the influenza CD8 T cell response.

In mass cytometry, sample loss is of considerable concern due to the relative inefficiency of cell event collection compared to similar techniques such as flow cytometry. Cell stimulation and the harsh conditions required in the later stages of certain sample preparations also contribute to cell loss. Low starting cell numbers are especially susceptible to these effects, potentially limiting the ability to use mass cytometry. Here is presented a live cell barcoding scheme and additional efficiency methods to improve recovery and achieve consistent staining for small samples.

Somatic mutations in cancer are a potential source of cancer specific neoantigens. Acute myeloid leukemia (AML) has common recurrent mutations shared between patients in addition to private mutations specific to individuals. We hypothesized that neoantigens derived from recurrent shared mutations would be attractive targets for future immunotherapeutic approaches. Here we sought to study the HLA Class I and II immunopeptidome of thirteen primary AML tumor samples and two AML cell lines (OCI-AML3 and MV4-11) using mass spectrometry to evaluate for endogenous mutation-bearing HLA ligands from common shared AML mutations. We identified two endogenous, mutation-bearing HLA Class I ligands from nucleophosmin (NPM1). The ligands, AVEEVSLRK from two patient samples and C(cys)LAVEEVSL from OCI-AML3, are predicted to bind the common HLA haplotypes, HLA-A*03:01 and HLA-A*02:01 respectively. Since NPM1 is mutated in approximately one-third of patients with AML, the finding of endogenous HLA ligands from mutated NPM1 supports future studies evaluating immunotherapeutic approaches against this shared target, for this subset of patients with AML.

In mass cytometry, sample loss is of considerable concern due to the relative inefficiency of cell event collection compared to similar techniques such as flow cytometry. Cell stimulation and the harsh conditions required in the later stages of certain sample preparations also contribute to cell loss. Low starting cell numbers are especially susceptible to these effects, potentially limiting the ability to use mass cytometry. Here is presented a live cell barcoding scheme and additional efficiency methods to improve recovery and achieve consistent staining for small samples.

Summary B cells are capable of a wide range of effector functions including antibody secretion, antigen presentation, cytokine production, and generation of immunological memory. A consistent strategy for classifying human B cells by using surface molecules is essential to harness this functional diversity for clinical translation. We developed a highly multiplexed screen to quantify the co-expression of 351 surface molecules on millions of human B cells. We identified differentially expressed molecules and aligned their variance with isotype usage, VDJ sequence, metabolic profile, biosynthesis activity, and signaling response. Based on these analyses, we propose a classification scheme to segregate B cells from four lymphoid tissues into twelve unique subsets, including a CD45RB+CD27− early memory population, a class-switched CD39+ tonsil-resident population, and a CD19hiCD11c+ memory population that potently responds to immune activation. This classification framework and underlying datasets provide a resource for further investigations of human B cell identity and function., Graphical Abstract, Highlights • A mass cytometry screen reveals 98 surface molecules expressed by human B cells • High-dimensional analysis identifies twelve B cell subsets across four tissues • CD45RB, CD11c, CD39, CD73, and CD95 define subsets of antigen-experienced B cells • Isotype usage, signaling, and metabolism vary in accordance with cell surface phenotype, To identify molecules that distinguish functionally distinct subsets of human B cells, Glass et al. screened the expression of 351 surface molecules by mass cytometry. By combining identified molecules with functional readouts, they propose a new classification scheme to segregate B cells from four lymphoid tissues into twelve unique subsets.

Broadly effective vaccines against dengue virus (DENV) infection have remained elusive, despite rising infection rates in the developing world. Infection-specific peptide ligands presented on Major Histocompatibility Complexes (MHC) open new avenues for developing T-cell-based interventions. Past efforts towards mapping viral MHC epitopes were based on computational predictions that only partially reflected actual antigen presentation. To empirically identify DENV-specific MHC ligands, we developed an immuno-proteomics approach for interrogating DENV- and self-derived MHC ligands from infected B-lymphocytes. Here, we report four fundamental findings: First, over 700 infection-specific MHC-ligands reflected host cellular responses to DENV that were not apparent from the proteome. Second, we report 121 viral MHC-I ligands (108 novel) which clustered into discrete hotspots across the DENV polyprotein, some of which spanned DENV polyprotein components, described here as MHC ligands for the first time. Third, we found DENV ligands which were distinctly presented by MHC alleles previously associated with either high or low anti-DENV response. Fourth, we demonstrate that while our in vitro assay only overlapped with a small fraction of previously described DENV T-cell epitopes, several novel MHC ligands identified here were recognized by T-cells from DENV-infected patients despite having low binding affinities. Together, these discoveries suggest that virus and host-derived MHC ligands have under-exploited potential for describing the cell biology of DENV infection, and as candidates for designing effective DENV vaccines.

Antigenic exposures at epithelial sites in infancy and early childhood are thought to influence the maturation of humoral immunity and modulate the risk of developing immunoglobulin E (IgE)-mediated allergic disease. How different kinds of environmental exposures influence B cell isotype switching to IgE, IgG, or IgA, and the somatic mutation maturation of these antibody pools, is not fully understood. We sequenced antibody repertoires in longitudinal blood samples in a birth cohort from infancy through the first 3 years of life and found that, whereas IgG and IgA show linear increases in mutational maturation with age, IgM and IgD mutations are more closely tied to pathogen exposure. IgE mutation frequencies are primarily increased in children with impaired skin barrier conditions such as eczema, suggesting that IgE affinity maturation could provide a mechanistic link between epithelial barrier failure and allergy development.

The distal lung contains terminal bronchioles and alveoli that facilitate gas exchange and is affected by disorders including interstitial lung disease, cancer, and SARS-CoV-2-associated COVID-19 pneumonia. Investigations of these localized pathologies have been hindered by a lack of 3D in vitro human distal lung culture systems. Further, human distal lung stem cell identification has been impaired by quiescence, anatomic divergence from mouse and lack of lineage tracing and clonogenic culture. Here, we developed robust feeder-free, chemically-defined culture of distal human lung progenitors as organoids derived clonally from single adult human alveolar epithelial type II (AT2) or KRT5 (+) basal cells. AT2 organoids exhibited AT1 transdifferentiation potential, while basal cell organoids progressively developed lumens lined by differentiated club and ciliated cells. Organoids consisting solely of club cells were not observed. Upon single cell RNA-sequencing (scRNA-seq), alveolar organoids were composed of proliferative AT2 cells; however, basal organoid KRT5 (+) cells contained a distinct ITGA6 (+) ITGB4 (+) mitotic population whose proliferation segregated to a TNFRSF12A (hi) subfraction. Clonogenic organoid growth was markedly enriched within the TNFRSF12A (hi) subset of FACS-purified ITGA6 (+) ITGB4 (+) basal cells from human lung or derivative organoids. In vivo, TNFRSF12A (+) cells comprised ~10% of KRT5 (+) basal cells and resided in clusters within terminal bronchioles. To model COVID-19 distal lung disease, we everted the polarity of basal and alveolar organoids to rapidly relocate differentiated club and ciliated cells from the organoid lumen to the exterior surface, thus displaying the SARS-CoV-2 receptor ACE2 on the outwardly-facing apical aspect. Accordingly, basal and AT2 apical-out organoids were infected by SARS-CoV-2, identifying club cells as a novel target population. This long-term, feeder-free organoid culture of human distal lung alveolar and basal stem cells, coupled with single cell analysis, identifies unsuspected basal cell functional heterogeneity and exemplifies progenitor identification within a slowly proliferating human tissue. Further, our studies establish a facile in vitro organoid model for human distal lung infectious diseases including COVID-19-associated pneumonia.

Given the limited efficacy of clinical approaches that rely on ex vivo generated dendritic cells (DCs), it is imperative to design strategies that harness specialized DC subsets in situ. This requires delineating the expression of surface markers by DC subsets among individuals and tissues. Here, we performed a multiparametric phenotypic characterization and unbiased analysis of human DC subsets in blood, tonsil, spleen, and skin. We uncovered previously unreported phenotypic heterogeneity of human cDC2s among individuals, including variable expression of functional receptors such as CD172a. We found marked differences in DC subsets localized in blood and lymphoid tissues versus skin, and a striking absence of the newly discovered Axl+ DCs in the skin. Finally, we evaluated the capacity of anti-receptor monoclonal antibodies to deliver vaccine components to skin DC subsets. These results offer a promising path for developing DC subset-specific immunotherapies that cannot be provided by transcriptomic analysis alone.

T cell receptor (TCR) sequences are very diverse, with many more possible sequence combinations than T cells in any one individual1–4. Here we define the minimal requirements for TCR antigen specificity, through an analysis of TCR sequences using a panel of peptide and major histocompatibility complex (pMHC)-tetramer-sorted cells and structural data. From this analysis we developed an algorithm that we term GLIPH (grouping of lymphocyte interactions by paratope hotspots) to cluster TCRs with a high probability of sharing specificity owing to both conserved motifs and global similarity of complementarity-determining region 3 (CDR3) sequences. We show that GLIPH can reliably group TCRs of common specificity from different donors, and that conserved CDR3 motifs help to define the TCR clusters that are often contact points with the antigenic peptides. As an independent validation, we analysed 5,711 TCRβ chain sequences from reactive CD4 T cells from 22 individuals with latent Mycobacterium tuberculosis infection. We found 141 TCR specificity groups, including 16 distinct groups containing TCRs from multiple individuals. These TCR groups typically shared HLA alleles, allowing prediction of the likely HLA restriction, and a large number of M. tuberculosis T cell epitopes enabled us to identify pMHC ligands for all five of the groups tested. Mutagenesis and de novo TCR design confirmed that the GLIPH-identified motifs were critical and sufficient for shared-antigen recognition. Thus the GLIPH algorithm can analyse large numbers of TCR sequences and define TCR specificity groups shared by TCRs and individuals, which should greatly accelerate the analysis of T cell responses and expedite the identification of specific ligands.

Background: Current in vitro lymphoma models, including three-dimensional organoids, generally contain exclusively neoplastic lymphocytes and require artificial reconstitution to recapitulate the tumor microenvironment (TME). The co-culture of syngeneic tumor-infiltrating lymphocytes (TILs) alongside endogenous primary malignant lymphocytes could be useful for modeling complex interactions in the TME, and for immunological maneuvers and therapies relying on TILs. However, such conditions for maintaining lymphomas in their syngeneic TME as a cohesive unit have remained elusive. Methods: We adapted an air-liquid interface (ALI) method that we previously described (Neal JT et al 2019 Cell) for propagating patient-derived organoids (PDOs) from primary human follicular lymphomas. Surgically excised lymphoma samples were tested for the ability to maintain lymphoma cell viability in vitro using a lymph node organoid technique. Lymph nodes containing lymphoma cells (and in one case, a PBMC sample including circulating lymphoma cells) were processed into a single cell suspension and frozen until use. Samples were thawed and prepared into immune organoids (see figure). We assessed cell composition by flow cytometry on day 7, and in a subset of samples, up to 21 days post-thaw. Results: A total of 6 patients were profiled for PDO formation, PDO composition and stability, and PDO longevity. 4 of 6 samples showed good cell viability at day 7 post-culture and in a subset of samples, up to 21 days post-culture. Cell composition was well-maintained over time, with presence of lymphoma cells (CD19+ CD10+ CD5-) easily detectable and maintenance of supporting cells of the lymph node such as T follicular helper cells (CD3+ CD4+ CXCR5+ PD-1+) and non-B, non-T cells. Supporting lymph node cells were not detected in the PBMC sample, suggesting the cell composition is related to the initial composition and not due to differentiation in vitro. Genotyping, gene expression phenotyping, and T-cell/B-cell receptor profiling data will be presented at the meeting, including accuracy of PDOs for preserving the original spectrum of these indices. Conclusions: Propagation of PDOs of primary lymphomas with endogenous immune stroma is feasible and maintains cohesive elements of the TME. This system should allow immunoncology investigations within the TME and to facilitate personalized immunotherapy testing. Fig 1: Human lymphoma organoid cultures as a model to study tumor microenvironments and immune responses in vitro. (A) Experimental schema for preparing lymphoma organoids from tumor explants. In an adapted workflow optimized for ex vivo culture of human tonsillar germinal centers (Wagar L et al, submitted), we subject cryopreserved FL samples to organoid culture. (B) An example of follicular lymphoma organoid reorganization in vitro after four days in culture. (C) Total cell viability in FL organoids for up to 21 days in culture. Six samples were tested. (D) Frequency of major cell types in FL samples after organoid culture. Although there is variation among donors' samples, an individual's cell composition is well maintained for at least 14 days in most organoids. Figure 1 Disclosures Khodadoust: Corvus Pharmaceuticals: Research Funding. Davis:Vir Biotechnology: Consultancy, Equity Ownership, Honoraria; PACT Bio: Consultancy, Equity Ownership, Honoraria; Adicet Inc: Consultancy, Equity Ownership, Honoraria; Chuga Pharmabody: Consultancy, Honoraria; Amgen: Consultancy, Research Funding; Atreca: Consultancy, Equity Ownership, Honoraria; Juno: Consultancy, Equity Ownership, Honoraria. Alizadeh:Pfizer: Research Funding; Chugai: Consultancy; Celgene: Consultancy; Gilead: Consultancy; Pharmacyclics: Consultancy; Janssen: Consultancy; Genentech: Consultancy; Roche: Consultancy.

Vaccination with attenuated live varicella zoster virus (VZV) can prevent zoster reactivation, but protection is incomplete especially in an older population. To decipher the molecular mechanisms underlying variable vaccine responses, T- and B-cell responses to VZV vaccination were examined in individuals of different ages including identical twin pairs. Contrary to the induction of VZV-specific antibodies, antigen-specific T cell responses were significantly influenced by inherited factors. Diminished generation of long-lived memory T cells in older individuals was mainly caused by increased T cell loss after the peak response while the expansion of antigen-specific T cells was not affected by age. Gene expression in activated CD4 T cells at the time of the peak response identified gene modules related to cell cycle regulation and DNA repair that correlated with the contraction phase of the T cell response and consequently the generation of long-lived memory cells. These data identify cell cycle regulatory mechanisms as targets to reduce T cell attrition in a vaccine response and to improve the generation of antigen-specific T cell memory, in particular in an older population., Author Summary Vaccination is one of the most successful medical interventions, but it loses its effectiveness in an older population that is of particular risk for infectious diseases. Shingles, caused by the reactivation of the chickenpox virus, is a prime example. Nearly every second individual has experienced shingles by the age of 80 years, and the shingles vaccine is only partially protective. Attempts to improve the vaccine response are mostly empiric. Vaccinations induce a rapid expansion of antigen-specific T cells with frequencies peaking after one to two weeks. Most expanded T cells die after the peak response, and only few T cells survive to provide protection from infection or, as in case of shingles, from reactivation of latent viruses. Most vaccine studies have focused on the early stages of the response; how T cells are activated and expand. Surprisingly, in our study with the shingle vaccine, T cell survival after the peak response was the major factor determining memory T cell frequencies. T cell attrition was increased with age, independent of genetic predisposition. Using systems biology tools we found several pathways involved in T cell division and DNA repair that could be targeted to improve T cell survival and thereby increase the effectiveness of vaccination.

Copyright © 2018 by The American Association of Immunologists, Inc. IL-7 therapy has been evaluated in patients who do not regain normal CD4 T cell counts after virologically successful antiretroviral therapy. IL-7 increases total circulating CD4 and CD8 T cell counts; however, its effect on HIV-specific CD8 T cells has not been fully examined. TRAF1, a prosurvival signaling adaptor required for 4-1BB-mediated costimulation, is lost from chronically stimulated virus-specific CD8 T cells with progression of HIV infection in humans and during chronic lymphocytic choriomeningitis infection in mice. Previous results showed that IL-7 can restore TRAF1 expression in virus-specific CD8 T cells in mice, rendering them sensitive to anti-4-1BB agonist therapy. In this article, we show that IL-7 therapy in humans increases the number of circulating HIV-specific CD8 T cells. For a subset of patients, we also observed an increased frequency of TRAF1+HIV-specific CD8 T cells 10 wk after completion of IL-7 treatment. IL-7 treatment increased levels of phospho-ribosomal protein S6 in HIV-specific CD8 T cells, suggesting increased activation of the metabolic checkpoint kinase mTORC1. Thus, IL-7 therapy in antiretroviral therapy-treated patients induces sustained changes in the number and phenotype of HIV-specific T cells.

Astronauts face numerous health challenges during long-duration space missions, including diminished immunity, bone loss and increased risk of radiation-induced carcinogenesis. Changes in the intestinal flora of astronauts may contribute to these problems. Soy-based fermented food products could provide a nutritional strategy to help alleviate these challenges by incorporating beneficial lactic acid bacteria, while reaping the benefits of soy isoflavones. We carried out strain selection for the development of soy ferments, selecting strains of lactic acid bacteria showing the most effective growth and fermentation ability in soy milk (Streptococcus thermophilus ST5, Bifidobacterium longum R0175 and Lactobacillus helveticus R0052). Immunomodulatory bioactivity of selected ferments was assessed using an in vitro challenge system with human intestinal epithelial and macrophage cell lines, and selected ferments show the ability to down-regulate production of the pro-inflammatory cytokine interleukin-8 following challenge with tumour necrosis factor-alpha. The impact of fermentation on vitamin B1 and B6 levels and on isoflavone biotransformation to agluconic forms was also assessed, with strain variation-dependent biotransformation ability detected. Overall this suggests that probiotic bacteria can be successfully utilized to develop soy-based fermented products targeted against health problems associated with long-term space travel.

Fermented soy and dairy milk preparations provide a means for delivering lactic acid bacteria and their fermentation products into the diet. Our aims were to test immunomodulatory bioactivity of fermented soy beverage (SB) and dairy milk blend (MB) preparations on human intestinal epithelial cells (IEC) and to determine the impact of freezing medium on culture survival prior to bioactivity analyses. Fermented SB and MB were prepared using pure or mixed cultures of Streptococcus thermophilus ST5, Bifidobacterium longum R0175, and Lactobacillus helveticus R0052. Immunomodulatory bioactivity was assessed by testing selected SB and MB ferments on tumor necrosis factor alpha (TNFalpha)-treated IEC and measuring effects on Interleukin-8 (IL-8) production. Impact of timing of ferment administration relative to this pro-inflammatory challenge was investigated. The most pronounced reductions in IEC IL-8 production were observed when IEC were treated with either SB or MB ferment preparations prior to TNFalpha challenge. These results indicate that freezing-stable MB and SB ferments prepared with selected strains can modulate IEC IL-8 production in vitro, and suggest that yogurt-like fermented soy formulations could provide a functional food alternative to milk-based fermented products.

Mutations in mitochondrial (mt) DNA accumulate with age and can result in the generation of neopeptides. Immune surveillance of such neopeptides may allow suboptimal mitochondria to be eliminated, thereby avoiding mt-related diseases, but may also contribute to autoimmunity in susceptible individuals. To date, the direct recognition of neo-mtpeptides by the adaptive immune system has not been demonstrated. In this study we used bioinformatics approaches to predict MHC binding of neopeptides identified from known deletions in mtDNA. Six such peptides were confirmed experimentally to bind to HLA-A*02. Pre-existing human CD4+ and CD8+ T cells from healthy donors were shown to recognize and respond to these neopeptides. One remarkably promiscuous immunodominant peptide (P9) could be presented by diverse MHC molecules to CD4+ and/or CD8+ T cells from 75% of the healthy donors tested. The common soil microbe, Bacillus pumilus, encodes a 9-mer that differs by one amino acid from P9. Similarly, the ATP synthase F0 subunit 6 from normal human mitochondria encodes a 9-mer with a single amino acid difference from P9 with 89% homology to P9. T cells expanded from human PBMCs using the B. pumilus or self-mt peptide bound to P9/HLA-A2 tetramers, arguing for cross-reactivity between T cells with specificity for self and foreign homologs of the altered mt peptide. These findings provide proof of principal that the immune system can recognize peptides arising from spontaneous somatic mutations and that such responses might be primed by foreign peptides and/or be cross-reactive with self.

Neoantigens arising through somatic mutations are increasingly recognized as key tumor antigens driving clinical immune responses. We sought to identify human lymphoma neoantigens through a genomic and proteomic characterization of peptide ligands of major histocompatibility complex class I (MHC-I) and class II (MHC-II) of a cohort of patients with untreated mantle cell lymphoma. Peptide identification was performed through analysis of MHC-I and MHC-II peptide ligands using liquid chromatography and tandem mass spectrometry (LC/MS-MS), and augmented by generating patient-specific proteome databases through tumor:normal whole exome sequencing and targeted immunoglobulin gene sequencing in 17 patients. A total of 66 neoantigen peptides from 9 patients were found to be presented by MHC, and surprisingly all neoantigenic peptides were derived from either the lymphoma immunoglobulin heavy or light chain genes. There was no detectable MHC presentation of hundreds of candidate somatic neoantigenic peptides from other genes despite widespread antigen presentation of the proteome including proteins encoded by germline heterozygous single nucleotide polymorphisms. Clonotypic lymphoma immunoglobulin sequences were among the most commonly presented proteins by both MHC-I and MHC-II, yet MHC-I presentation of variable regions was nearly absent and included only a single neoantigen derived from IGVH. In contrast, the variable region was frequently presented by MHC-II and included neoantigen peptides created through somatic hypermutation or VDJ recombination. Autologous circulating T-cells specific for immunoglobulin-derived neoantigens could be detected using MHC-II tetramers and their frequency dynamically changed with vaccination and therapy. These findings suggest that immunoglobulin derived neoantigens are frequently presented by lymphoma, and represent potential targets for CD4, but not CD8, T-cell mediated therapies. Figure. MHC-I and MHC-II presentation of lymphoma immunoglobulin. (A) MHC-I (left) and MHC-II (right) presented peptides derived from patients' immunoglobulin were mapped to the immunoglobulin domains. Peptides recovered from all seventeen patients are mapped to a schematic of the IgM molecule with a heat map indicating the number of recovered peptides covering each position. (B) Expanded view of the immunoglobulin heavy chain variable region. The heatmap represents the total number of MHC-presented peptides that span that position, including non-neoantigens for MHC-I (above) and MHC-II (below). Neoantigen peptides created by either somatic hypermutation or VDJ rearrangement are mapped above or below the heat map. Red, positions within the recovered peptides which are altered from germline variable genes thus creating neoantigens. Boxes, peptides derived from a single patient. Figure. MHC-I and MHC-II presentation of lymphoma immunoglobulin. (A) MHC-I (left) and MHC-II (right) presented peptides derived from patients' immunoglobulin were mapped to the immunoglobulin domains. Peptides recovered from all seventeen patients are mapped to a schematic of the IgM molecule with a heat map indicating the number of recovered peptides covering each position. (B) Expanded view of the immunoglobulin heavy chain variable region. The heatmap represents the total number of MHC-presented peptides that span that position, including non-neoantigens for MHC-I (above) and MHC-II (below). Neoantigen peptides created by either somatic hypermutation or VDJ rearrangement are mapped above or below the heat map. Red, positions within the recovered peptides which are altered from germline variable genes thus creating neoantigens. Boxes, peptides derived from a single patient. Disclosures Newman: Roche: Consultancy. Carlton:Adaptive Biotechnologies: Employment, Equity Ownership. Moorhead:Adaptive Biotechnologies: Employment. Faham:Adaptive Biotechnologies Corp: Employment.

T cells specific for persistent pathogens accumulate with age and express markers of immune senescence. In contrast, much less is known about the state of T cell memory for acutely infecting pathogens. Here we examined T cell responses to influenza in human peripheral blood mononuclear cells from older (>64) and younger (

We evaluated a cohort of Canadian donors for T cell and antibody responses against influenza A/California/7/2009 (pH1N1) at 8-10 months after the 2nd pandemic wave by flow cytometry and microneutralization assays. Memory CD8 T cell responses to pH1N1 were detectable in 58% (61/105) of donors. These responses were largely due to cross-reactive CD8 T cell epitopes as, for those donors tested, similar recall responses were obtained to A/California 2009 and A/PR8 1934 H1N1 Hviruses. Longitudinal analysis of a single infected individual showed only a small and transient increase in neutralizing antibody levels, but a robust CD8 T cell response that rose rapidly post symptom onset, peaking at 3 weeks, followed by a gradual decline to the baseline levels seen in a seroprevalence cohort post-pandemic. The magnitude of the influenza-specific CD8 T cell memory response at one year post-pandemic was similar in cases and controls as well as in vaccinated and unvaccinated donors, suggesting that any T cell boosting from infection was transient. Pandemic H1-specific antibodies were only detectable in approximately half of vaccinated donors. However, those who were vaccinated within a few months following infection had the highest persisting antibody titers, suggesting that vaccination shortly after influenza infection can boost or sustain antibody levels. For the most part the circulating influenza-specific T cell and serum antibody levels in the population at one year post-pandemic were not different between cases and controls, suggesting that natural infection does not lead to higher long term T cell and antibody responses in donors with pre-existing immunity to influenza. However, based on the responses of one longitudinal donor, it is possible for a small population of pre-existing cross-reactive memory CD8 T cells to expand rapidly following infection and this response may aid in viral clearance and contribute to a lessening of disease severity.