Secondary Response Research Articles

Recruitment of epitope-specific T cell clones with a low-avidity threshold supports efficacy against mutational escape upon re-infection

Repetitive pathogen exposure leads to the dominant outgrowth of Tcell clones with high Tcell receptor (TCR) affinity to the relevant pathogen-associated antigens. However, low-affinity clones are also known to expand and form immunological memory. While these low-affinity clones contribute less immunity to the original pathogen, their role in protection against pathogens harboring immune escape mutations remains unclear. Based on identification of the TCR repertoire and functionality landscape of naive epitope-specific CD8+ Tcells, we reconstructed defined repertoires that could be followed as polyclonal populations during immune responses invivo. We found that selective clonal expansion is governed by clear TCR avidity thresholds. Simultaneously, initial recruitment of broad TCR repertoires provided a polyclonal niche from which flexible secondary responses to mutant epitopes could be recalled. Elucidating how Tcell responses develop "from scratch" is informative for the development of enhanced immunotherapies and vaccines.

Loss of HLA-DO function impairs the development of memory CD4 T cells

Abstract 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 vitroand in vivorecall 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

Responders and nonresponders to topical capsaicin display distinct temporal summation of pain profiles.

Topical application of capsaicin can produce an ongoing pain state in healthy participants. However, approximately one-third report no pain response (ie, nonresponders), and the reasons for this are poorly understood. In this study, we investigated temporal summation of pain (TSP) profiles, pain ratings and secondary hyperalgesia responses in responders and nonresponders to 1% topical capsaicin cream. Assessments were made at baseline and then during an early (ie, 15 minutes) and late (ie, 45 minutes) time points post-capsaicin in 37 healthy participants. Participants reporting a visual analogue scale (VAS) rating of >50 were defined as responders (n = 24) and those with <50 VAS rating were defined as nonresponders (n = 13). There was a facilitation of TSP during the transition from an early to the late time point post-capsaicin (P<0.001) and the development of secondary hyperalgesia (P<0.05) in the responder group. Nonresponders showed no changes in TSP or secondary hyperalgesia during the early and late time points. There was an association between baseline TSP scores and the later development of a responder or nonresponder phenotype (r = 0.36; P = 0.03). Receiver operating characteristic analysis revealed that baseline TSP works as a good response predictor at an individual level (area under the curve = 0.75). These data suggest that responders and nonresponders have different facilitatory pain mechanisms. The assessment of TSP may help to identify participants with stronger endogenous pain facilitation who may be more likely to respond to topical capsaicin.

MANGO: Inferring gene regulatory networks from single cell multiomics

Abstract Transcription factors (TFs) and their target gene regulatory networks (GRNs) control immune cell fate decisions during differentiation and responses to environmental cues and signals. To better understand how GRN control immune outcomes and are dysregulated in disease settings, we developed MANGO (Multiomics Aided Neural Graph Ontology), a computational method that integrates single cell gene expression and chromatin accessibility data to infer genome-wide, cell-type specific GRN. MANGO harnesses graph neural networks to generate representations of gene and regulatory link behavior and uses existing interpretable methods, like UMAPs, to visualize the GRN and gather insights about functional neighborhoods of regulators or programs of regulation. By scoring each regulatory interaction and aggregating those with common sources, TF activity can be quantified and compared between networks. We applied MANGO to a human PBMC dataset to define GRN and identify key regulatory, cell-type specific TFs, despite sometimes having low changes in expression. Additionally, we show that MANGO can distinguish cells by regulation network at single cell resolution by feeding each cell's expression and accessibility through its respective cell type's regulation network and projecting the resulting embedding in lower dimensions with a UMAP. Finally, MANGO was used to uncover memory B and T cell GRNs that prime rapid secondary immune responses. Given the increase in single cell multiome datasets, we anticipate MANGO will provide unique insights into GRN in normal and diseased immune responses, and ultimately give rise to patient specific understanding of the immune system and precision medicine. Supported by grants from NIH/NIAID (R01 AI148471) to CDS

Virtual Inertia Response and Frequency Control Ancillary Services From Hydrogen Electrolyzers

This paper presents the modeling foundations to study the capabilities of hydrogen electrolyzers (HEs) to provide frequency control ancillary services (FCAS), including virtual inertia and primary and secondary frequency response. To do so, we propose a general, unified HE dynamic model for the electrolyzer stack circuit, power-electronics interface (PEI), and relevant converter-level control loops. The equivalent circuit of the stack is derived from its current transfer function, with poles and zero obtained from its step response characteristics (e.g., rise&#x002F;settling time). The stack model also considers relevant physical nonlinearities and downstream hydrogen buffer&#x002F;process operational constraints. The PEI control loops account for stack model parameters, hydrogen production operational constraints, stack temperature dynamics, and active power reference generation strategy for contingency and regulation frequency support services. Further, we propose a virtual synchronous machine (VSM) control approach to study the VSM HE capabilities to also provide virtual inertia response. We apply the modeling to both alkaline and proton exchange membrane (PEM) technologies, including design of appropriate control schemes. Finally, we assess the FCAS performance of alkaline and PEM HEs via dynamic simulation of the Australian south-east interconnection in a 50&#x0025;-renewable scenario, also discussing comparison and cooperation with battery energy storage systems.

National Early Warning Scores Following Emergency Hospital Transfer: Implications for Care Home Residents.

Care home residents have high rates of hospital admission. The UK National Early Warning Score (NEWS2) standardizes the secondary care response to acute illness. However, the ability of NEWS2 to predict adverse health outcomes specifically for care home residents is unknown. This study explored the relationship between NEWS2 on admission to hospital and resident outcome 7days later. Repeated cross-sectional study. Data on UK care home residents admitted to 160 hospitals in two 24-hour periods (2019 and 2020). Chi-squared and Kruskal-Wallis tests, and multinomial regression were used to explore the association between low (score ≤2), intermediate (3-4), high (5-6), and critically high (≥7) NEWS2 on admission and each of the following: discharge on day of admission, admission and discharge within 7days, prolonged hospital admission (>7days), and death. From 665 resident admissions across 160 hospital sites, NEWS2 was low for 54%, intermediate for 18%, high for 13%, and critically high for 16%. The 7-day outcome was 10% same-day discharge, 47% admitted and subsequently discharged, 34% remained inpatients, and 8% died. There is a significant association between NEWS2 and these outcomes (P < .001). Compared with those with low NEWS2, residents with high and critically high NEWS2 had 3.6 and 9.5 times increased risk of prolonged hospitalization [relative risk ratio (RRR) 3.56; 95% CI 1.02-12.37; RRR 9.47; CI 2.20-40.67], respectively. The risk of death was approximately 14 times higher for residents with high NEWS2 (RRR 13.62; CI 3.17-58.49) and 54 times higher (RRR 53.50; CI 11.03-259.54) for critically high NEWS2. Higher NEWS2 measurements on admission are associated with an increased risk of hospitalization up to 7days duration, prolonged admission, and mortality for care home residents. NEWS2 may have a role as an adjunct to acute care decision making for hospitalized residents.

Simulated lab module to teach vaccine biology: From disease transmission to vaccine efficacy, all in a matter of three lab sessions.

Abstract Since the COVID-19 pandemic, it has become very important to teach undergraduate students at all levels, about disease transmission and vaccine biology. Just using lectures and reading assignments on these topics often will not be very effective for the student learning. In resource limited undergraduate teaching environment, it is often not possible to use advanced immunology lab techniques. To combat this problem, a lab-module was incorporated into the undergraduate immunology course. In this module, students will follow the process of vaccine development from the identification of a novel disease through the creation and clinical testing of a vaccine This hands-on module was run over the course of three-week lab period, including important concepts of disease transmission, disease diagnosis, mRNA vaccine development, vaccine efficacy and herd immunity. All the exercises included paper-simulations and simulated samples using sodium hydroxide pellets. Overall concepts of innate and adaptive immunity, and detailed concepts like antigenic epitopes, primary and secondary immune responses, and booster doses are emphasized. Students also learn to design mRNA vaccines using gene sequences of Coronavirus spike protein. They also learn principles of ELISA and phases of clinical trial of a novel vaccine. A survey was conducted at the end of the module to evaluate the student learning and student responses to this module. 90% of the students in class were very pleased with this module and stated that this module helped them consolidate their understanding of vaccines. This module can also be modified to be used in a molecular biology or upper-level microbiology courses, with minimum resources. None

Imaging of lung resident memory CD8 +T cells reveals antigen-specific early protective responses during secondary infection

Abstract Tissue-resident memory T (T RM) cells are poised at mucosal sites to confer robust protection against secondary infection. In contrast to skin and intestine, lung T RMcells are short lived with a decline in number coinciding with a decline in protective immunity against subsequent heterosubtypic influenza infections. Despite this causal relationship, the localization, dynamics and function of lung Trm during secondary responses remains poorly defined. To address this, we used two-photon intravital microscopy of lungs to assess in real time the spatiotemporal dynamics of lung T RMcells during recall infection. At a memory time point, lung T RMcells (representing both CD103 +and CD103 −subsets) exhibit significantly lower motility and less displacement compared to an CD8 +effector T cells at an earlier time point post infection. Depletion of circulating CD8 +T cells using a low dose antibody administered systemically led to enrichment of lung T RMcells that exhibited slow to moderate speed scanning patterns. A fate mapping model for CD103 +T RMcells specifically revealed limited speed (&amp;lt;5 um/min) and patrolling behavior compared to CD103 −T RMcells. Within 24 hr of rechallenge, CD103 +T RMcells co-localized with infected cells and by 48 hr, lung-resident CD8 +T cells exhibit significantly lower motility with recruitment and clustering in infected regions in an antigen-specific manner. Protective immune responses by lung T RMcells is further accompanied by CXCR3-dependent migration of dendritic cells to draining lymph nodes as well as recruitment of inflammatory monocytes. Overall, these data unravel the spatiotemporal dynamics of lung T RMcells and interactions with recruited immune cells for early protective immunity. Supported by grants from NIH (GM134880, AI42767, AI114543, AI151183, AI167249, the Holden Cancer Center at the University of Iowa and its National Cancer Institute Award P30CA086862

SARS-CoV-2 Envelope Protein Tolerizes Macrophage Response to Secondary Inflammatory Stimuli

Abstract Exposure of innate immune cells to bacteria or viruses may result in epigenetic rewiring that either enhances or weakens innate immune responses to a secondary challenge. One primary mechanism of innate immune cell function is through toll-like receptors (TLRs) that sense a variety of bacterial and viral molecules and cause immune activation. SARS-CoV-2 Envelope protein (E protein) has been shown to mediate innate immune activation, through engagement of TLR2. In this study, we determined the secretome of chemokines, cytokines and growth factors from monocytes in response to E protein stimulation and compared this response with other viral antigens and TLR agonists. Next, we stimulated monocytes with E protein followed by secondary stimulation with lipopolysaccharide (LPS) on monocyte-derived macrophages (MDM) 1 week later. Reduced expression of CCL3, CCL4, CCL5, CXCL10, TNF-A, and IL-12 upon secondary LPS or autologous E protein stimulation were statistically reflecting a more tolerant MDM state that is imprinted after E protein exposure. Finally, we demonstrated that immunizing neonatal mice with SARS-CoV-2 E protein induced proinflammatory cytokine secretion and lung tissue inflammation pathology and demonstrated a long-term impact on immune cell function and secondary response to LPS. Thus, E protein induces macrophage proinflammatory networks that lead to tissue inflammation, but also induce a more tolerant state after return to a resting state that could impact the ability to respond to secondary infection. Further understanding of the molecular pathways that drive this immune activation and tolerance could identify treatment targets to restore immunity after SARS-CoV-2 infection.

Human Memory T cells show differential metabolic preferences

Abstract Metabolic reprogramming regulates survival and function of immune cells, including T cells. Previous studies revealed that memory T cells are quiescent populations that primarily use OXPHOS for their metabolic needs. However, antigen rechallenge induces metabolic reprogramming including the engagement of aerobic glycolysis in which glucose is converted into lactate rather than oxidized into mitochondria. Here, we compared the metabolic characteristics of human naïve and memory T cells (TCM, TEM, and TEMRA) that are characterized by the cell surface proteins CCR7, and CD45RA. T cells were enriched from human PBMC samples, and then the subsets were sorted by flow cytometry. RNA-seq libraries constructed from each population were sequenced. Comparison of metabolism related transcript levels of the subsets revealed a distinct metabolic state in each cell type. Seahorse assays, mitochondrial staining flow cytometry assays and the SCENITH (Single Cell ENergetIc metabolism by profil Ing Translation in Hibition) assays were performed to assess the metabolisms of the individual populations. Our data indicated that resting CD4 and CD8 memory T cell populations have higher glycolytic and fatty acid capacity than the naïve T cells, consistent with the RNA-seq data. Resting naïve T cells depend on mitochondrial ATP production more than the memory T cells. However, when the cells are stimulated, the glycolytic capacity increased in all cell types and mitochondrial dependence decreased without a significant difference between subsets. Overall, our data highlights how memory T cell subsets retain greater metabolic capacity than naive T cell, potentially contributing to more efficient secondary responses.

Anti-CD3 inhibits circulatory and tissue-resident memory CD4 T cells that drive asthma exacerbations in mice.

Exacerbations of asthma are thought to be strongly dependent on reactivation of allergen-induced lung tissue-resident and circulatory memory CD4 T cells. Strategies that broadly inhibit multiple T cell populations might then be useful to limit asthma. Accordingly, we tested whether targeting CD3 during exposure to inhaled allergen could prevent the accumulation of lung-localized effector memory CD4 T cells and block exacerbations of asthmatic inflammation. House dust mite-sensitized and repetitively challenged BL/6 mice were transiently treated therapeutically with F(ab')2 anti-CD3ε and memory T cell responses and lung inflammation were assessed. PBMCs from HDM-allergic donors were examined for the effect of anti-CD3 on expansion of allergen-reactive T cells. Allergen-sensitized mice undergoing exacerbations of asthma were protected from lung inflammation by transient therapeutic treatment with F(ab')2 anti-CD3. Regardless of whether sensitized mice underwent a secondary or tertiary recall response to inhaled allergen, anti-CD3 inhibited all phenotypes of effector memory CD4 T cells in the lung tissue and lung vasculature by 80%-90%, including those derived from tissue-resident and circulatory memory T cells. This did not depend on Treg cells suggesting it was primarily a blocking effect on memory T cell signaling. Correspondingly, anti-CD3 also strongly inhibited proliferation of human allergen-reactive memory CD4 T cells from allergic individuals. In contrast, the number of surviving tissue-resident memory CD4 T cells that were maintained in the lungs at later times was not robustly reduced by anti-CD3. Anti-CD3 F(ab')2 administration at the time of allergen exposure represents a viable strategy for limiting the immediate activity of allergen-responding memory T cells and asthma exacerbations.

Local Complement Contributes to Pathogenic Activation of Lung Endothelial Cells in SARS-CoV-2 Infection.

Endothelial dysfunction and inflammation contribute to the vascular pathology of coronavirus disease (COVID-19). However, emerging evidence does not support direct infection of endothelial or other vascular wall cells, and thus inflammation may be better explained as a secondary response to epithelial cell infection. In this study, we sought to determine whether lung endothelial or other resident vascular cells are susceptible to productive severe acute respiratory syndrome coronavirus 2(SARS-CoV-2) infection and how local complement activation contributes to endothelial dysfunction and inflammation in response to hypoxia and SARS-CoV-2-infected lung alveolar epithelial cells. We found that ACE2 (angiotensin-converting enzyme 2) and TMPRSS2 (transmembrane serine protease 2) mRNA expression in lung vascular cells, including primary human lung microvascular endothelial cells (HLMVECs), pericytes, smooth muscle cells, and fibroblasts, was 20- to 90-fold lower compared with primary human alveolar epithelial type II cells. Consistently, we found that HLMVECs and other resident vascular cells were not susceptible to productive SARS-CoV-2 infection under either normoxic or hypoxic conditions. However, viral uptake without replication (abortive infection) was observed in HLMVECs when exposed to conditioned medium from SARS-CoV-2-infected human ACE2 stably transfected A549 epithelial cells. Furthermore, we demonstrated that exposure of HLMVECs to conditioned medium from SARS-CoV-2-infected human ACE2 stably transfected A549 epithelial cells and hypoxia resulted in upregulation of inflammatory factors such as ICAM-1 (intercellular adhesion molecule 1), VCAM-1 (vascular cell adhesion molecule 1), and IL-6 (interleukin 6) as well as complement components such as C3 (complement C3), C3AR1 (complement C3a receptor 1), C1QA (complement C1q A chain), and CFB (complement factor B). Taken together, our data support a model in which lung endothelial and vascular dysfunction during COVID-19 involves the activation of complement and inflammatory signaling and does not involve productive viral infection of endothelial cells.

A finite element model of contusion spinal cord injury in rodents

Traumatic spinal cord injuries result from high impact forces acting on the spine and are proceeded by an extensive secondary inflammatory response resulting in motor, sensory, and autonomic dysfunction. Experimental in vivo traumatic spinal cord injuries in rodents using a contusion model have been extremely useful in elucidating the underlying pathophysiology of these injuries. However, the relationship between the pathophysiology and the biomechanical factors is still not well understood. Therefore, the aim of this research is to provide a comprehensive analysis of the biomechanics of traumatic spinal cord injury in a rat contusion model. This is achieved through the development and validation of a finite element model of the thoracic rat spinal cord and subsequently simulating controlled cortical impact-induced traumatic spinal cord injury. The effects of impactor velocity, depth, and geometry on the resulting stresses and strains within the spinal cord are investigated. Our results show that increasing impactor depth results in larger stresses and strains within the spinal cord tissue as expected. Further, for the first time ever our results show that impactor geometry (spherical versus cylindrical) plays an important role in the distribution and magnitude of stresses and strains within the cord. Therefore, finite element modelling can be a powerful tool used to predict stresses and strains that occur in spinal cord tissue during trauma.

Engineered murine IL-21-secreting leukemia cells induce granzyme B+ T cells and CD4+CD44+CD62L- effector memory cells while suppressing regulatory T cells, leading to long-term survival.

We have explored the use of an IL-21 cell-based anti-leukemia treatment in a mouse model of acute lymphoblastic leukemia. 70Z/3 leukemia cells, engineered to secrete IL-21 and injected into the peritoneum of syngeneic mice, induced a strong anti-leukemia response resulting in 100% survival. Mice that mounted an IL-21-induced anti-leukemia immune response were immune to the parent cell line (no IL-21) when rechallenged.Above a certain threshold, IL-21 secretion correlated with improved survival compared to mice injected with parent 70Z/3 cells. IL-21 was detected in serum with peak levels on day 7, correlating with the maximum expansion of IL-21-secreting 70Z/3 cells which subsequently were eliminated. Mice injected with IL-21-secreting leukemia cells had elevated numbers of granzyme B+ CD4+ and CD8+ T cells in the peritoneum, compared to mice injected with the parent cell line. Regulatory T cells, which increased greatly in 70Z/3-injected mice, failed to do so in mice injected with IL-21-secreting cells. Upon rechallenge, IL-21-primed mice went through a secondary immune response, primarily requiring CD4+ T cells, triggering a significant increase of CD4+CD44+CD62L- effector memory T cells. Adoptive transfer of T cells from IL21-primed/rechallenged hosts into naïve mice was successful, indicating that IL-21-primed antigen-experienced T cells convey immunity to naïve mice.Our study shows that delivery of IL-21 in a cell-based anti-leukemia protocol has the potential to induce a potent immune response leading to cancer elimination and long-term immunity-properties which make IL-21 an attractive candidate for cancer immunotherapy. Protecting against tumor antigens as well as improving cancer immunity is justified, as current strategies are limited.

Designing of multi-epitope peptide vaccine against Acinetobacter baumannii through combined immunoinformatics and protein interaction-based approaches.

Acinetobacter baumannii is one of the major pathogenic ESKAPE bacterium, which is responsible for about more than 722,000 cases in a year, globally. Despite the alarming increase in multidrug resistance, a safe and effective vaccine for Acinetobacter infections is still not available. Hence in the current study, a multiepitope vaccine construct was developed using linear B cell, cytotoxic T cell, and helper T cell epitopes from the antigenic and well-conserved lipopolysaccharide assembly proteins employing systematic immunoinformatics and structural vaccinology strategies. The multi-peptide vaccine was predicted to be highly antigenic, non-allergenic, non-toxic, and cover maximum population coverage worldwide. Further, the vaccine construct was modeled along with adjuvant and peptide linkers and validated to achieve a high-quality three-dimensional structure which was subsequently utilized for cytokine prediction, disulfide engineering, and docking analyses with Toll-like receptor (TLR4). Ramachandran plot showed 98.3% of the residues were located in the most favorable and permitted regions, thereby corroborating the feasibility of the modeled vaccine construct. Molecular dynamics simulation for a 100 ns timeframe further confirmed the stability of the binding vaccine-receptor complex. Finally, in silico cloning and codon adaptation were also performed with the pET28a (+) plasmid vector to determine the efficiency of expression and translation of the vaccine. Immune simulation studies demonstrated that the vaccine could trigger both B and T cell responses and can elicit strong primary, secondary, and tertiary immune responses. The designed multi-peptide subunit vaccine would certainly expedite the experimental approach for the development of a vaccine against A. baumannii infection.

Abstract 4162: T-cell metabolic profiling with bioluminescent assays

Abstract Cellular energy metabolism is crucial for cell survival and the balance of metabolic pathways determines the health of the cell. Determining and understanding metabolic profiles of cells can aid in overall insight of cell health and improvement of cell therapeutics, especially for immune interventions. T-cells actively regulate their metabolic profile from quiescent to a highly metabolically active state after activation. Activation rewires the cell to drive glycolysis and oxidative phosphorylation for production of reducing power and ATP for cell growth and proliferation. A small population of these cells then transition to a more quiescent state and persist as memory cells to aid in secondary responses. Although the trend of T-cell metabolism after activation has been studied, the method of investigation is cumbersome and lacks depth of information of overall cell health. To address this, we developed a bioluminescence NAD(P)H core technology and applied it to build a set of cell-based assays covering diverse metabolic pathways including glucose, glycogen, and amino acid metabolism, and the TCA cycle. Here we utilize those assays to investigate changes in metabolic profiles of naïve, effector, and memory T-cells as they transition from a state of oxidative phosphorylation to increased glycolytic rates, dependence on different fuel consumption and storage, including an increase in fatty acid oxidation and glycogen accumulation. We demonstrate flexibility of the assays for detecting multiple metabolites from the same sample and monitoring metabolite changes over time by assaying multiple samples. We show that the assays are amenable to various volumes (96-,384-well plates) and low cell numbers. For example, activity of dehydrogenases involved in glycolysis, pentose phosphate and TCA cycles can be detected with less than 400 cells/well. In conclusion, understanding the processes governing dynamic metabolic changes of T-cells during their transition from naïve to activated to memory phenotypes is required for next-generation cell therapy development. The developed bioluminescence cell-based assays provide the sensitivity, throughput and robustness required for rapid evaluation of such changes. Citation Format: Maggie Bach, Natasha Karassina, Julia Gilden, Donna Leippe, Mike Valley, Kayla Sylvester, Jolanta Vidugiriene. T-cell metabolic profiling with bioluminescent assays. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4162.

Abstract 3464: Dynamics of resident memory (Trm) cell responses between skin and draining lymph nodes during primary and recall responses to melanoma

Abstract Tissue resident memory (TRM) cells reside durably in tissues where they serve as a vital component of protective anti-tumor immunity. In mice, TRM cells are defined by their expression of tissue retention markers such as CD69 and CD103, and lack of egress markers such as CD62L, CCR7 and S1PR1. We previously showed that depletion of regulatory T cells induces the generation of protective CD8 Trm cells in skin and draining LNs of B16 melanoma tumor-excised mice. While tumor specific Trm cells were shown to stably reside in tissues at steady state, little is known about their migration between tissue compartments and circulation during primary and recall responses to melanoma. Using gp100-specific transgenic pmel CD8 T cells expressing a Kaede-GFP photoconvertible marker, we investigated the trafficking patterns of tumor Ag specific CD8+ T-cells that were labeled in situ by exposure to UV light in the skin. During primary tumor growth, we observed pronounced movement of antigen-experienced pmel cells through skin to tumor-draining LNs. This trafficking behavior could be inhibited by treatment with FTY720, indicating its S1PR-dependence. Such movement was no longer evident at memory timepoints. However, re-challenge with B16 cells in the dermis induced rapid pmel T cell egress to both LNs and circulation. Recallenge-mediated Trm cell skin egress was confirmed using anti-Thy1.1 monoclonal antibody to deplete circulating and lymphoid memory, while preserving skin TRM. Here we found that skin TRM proliferate and transiently increase expression of Tbet, but go on to generate a durable secondary Trm response in lymph nodes. These data highlight T cell movement between tissues during Trm programming and recall responses to cancer, and shed light on a potential mechanism for the generation TRM populations in tumor-draining lymph nodes. Moreover, our results highlight the potential for retrograde movement of Trm responses from peripheral tissue reservoirs to participate more broadly in systemic anti-tumor immunity. Citation Format: Cameron Messier, Nikhil Khatwani, Aaron Hawkes, Yuyang (Poppy) Huang, Mary Jo Turk. Dynamics of resident memory (Trm) cell responses between skin and draining lymph nodes during primary and recall responses to melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3464.

Abstract 3591: Does the metastatic immunoenvironment determine poor immunotherapy responses

Abstract Despite new treatments, cancer remains the second leading cause of death worldwide with breast cancer being the most common type. Of these deaths, the majority are due to the formation of distant metastases. While new immunotherapies have shown some success in primary tumors, their efficacy in combating metastases has been lacking. Thus we aim to investigate the hypothesis that immune responses at the metastatic site contribute to limitations of current therapies.Within the last decade, new microscopy technologies now enable real-time visualization of immune responses at primary and secondary sites. Here, we present use of these microscope technologies, along with the development of novel probes to detect apoptotic cancer cells, to investigate the success of checkpoint inhibitors in the primary and secondary sites. We validate, in multiple in vitro and in vivo assays (including intravital imaging), that fluorescent cyclic amphipathic peptide probes specifically label cells undergoing therapy-induced cancer death. In addition, we introduce a newly developed fluorescent, enzyme-activatable, chemokine-conjugate capable of targeting defined macrophage subsets in live tumors. This chemokine-conjugate exploits the high expression of chemokine receptors (e.g. CCR2) and the activity of cysteine cathepsins in tumor associated macrophages to selectively target and eradicate them without affecting other macrophages or immune cells (e.g., neutrophils, T cells, B cells). This research will help to elucidate the role of combination therapy on the metastatic foci and will provide the first insights into the benefit and limitations of combination therapy on secondary site immune responses. Citation Format: Nicole D. Barth, Floris J. van Dalen, Camille Duran, Utsa Karmakar, Lorena Mendive-Tapia, Takanori Kitamura, Martjin Verdoes, John S. Condeelis, Maja Oktay, Marc Vendrell, Valerie Brunton, David Entenberg. Does the metastatic immunoenvironment determine poor immunotherapy responses. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3591.

The NAC transcription factor family in Eucommia ulmoides: Genome-wide identification, characterization, and network analysis in relation to the rubber biosynthetic genes.

The NAC transcription factor family is a large plant gene family, participating in plant growth and development, secondary metabolite synthesis, biotic and abiotic stresses responses, and hormone signaling. Eucommia ulmoides is a widely planted economic tree species in China that can produce trans-polyisoprene: Eucommia rubber (Eu-rubber). However, genome-wide identification of the NAC gene family has not been reported in E. ulmoides. In this study, 71 NAC proteins were identified based on genomic database of E. ulmoides. Phylogenetic analysis showed that the EuNAC proteins were distributed in 17 subgroups based on homology with NAC proteins in Arabidopsis, including the E. ulmoides-specific subgroup Eu_NAC. Gene structure analysis suggested that the number of exons varied from 1 to 7, and multitudinous EuNAC genes contained two or three exons. Chromosomal location analysis revealed that the EuNAC genes were unevenly distributed on 16 chromosomes. Three pairs of genes of tandem duplicates genes and 12 segmental duplications were detected, which indicated that segmental duplications may provide the primary driving force of expansion of EuNAC. Prediction of cis-regulatory elements indicated that the EuNAC genes were involved in development, light response, stress response and hormone response. For the gene expression analysis, the expression levels of EuNAC genes in various tissues were quite different. To explore the effect of EuNAC genes on Eu-rubber biosynthesis, a co-expression regulatory network between Eu-rubber biosynthesis genes and EuNAC genes was constructed, which indicated that six EuNAC genes may play an important role in the regulation of Eu-rubber biosynthesis. In addition, this six EuNAC genes expression profiles in E. ulmoides different tissues were consistent with the trend in Eu-rubber content. Quantitative real-time PCR analysis showed that EuNAC genes were responsive to different hormone treatment. These results will provide a useful reference for further studies addressing the functional characteristics of the NAC genes and its potential role in Eu-rubber biosynthesis.

Chronic stress negatively impacts wound healing, welfare, and stress regulation in internally tagged Atlantic salmon (Salmo salar).

The desire to understand fish welfare better has led to the development of live monitoring sensor tags embedded within individuals for long periods. Improving and understanding welfare must not come at the cost of impaired welfare due to a tag's presence and implantation process. When welfare is compromised, the individual will experience negative emotions such as fear, pain, and distress, impacting the stress response. In this study, Atlantic salmon (Salmo salar) underwent surgical implantation of a dummy tag. Additionally, half of this group was introduced to daily crowding stress. Both groups and an untagged group were followed for 8weeks using triplicate tanks per group. Sampling took place once a week, and where stress was given, it was conducted 24h before sampling. Stress-related measurements were taken to understand if tagging caused chronic stress and explore the chronic stress response and its impact on wound healing. Primary stress response hormones measured included CRH, dopamine, adrenocorticotropic hormone, and cortisol. Secondary stress response parameters measured included glucose, lactate, magnesium, calcium, chloride, and osmolality. Tertiary stress response parameters measured included weight, length, and five fins for fin erosion. Wound healing was calculated by taking the incision length and width, the inflammation length and width, and the inside wound length and width. The wound healing process showed that stressed fish have a larger and longer-lasting inflammation period and a slower wound healing process, as seen from the inside wound. The tagging of Atlantic salmon did not cause chronic stress. In contrast, daily stress led to an allostatic overload type two response. ACTH was elevated in the plasma after 4weeks, and cortisol followed elevation after 6weeks, highlighting a breakdown of the stress regulation. Fin erosion was elevated alongside cortisol increase in the stressed group. This data suggests that tagging previously unstressed fish in a controlled environment does not negatively affect welfare regarding stress responses. It also indicates that stress delays wound healing and increases the inflammatory response, highlighting how continued stress causes a breakdown in some stress responses. Ultimately, the tagging of Atlantic salmon can be successful under certain conditions where proper healing is observed, tag retention is high, and chronic stress is not present, which could allow for the possible measurement of welfare indicators via smart-tags.