Abstract Due to their position in the pulmonary mucosa, CD8 +tissue resident memory T cells (T RM) act as sentinels that rapidly respond to, and mediate protection against, respiratory viruses. In mice, T RMhave been shown to mediate protection at barrier sites by producing cytokines, chemokines, and performing cell lysis. However, in humans, less is known about the effector functions of virus-specific lung CD8 +T RM. Using cells from healthy human lung donors, we first identified and quantified the frequency of antigen-specific cells against common respiratory viruses by performing intracellular cytokine staining. We next investigated the polyfunctionality and residency profiles of responding antigen-specific CD8 +T cells and found that that CD69 +CD103 +T RMcomprise a larger portion of the antigen-specific CD8 +T cells for viruses largely restricted to the respiratory epithelium when compared to cells specific for viruses with a broader tissue tropism. Next, by performing a series of in vitro peptide stimulation and cytokine neutralization experiments, we investigated how antigen-specific CD8 +T cells impact local innate and epithelial cells. When stimulated with their cognate antigen, lung CD8 +T RM-derived IFNγ strongly correlated with epithelial cell activation. Finally, we stimulated lung CD8 +T RMwith cognate antigen +/− anti-IFNγ and performed RNAseq on responding antigen-specific CD8 +T RM(CD25 +CD137 +) and epithelial cells to determine exactly how T RM-derived IFNγ alters the cellular program of these cells. Results of this study suggest that human lung CD8 +T RMact to rapidly reprogram local immune cells, and these data will ultimately help us understand how CD8 +T RMfit into the overall immune response to respiratory viruses. This project is supported by R35 HL150803 and the Emory Center of Excellence for Influenza Research and Response (CEIRR) 75N93019R0028.
The current pandemic of respiratory illness, COVID-19, is caused by a recently emerged coronavirus named SARS-CoV-2. This virus infects airway and lung cells causing fever, dry cough, and shortness of breath. Severe cases of COVID-19 can result in lung damage, low blood oxygen levels, and even death. As there are currently no vaccines approved for use in humans, studies of the mechanisms of SARS-CoV-2 infection are urgently needed. Our research identifies an excellent system to model SARS-CoV-2 infection of the human airways that can be used to test various treatments. Analysis of infection in this model system found that human airway epithelial cell cultures induce a strong proinflammatory cytokine response yet block the production of type I and III IFNs to SARS-CoV-2. However, treatment of airway cultures with the immune molecules type I or type III interferon (IFN) was able to inhibit SARS-CoV-2 infection. Thus, our model system identified type I or type III IFN as potential antiviral treatments for COVID-19 patients.
Abstract One potential advantage of live attenuated influenza vaccines (LAIVs) is their ability to establish both virus-specific Ab and tissue-resident memory T cells (TRM) in the respiratory mucosa. However, it is hypothesized that pre-existing immunity from past infections and/or immunizations prevents LAIV from boosting or generating de novo CD8+ T cell responses. To determine whether we can overcome this limitation, we generated a series of drifted influenza A/PR8 LAIVs with successive mutations in the hemagglutinin protein, allowing for increasing levels of escape from pre-existing Ab. We also inserted a CD8+ T cell epitope from the Sendai virus nucleoprotein (NP) to assess both generation of a de novo T cell response and boosting of pre-existing influenza-specific CD8+ T cells following LAIV immunization. Increasing the level of escape from Ab enabled boosting of pre-existing TRM, but we were unable to generate de novo Sendai virus NP+ CD8+ TRM following LAIV immunization in PR8 influenza-immune mice, even with LAIV strains that can fully escape pre-existing Ab. As these data suggested a role for cell-mediated immunity in limiting LAIV efficacy, we investigated several scenarios to assess the impact of pre-existing LAIV-specific TRM in the upper and lower respiratory tract. Ultimately, we found that deletion of the immunodominant influenza NP366–374 epitope allowed for sufficient escape from cellular immunity to establish de novo CD8+ TRM. When combined, these studies demonstrate that both pre-existing humoral and cellular immunity can limit the effectiveness of LAIV, which is an important consideration for future design of vaccine vectors against respiratory pathogens.
Marginal zone B cells (MZB) are a mature B cell subset that rapidly respond to blood-borne pathogens. Although the transcriptional changes that occur throughout MZB development are known, the corresponding epigenetic changes and epigenetic modifying proteins that facilitate these changes are poorly understood. The histone demethylase LSD1 is an epigenetic modifier that promotes plasmablast formation, but its role in B cell development has not been explored. In this study, a role for LSD1 in the development of B cell subsets was examined. B cell-conditional deletion of LSD1 in mice resulted in a decrease in MZB whereas follicular B cells and bone marrow B cell populations were minimally affected. LSD1 repressed genes in MZB that were normally upregulated in the myeloid and follicular B cell lineages. Correspondingly, LSD1 regulated chromatin accessibility at the motifs of transcription factors known to regulate splenic B cell development, including NF-κB motifs. The importance of NF-κB signaling was examined through an ex vivo MZB development assay, which showed that both LSD1-deficient and NF-κB-inhibited transitional B cells failed to undergo full MZB development. Gene expression and chromatin accessibility analyses of in vivo- and ex vivo-generated LSD1-deficient MZB indicated that LSD1 regulated the downstream target genes of noncanonical NF-κB signaling. Additionally LSD1 was found to interact with the noncanonical NF-κB transcription factor p52. Together, these data reveal that the epigenetic modulation of the noncanonical NF-κB signaling pathway by LSD1 is an essential process during the development of MZB.
Abstract Background: Neuroblastoma is a childhood tumor characterized by relatively few somatic mutations and low MHC expression, which has thus far largely precluded it from investigation using adoptive immunotherapy. Methods: We characterized MHC-presented antigens in 8 patient derived xenograft (PDX) tumors using LC/MS/MS immunopeptidomics. We developed a method to identify antigens derived from differentially expressed proteins by combining RNA-seq data from 153 neuroblastoma and 1641 healthy tissues, and ligandomic data from 190 healthy tissues. We also performed functional characterization on the ability HLA-A2 neuroblastoma lines to elicit a T-cell response using CEF1 antigen-specific T-cell hyrbidoma when challenged with flu virus, and performed bioinformatic analysis of the TME. Results: From 8 PDX tumors, we identified a total of 14119 MHC-presented antigens. Interestingly, we observed antigens from all tumors which stained negative for MHC by IHC, suggesting that antigens can be identified from tumors below the detection limits of the standard staining protocols. We first searched the ligandome dataset for all possible neoantigens of 8-14aa arising from known mutations. Not having found any mutated neoantigens, we developed a method to search for tumor antigens derived neuroblastoma-specific proteins. We discovered 83 MHC ligands that derive from neuroblastoma-specific proteins, which we expect to be promising targets for adoptive T-cell therapy. We also found a number of recurrent antigens across tumor samples, suggesting that the proteins from which these antigens derive could be used in HLA-agnostic tumors vaccines. To test whether the MHC expression in neuroblastoma is sufficient to induce a T-cell response, we characterized the ability of neuroblastoma cells to elicit a CD8 response to CEF1 antigen. We demonstrate that despite low MHC expression in all lines tested, 4/7 lines are able to induce a robust T-cell response to flu antigen greater than HLA-A2 melanoma cells (>20pg/mL IL-2 release in all responsive lines). We also characterized immune activity in patient tumors using Granzyme A (GZMA) and Perforin (PRF1) as surrogates for T-cell activity. We observed a strong correlation between known T-cell-recruiting cytokines (particularly CXCL5/9/10) and GZMA/PRF1 (p=2.51x10-35), providing further evidence that T-cells are active in the tumor microenvironment despite low MHC expression on tumor cells. Conclusions: We identify neuroblastoma-specific MHC class I antigens in each of the 8 PDX tumors we tested, yielding a total of 83 novel antigens. We conclude that neuroblastoma tumors harbor promising tumor-specific targets for immunotherapy despite low rates of somatic mutation and low MHC expression, and that at least a subset of these tumors are able to induce a robust response using antigen-specific T-cell hybridomas. We also describe methods of identifying tumor-specific MHC antigens in other tumors with low mutational burden. Note: This abstract was not presented at the meeting. Citation Format: Mark Yarmarkovich, Moreno Di Marco, Olivia Padovan, Jenna Lobby, Laurence Eisenlohr, Dimitrios Monos, Stefan Stevanovic, John M. Maris. MHC class I immunogenicity and novel tumor antigen discovery in neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5824. doi:10.1158/1538-7445.AM2017-5824
Abstract Lung-resident memory T cells (lung TRM) are critical for protective heterosubtypic immunity against influenza viruses. However, the efficacy of cellular immunity against respiratory pathogens such as influenza wanes over time due to the gradual loss of flu-specific lung TRM. One possible mechanism for this decline is frequent exposure to environmental and biological insults resulting in localized inflammation that promotes the death of established lung TRM. We investigated whether unrelated infections could exacerbate the loss of pre-existing, flu-specific lung TRM and reduce the efficacy of cellular immunity to subsequent influenza challenge. Infection of influenza-immune mice with Sendai virus, an unrelated murine parainfluenza virus, resulted in significantly higher viral titers and greater morbidity following influenza challenge compared to PBS-treated controls. This loss of protective cellular immunity corresponded to a significant decrease in the number of pre-existing flu-specific lung CD8 TRM compared to PBS controls due to increased apoptosis. This loss of pre-existing lung TRM following Sendai infection was not due to competition for limited resources or tissue niches between flu- and Sendai-specific lung TRM. Sendai infection had no impact on the number of systemic flu-specific memory CD8 T cells in the spleen. The loss of lung TRM required a respiratory infection, as LCMV infection and intranasal delivery of TLR agonists did not significantly reduce the number of pre-existing flu-specific lung CD8 TRM. Together, these data suggest that tissue damage induced by unrelated respiratory infections can promote the loss of pre-existing lung TRM and compromise cellular immunity against respiratory pathogens.
Abstract Lung tissue-resident memory T cells are crucial mediators of cellular immunity against respiratory viruses; however, their gradual decline hinders the development of T cell–based vaccines against respiratory pathogens. Recently, studies using adenovirus (Ad)-based vaccine vectors have shown that the number of protective lung-resident CD8+ TRMs can be maintained long term. In this article, we show that immunization of mice with a replication-deficient Ad serotype 5 expressing influenza (A/Puerto Rico/8/34) nucleoprotein (AdNP) generates a long-lived lung TRM pool that is transcriptionally indistinct from those generated during a primary influenza infection. In addition, we demonstrate that CD4+ T cells contribute to the long-term maintenance of AdNP-induced CD8+ TRMs. Using a lineage tracing approach, we identify alveolar macrophages as a cell source of persistent NP Ag after immunization with AdNP. Importantly, depletion of alveolar macrophages after AdNP immunization resulted in significantly reduced numbers of NP-specific CD8+ TRMs in the lungs and airways. Combined, our results provide further insight to the mechanisms governing the enhanced longevity of Ag-specific CD8+ lung TRMs observed after immunization with recombinant Ad.
Abstract Previous reports have demonstrated the many effects of IL-27 on CD4 T cell activation and differentiation, in addition to a role for IL-27 in cancer therapies mediated by cytolytic CD8 T cells. However, as IL-27 is produced by many cell types during infection, and the IL-27 receptor (IL-27R) is widely expressed on NK cells, as well as on activated CD4 and CD8 T cells, the direct effects of IL-27 signaling on CD8 T cell function are less well understood. We used a murine model of influenza infection in combination with co-transfer of congenic wild type (WT) and IL-27R−/− OT-I CD8 T cells to investigate whether IL-27 signaling had a direct effect on virus-specific CD8 T cell function in vivo. We observed a dramatic reduction in the frequency and number of IL-27R−/− OT-I compared to their WT OT-I counterparts within the same host following influenza infection, indicating a CD8 T cell-intrinsic role for IL-27 in the generation of an acute anti-viral response. This defect was observed in the spleen, lung interstitium, airways and the lung-draining mediastinal lymph node, indicating a systemic defect in development of the CD8 T cell effector response in the absence of IL-27R signaling. Both memory precursor and short lived effector cells were reduced in the absence of IL-27R signaling indicating potential defects in both effector and memory generation. We are currently exploring the mechanism of defective effector CD8 T cell generation in the absence of cell-intrinsic IL-27R signaling. Overall, these findings show a cell-intrinsic role for IL-27 signaling in the generation of a robust effector CD8 T cell response following influenza infection.
Abstract Tissue-resident memory CD8+ T cells (TRM) are strategically located in peripheral tissues, especially at mucosal surfaces, where they can provide protection against invading pathogenic microbes. In the lungs, TRM play a critical role in limiting disease and transmission of respiratory viruses; however, the heterogeneity present in TRM populations in the human lung remains largely unexplored. Here we show that human lungs harbor transcriptionally, epigenetically, and phenotypically distinct populations of memory CD8+ T cells expressing the tissue residency-associated markers CD69 and CD103. High-dimensional flow cytometry and single-cell RNA sequencing of memory CD8+ T cells isolated from human lungs shows that heterogeneity exists both between and within CD69+ CD103− and CD69+ CD103+ subsets, with transcriptional diversity among the CD69+ CD103− subset being most prominent. Single-cell ATACseq demonstrates that similar levels of epigenetic diversity exist between and within these different TRM subsets. However, flow cytometry and single-cell RNAseq of influenza- and SARS-CoV-2-specific lung TRM demonstrates that heterogeneity between CD69+ CD103− and CD69+ CD103+ subsets is largely eliminated when the cells are specific for a common antigen. Together, these data illuminate underappreciated and unexplored aspects of heterogeneity in TRM populations in humans. Supported by grants from NIH/NIAID (75N93019R00028) and NIH/NHLBI (R35 HL150803)
