bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of the cell-mediated immune response to the NIH tetravalent dengue vaccine in an endemic population
Authors: Torrey Jessica , Nadeem Iqra , Gray Lauren , Omidi Nazinin , Fernandez Stefan , Whitehead Steve , Currier Jeffrey R , Friberg Heather
Published: August 2023

Dengue is now endemic in more than 100 countries, with Southeast Asian and Pacific Island regions being the most seriously affected. The Department of Defense has long recognized the threat of dengue and has made great contributions to understanding it. The ideal dengue vaccine will provide long-lasting protection against all four serotypes of dengue virus (DENV1-4) and will be effective in both endemic and non-endemic populations. Several tetravalent dengue vaccines are either licensed or in advanced stages of clinical development, including the live attenuated vaccine candidate developed by the NIH. The purpose of this study is to characterize the cellular immune responses to the NIH vaccine, with particular attention to the activation and memory profiles of DENV-specific T and B cell populations. Our hypothesis is that the NIH vaccine induces a durable and tetravalent DENV-specific adaptive immune response, which varies in its magnitude and phenotype over time (3 years post-vaccination) and across age strata. To assess the magnitude, breadth, and evolution of DENV-specific T and B cells following vaccination, we use a suite of different assays, including IFNγ ELISpot, memory B cell fluorospot, and flow cytometry-based assays. This study evaluates the ability of the NIH vaccine to induce durable and balanced immune responses to all four DENV serotypes, and how that compares to naturally acquired immunity.
Innate Immune Training Enhances the Reactivation of Latently Infected HIV-1 from Monocytic Cell lines
Authors: James Heera , Forbes Greta E , John Sinu P , Fraser Iain D C
Published: August 2023

HIV virus can persist in a latent but activatable chronic state in resting CD4+ T-cells and macrophages that are more persistent, leading to the requirement of lifelong combination antiretroviral therapy (cART). The latency of HIV-1 in macrophage cells is associated with increased chromatin condensation induced by histone methylation at HIV-1 integration sites. Innate immune training of macrophages has been shown to relieve chromosome condensation through epigenetic rewiring. We hypothesized that training of macrophages could enhance the reactivation of HIV-1 by opening the chromatin to facilitate transcription in response to Latency Reversing Agents (LRAs). We used beta-glucan, Syk inhibitor and Rutaecarpine to train the THP89GFP cell line, which is an experimental model for latently infected HIV-1 monocytes. We found that these trained THP89GFP cells have higher transcription of HIV-1 specific genes in response to PMA stimulation. To check the epigenetic changes associated with training in the proviral HIV-1 DNA of THP89GFP, we performed a chromatin immunoprecipitation for the histone mark, H3K27Ac. Consistent with the higher induction of transcription of HIV-1 genes, we observed that multiple regions of the HIV-1 LTR had higher deposition of H3K27Ac in the trained macrophages. Our studies thus show that induction of trained innate immunity may be a viable approach to the reactivation of latently infected HIV-1. This finding supports the hypothesis that innate immune training stimuli could be developed as novel candidates for enhancing reactivation of latently infected HIV-1, which may facilitate elimination of macrophage reservoirs.

This work was supported by the Intramural Research Program of NIAID, NIH

This work was supported by the Intramural Research Program of NIAID, NIH and Office of AIDS research.
Longitudinal analysis of the LA-SPARTA cohort of high-risk individuals after SARS-CoV-2 infection and vaccination
Authors: Jenkins Meagan M , Tran Donna Phan , Flores Evelyn A , Kupferwasser Deborah , Pickering Harry , Zheng Ying , Gjertson David W , Ross Ted M , Schaenman Joanna M , Miller Loren G , Yeaman Michael R , Reed Elaine F
Published: August 2023

Questions remain regarding correlates of risk and immune protection against SARS-CoV-2 infection and vaccination response. Thus, we prospectively enrolled 200 participants with a high risk of SARS-CoV-2 occupational exposure. Longitudinal analysis of participant exposure risks, vaccination/infection status, and symptoms were assessed at 3, 6, and 12 months, with blood and saliva collection. Quantitative ELISA assay was used to assess the serological response to the SARS-CoV-2 spike holoprotein (S), S-receptor binding domain (RBD) and nucleocapsid protein (NP). 40 of 200 (20%) participants were found to be infected via serology. Equal incidence of infection was found in healthcare and non-healthcare occupations. In those infected, only 79.5% seroconverted for NP, and 11.5% were unaware they had been infected. The antibody response to S was greater than to RBD. Additionally, Hispanic ethnicity was associated with 2-fold greater incidence of infection despite vaccination. Booster vaccination significantly increased the avidity of RBD-specific responses to variant spike proteins. No differences in avidity were found if a participant was boosted via vaccination or natural infection. Collectively, our results indicate that antibody quality and holoprotein response may reveal meaningful determinants of protection beyond RBD antibody titer alone. Additionally, determinants of infection risk may include race/ethnicity, and remain independent of comorbidity and humoral response in this cohort.

This study received funding from Center for Influenza Vaccine Research for High Risk Populations- CIVIC 75N93019C00052 (EFR), Lundquist Institute Institutional Funds (MRY), UCLA W. M. Keck Foundation COVID 19 Research Award Program (EFR), and the Community Engagement Alliance Against COVID-19 (CEAL) (MRY, EFR). The project described was supported by the National Center for Advancing Translational Sciences through the UCLA CTSI Grant UL1TR001881. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH or other funding sources. MMJ is supported by the NIH grants TL 1 DK132768 and U2C DK129496.
The Dendritic Cell Strikes Back In Coxsackievirus A10 Infection
Authors: Zhang Xin , Liu Longding
Published: August 2023

Coxsackievirus A10 (CV-A10) is a major causative pathogen of hand, foot, and mouth diseases (HFMDs). Currently, there is no effective vaccine for CV-A10. Dendritic cells (DCs) support antiviral immunity by linking innate and adaptive immune responses. DCs stimulated with different kinds of antigens have been used in recent vaccine studies for cancer with promising results so far. Here we examined DC dynamics during CV-A10 infection to uncover how they exert their functions and elucidate the significance and value of assessing the immunogenicity and immunoprotection of dendritic cell vaccination candidates on CV-A10-infected rhesus monkeys. We found that, dendritic cells expressing the surface marker CD11c showed an abnormal biological phenotype, including the altered transcription of various immune signaling molecules and inflammatory factors associated with innate immunity after viral replication. Furthermore, these CV-A10-infected dendritic cells have the capacity to stimulate the proliferation of T cells and to enhance the release of certain functional cytokines. An adaptive immune response induced by the back-transfusion of CV-A10-infected dendritic cells was observed in donor neonatal rhesus monkeys. Collectively, these data proposed that CV-A10 infection induced a strong innate immune response in rhesus monkey DCs, performing the function of the antigen-presenting response of this cell, which may provide helpful insights into the pathogenesis and vaccine research of CV-A10 in human.
PIM kinases are essential for CD8 T cell effector function and metabolism during chronic viral infection
Authors: Brown Ashley Kaye , Shen Jian , Volberding Peter J , Cui Weiguo
Published: August 2023

The production of IL-21 by CD4 T cells is one mechanism by which CD8 T cells can sustain their effector functions, including increased metabolic demand, during chronic viral infection. However, signaling cascades downstream of IL-21 that control effector function in CD8 T cells remain incompletely understood. One such set of targets are the PIM kinases. This family of serine/threonine kinases functions downstream of IL-21 via JAK-STAT signaling and are involved in diverse cellular processes. Treatment of activated CD8 T cells with IL-21 induces expression of PIM1 kinase and phosphorylation of its targets S6T372, 4EBPT37/42, and BADS112, which promote transcription and inhibit apoptosis. Using the lymphocytic choriomeningitis virus (LCMV) Clone 13 mouse model of chronic infection, we show that virus-specific effector CD8 T cells display the highest expression of Pim1 compared to exhausted and progenitor CD8 T cells at day 21 post-infection (p.i.). Infection of Pim1−/− mice with LCMV Clone 13 results in reduced virus-specific effector CD8 T cells and increased exhausted CD8 T cells at days 21–23 p.i. compared to wildtype controls. In addition, pharmacological inhibition of PIM kinases with AZD1208, a pan-PIM kinase inhibitor, in activated CD8 T cells results in compromised mitochondrial metabolism, which is essential for effector CD8 T cell function. Overall, our results suggest that PIM kinases are critical for effector CD8 T cell differentiation, function, and metabolism in the control of chronic viral infection.

Supported by grants from NIH (R01 AI148403) and by an award from the Medical College of Wisconsin Center for Immunology.
Beta cell expression of ST8Sia6 protects from autoimmune diabetes in the murine NOD model
Authors: Choe Justin , Rajcula Matthew , Hammer Brady , Mai Elissa , Shapiro Michael , Shapiro Virginia
Published: August 2023

In this study, we leveraged immunomodulatory Siglec-sialic acid interactions to protect beta-cells against autoimmune rejection in Type 1 Diabetes (T1D). Our lab previously demonstrated that the sialyltransferase ST8Sia6 generates ligands that bind the Siglec-E receptor expressed on murine islet-resident macrophages. Here, mice were engineered to express ST8Sia6 in beta cells (βST mice), backcrossed to the non-obese diabetic (NOD) mouse model of T1D, and followed for disease. 60% of female littermates spontaneously developed disease by 300 days, compared to only 6% in female βST mice. Histologic analysis of pancreas sections from non-diabetic βST and littermate mice suggested a similar degree of immune infiltration in early life, but drastically reduced insulitis only in βST mice at later timepoints. This was not due to an absence of autoreactive T cells, as an anti-PD-L1 challenge resulted in similarly rapid induction of disease in both βST and littermate mice. Our genetic model allows for temporal control of ST8Sia6 expression in βST mice using the tet-off system. Treating our mice with doxycycline at 20 weeks of age resulted in persistent protection compared to age matched littermates, while the same treatment at 8 weeks of age led to similar incidence of diabetes between βST mice and littermate controls. Flow cytometric analysis of islet infiltrating immune cells from euglycemic mice at 14 weeks of age demonstrated reductions in short-lived effector CD8 T cells and increased thymically derived Tregs in βST mice compared to littermate controls. These results highlight the therapeutic potential of sialyltransferase mediated immune regulation and present a potential path toward improved curative approaches for T1D.

Supported by grants from NIH (5R21AI135858-02, 1F30DK127564)
α-GalCer administration expands virtual memory CD8+ T cells through IL-4 secretion
Authors: Xie Jia-Xun , Tai Chi , Lin Jr-Shiuan
Published: August 2023

Virtual memory CD8+ T (CD8+ TVM) cells, a unique subset of CD8+ T cells bearing memory phenotypes, exist in naïve and even in germ-free mice. Distinct from the conventional memory T cells, TVM cells are antigen-inexperienced but able to respond quickly in a cognate antigen-independent manner, thus could provide a broad range of innate-like protective effects. Our previous study showed that TVM cells could be expanded by type 2/interleukin-4 (IL-4) responses. Thus, we used α-galactosylceramide (α-GalCer) to selectively activate invariant natural killer T (iNKT) cells and create a type 2/IL-4 response, which in turn induced robust TVM cell expansion. We further demonstrated that CD1d molecule and NKT cells were necessary for α-GalCer-induced TVM cell expansion, but the TVM cell expansion was only partially dependent on IL-4 in this scenario. It remains unclear how IL-4 induces TVM cell expansion, and what other cytokine(s) may contribute to IL-4-independent TVM cell expansion upon α-GalCer treatment.

Among multiple cytokines induced by α-GalCer treatment, we found that in addition to IL-4, IL-15 and type I interferons (IFNs), which have been reported to participate in the development and expansion of TVM cells, were elevated in response to α-GalCer treatment in vivo. We further found that IL-4 and IL-15 but not type I IFNs drove TVM cell proliferation and expansion in vitro. Thus. we speculated that one of the factors that involves in IL-4-independent α-GalCer-induced TVM cell expansion may be IL-15. We are currently testing this hypothesis. Taken together, our results suggested that the activation of iNKT cells by α-GalCer expands TVM cell pool through IL-4 production, thus may regulate the ability of TVM cells to provide broad protection.

Supported by grants from MOST (109-2320-B-002-074-MY3), NHRI (EX110/111-11033SI) and NTU (111L7829), Taiwan
Resident memory T cells in the lymph node balance localized and systemic protection
Authors: Heim Taylor , Delclaux Ines , Schultz Austin , Lund Amanda
Published: August 2023

The endogenous mechanisms that protect against pathogen infection in the lymph node (LN) and LN metastasis during early tumor development remain poorly understood. Resident memory T cells (Trm), which do not circulate, provide highly localized protection to both pathogens and cancer, and correlate with better clinical outcomes. Here we track LN Trm development using models of cutaneous viral infection to determine the kinetics and requirements for their differentiation. We show that after viral skin infection, Trm form in the draining LN concurrent with their establishment at the initial site of infection in skin.

LN Trm formed specifically in LNs draining infected skin expressing cognate antigen and did not form in contralateral controls. These data supported a hypothesis that T cell egress out of infected skin through lymphatic vessels was necessary for LN Trm formation. Consistent with this hypothesis, LN Trm were absent from mice lacking dermal lymphatic vessels indicating that either fluid transport or cellular transport from the skin to the LN was necessary for the establishment of LN Trm. To decouple LN priming from T cell egress we resected infected skin days 5 and 20 post infection. Ear resection at 5 days post infection impaired Trm formation but circulating memory T cell responses were unaffected. We demonstrated that LN Trm are poised for cell killing as evidenced by constitutive expression of granzymes, and were able to protect against viral rechallenge. These findings indicate that LN Trm are poised to provide rapid, localized protection specific to the draining LN. These data may suggest that LN’s harbor populations capable of controlling regional cancer metastasis and pathogenic infections.

Supported by grants from NIH (R01CA238163)
Decreased memory persistence in Piezo1 deficient T cells ameliorates disease progression in autoimmune mouse models
Authors: Aguilar Alicia Virginia , Choi Sung Hee , Myers Jay , Kingsley Daniel T , Huang Alex Y
Published: August 2023

To mount a successful immune response, T cells must successfully (1) migrate from the bloodstream into secondary lymphoid organs (naïve) or inflamed tissue (effector), and (2) form an effective immunological synapse for activation. Although the molecular interactions of these processes have been well described, the emergence of novel biophysical approaches has revealed that both critical processes require mechanosensing. Previously we have demonstrated that the mechanosensitive cation channel, Piezo1, forms a complex with activated integrin LFA-1, an important regulator of T cell migration and T cell receptor-triggered T cell activation. Therefore, we hypothesized that Piezo1 deficiency in T cells would diminish T cell activation and homing. To investigate Piezo1’s role on T cell function, we have generated T cell-specific Piezo1 knockout mice (P1cKO) by crossing Piezo1-floxed mice to CD4-Cre mice. Initial studies in vitro showed that under polarizing conditions P1cKO T cells produce more Th1 and Th17 inflammatory cytokines. However, when challenged with experimental autoimmune encephalomyelitis (EAE) and Graft-vs-Host Disease (GVHD), P1cKO mice showed a significant decrease in disease phenotype. Moreover, adoptive transfer mouse models of EAE show that activated P1cKO T cell transfer fails to establish disease phenotype. Upon further investigation, we found similar numbers of P1cKO T cells at sites of inflammation early in disease, but there was a decreased persistence of effector and central memory P1cKO T cells. Taken together, our data suggests that Piezo1 plays an important in role in the development and maintenance of T cell memory pools, while potentially contributing to T cell activation and migration.

This project was supported by grants from the NIH (TL1 TR002549, T32 GM007250, T32 AI089474), the St. Baldrick’s Foundation Innovation Fund, the Theresia G. & Stuart F. Kline Family Foundation, and the Karen & Alan Krause Pediatric Immunotherapy Innovation and Education Fund.
TCR signal strength and proliferation affect Runx2 expression and histone modifications in CD8+ T cells
Authors: Michaels-Foster Mary Jessamine , Berg Leslie
Published: August 2023

Runx family transcription factors are crucial for the establishment of genetic programs during important cell differentiation stages. Although the roles of Runx1 and Runx3 in T cells have been extensively studied, the role of Runx2 has not fully been explored. T cell specific Runx2 deletion leads to a cell intrinsic defect in the longevity of the CD8+ memory population and decreased expression of memory markers like TCF1 and Eomes. As histone modifiers are known Runx2 binding partners, we propose that Runx2 may bind CD8+ memory associated gene promotors, allowing for the maintenance of chromatin as memory cells undergo homeostatic proliferation. To further explore the function of Runx2 in T cells, we utilize a Runx2 fl/fl CD4-Cre model of T cell specific Runx2 deletion. We use an in vitro CD8+ T cell stimulation assay in which we co-culture DC2.4 (a mouse dendritic cell line) cells loaded with ovalbumin peptide variants (N4, T4, and V4) with OT-1 CD8+ T cells for 72 hours to observe the effects of Runx2 expression changes. Additionally, we utilize chromatin analyses in this system to examine histone modifications associated with Runx2 activity. In accordance with previous data, Runx2 expression is inversely correlated with TCR signal strength, as cells stimulated with low dose/affinity peptides upregulate higher levels of Runx2. Additionally, cell proliferation dye labeling reveals that Runx2 expression increases as cells divide during stimulation and continues to increase as activated CD8+ T cells are rested post-stimulation. Preliminary data also suggest that histone modifications associated with poised chromatin vary with Runx2 expression, indicating that Runx2 may play a role in chromatin modifications in CD8+ memory T cells.
Single-cell multi-omic analysis reveals serum amyloid A3 restrains alternative activations in macrophages derived from different origins
Authors: Lin Jian-Da , Chou Tzu-Yin , Ye Yu-Zhen , Lin I-Jung , Lien Chia-Jung , Wang Zhih-Yuan , Yang Chen-Hsuan , Chao Pei-An , Chen Yen-Ting , Loke P’ng
Published: August 2023

Alternatively activated macrophages (AAMs) play pivotal roles in regulating metabolism, anti-helminth infections, and tissue homeostasis. AAMs derived from different cellular origins are functionally and phenotypically distinct, but the regulatory networks mediating these differences remain unclear. Here, we use a combination of CITE-seq (Cellular Indexing of Transcriptomes and Epitopes by Sequencing; a 128 or 198 barcode-antibody panels) and genetic fate mapping, to profile F4/80int CD206+ macrophage (Mφ) and F4/80hi CD206-tissue-resident Mφ derived from CX3CR1+ precursors, in mice during homeostasis and Interleukin 4 (IL-4) or Heligmosomoides polygyrus initiated alternative activation. The analyses reveal greater complexity in embryonic-derived F4/80hi CD206-tissue-resident Mφ than F4/80int CD206+ Mφ or Mφ with tissue-resident phenotype from monocyte origins during homeostasis. We show that serum amyloid A3 (Saa3) is exclusively expressed in tissue-resident Mφ than other immune cell types and negatively associated with typical M2 markers such as Retnla, Mrc1, and Mgl2 expression in Interleukin 4 (IL-4) or Heligmosomoides polygyrus (H. Poly) initiated AAMs. The significant expansion of Retnla+ Mφ is observed in H. Poly- or IL-4 treated Saa3−/− mice and presents less worm burden and more M2 metabolic phenotypes than WT mice. Notably, we also identified a Folr2+ tissue-resident Mφ population that constitutively expresses Retnla and is associated with leukocyte and T-cell activation. Hence, single-cell multi-omic analysis allowed us to identify novel regulatory networks and distinct Mφ properties from different origins during alternative activation.
Deciphering the role of platelet alpha granules in alveolar-capillary breakdown in severe malaria
Authors: Reed Jenna , Deckers Margot , Van den Steen Philippe , Petrey Aaron , Evavold Brian D , Lamb Tracey
Published: August 2023

Malaria is considered the deadliest disease in human history, killing hundreds of thousands annually. A hallmark of severe malaria is the breakdown of blood-organ barriers, such as in the lungs and brain, but the precise mechanism of how this occurs remains unknown. We determined that, unlike wildtype mice (C57BL6), mice with platelet alpha-granule deficiencies (Nbeal2−/−) did not die of malaria-associated acute respiratory distress syndrome (MA-ARDS) when infected with Plasmodium berghei NK65-Edinburgh (PbE). While wildtype mice exhibited significant alveolar-capillary breakdown by 6 days post-infection, as measured by alveolar protein concentrations and Evans blue permeation, Nbeal2−/− mice did not develop severe pathology. Flow cytometric analysis revealed that PbE-infected Nbeal2−/− mice had reduced CD8 T cell lung recruitment and activation compared to wildtypes. From this data, we concluded that a component of platelet alpha granules is crucial for activating CD8 T cells that subsequently damage the alveolar barrier. We hypothesized that the enzyme hyaluronidase 2 (HYAL2) is that crucial component and plays a 2-fold role: 1) that it directly contributes to barrier breakdown by cleaving hyaluronan (HA) from the endothelial glycocalyx (eGC), and 2) that the HA fragments released after eGC cleavage promote inflammation via CD44-binding. We found that HA is elevated in the plasma of infected mice. Preliminary data suggest that CD44 splice variants are differentially expressed in the lungs of naïve vs. PbE-infected mice, which could alter the binding kinetics with HA. Together, these data support the hypothesis that platelet HYAL2 is a critical mediator of MA-ARDS.
Novel murine model reveals an early role for pertussis toxin in disrupting neonatal immunity to Bordetella pertussis
Authors: Sedney Colleen , Caulfield Amanda , Dewan Kalyan , Callender Maiya , Manley Nancy , Harvill Eric T
Published: August 2023

The increased susceptibility of neonates to some pathogens has previously been attributed to an underdeveloped immune system. More recent data suggest neonates have effective protection against most pathogens but are particularly susceptible to those that target immune functions specific to neonates. Bordetella pertussis (Bp), the causative agent of “whooping cough”, causes more serious disease in infants attributed to its production of pertussis toxin (PTx). Problematically, the rapid development of adult immunity in mice has confounded our ability to study the interaction of this agent with neonatal immune components, such as virtual memory T cells which are prominent prior to the maturation of the thymus. Here, we examine the rapid change in susceptibility of young mice and define a period from five- to eight-days-old during which mice are much more susceptible to Bp than mice even a few days older. These more narrowly defined “neonatal” mice display significantly increased susceptibility to wild type Bp but very rapidly and effectively respond to and control Bp lacking PTx, more rapidly even than adult mice. The clearance of the PTx mutant correlates with the early accumulation of neutrophils and T cells and suggests a role for PTx in disrupting their accumulation. These results define an early age of extreme susceptibility to Bp, and demonstrate that the neonatal response can be more efficient than the adult response in eliminating bacteria from the lungs, but these neonatal functions are substantially blocked by PTx. This refined definition of “neonatal” mice may be useful in the study of other pathogens that predate neonates, and PTx may prove a particularly valuable tool for probing the poorly understood neonatal immune system.

This work was supported by grants AI156293 and AI159347 of the National Institutes of Health to Eric Harvill and AI139449 of the National Institutes of Health to Nancy Manley.
Targeted activation of IL-22 producing innate lymphoid cells enhances host resistance against Clostridioides difficile infection
Authors: Abt Michael C , Denny Joshua E , Maslanka Jeffrey , Mdluli Nontokozo V , Mears Kevin S
Published: August 2023

Clostridioides difficile, the most common hospital acquired pathogen in the United States, infects the gastrointestinal tract following perturbation of the intestinal microbiota causing debilitating, potentially fatal colitis. Primary C. difficile infection has a high recurrence rate following antibiotic treatment, emphasizing the need to develop alternative treatment strategies to reduce the burden on the healthcare system. Innate lymphoid cells (ILCs) are necessary for acute host defense following C. difficile infection and represent a promising therapeutic target to harness the patient’s innate immune defenses to limit disease. Here, using a murine model of C. difficile infection, we report that oral administration of Resiquimod (R848), a TLR-7 agonist previously reported to activate type-3 ILCs (ILC3s), protects mice from lethal C. difficile challenge. R848 treatment did not alter intestinal microbial communities or impact C. difficile burden. R848 treatment conveyed protection via activation of the host immune system in a TLR-7 dependent manner. R848-mediated protection was independent of T and B cells as well as type-I IFN signaling. Mice lacking ILCs, however, did not respond to R848 treatment and remained susceptible to infection, identifying these cells as the key targets of R848 activity. Last, R848 treatment robustly induced ILC3s in the large intestine to produce IL-22 and genetic ablation of IL-22 abrogated R848-mediated protection from C. difficile infection. Combined, these data identify an immunostimulatory molecule that activates the IL-22 producing ILC3s to support host defense against C. difficile infection without causing further dysbiosis of the intestinal microbiota.

NIH R01AI158830, R21AI164385
Understanding Asymptomatic Malaria: The Utility of Wild-Derived Mouse Diversity
Authors: Cornwall Douglas , Srey Mellina T , Heins Kate , Maloba Franklin , Evavold Birk , Olatunde Adesola , Fogang Balotin , Jensen Anne , Ayong Lawrence , Evavold Brian D , Lamb Tracey J
Published: August 2023

With over 200 million clinical cases per year leading to over 600,000 deaths, malaria is still a significant global health problem. Up to 80% of individuals in some malaria-endemic areas can have asymptomatic carriage of Plasmodium infections (no overt symptoms) and are thought to be a significant reservoir for malaria transmission. Many asymptomatic Plasmodium-infected individuals show mild anemia and vascular activation and have an increased risk for co-morbidities such as typhoidal Salmonella. Currently, the immunological mechanisms governing asymptomatic Plasmodium carriage are poorly understood, partly due to a lack of immunologically intact asymptomatic animal models. Here, utilizing a unique genetically diverse SPF wild-derived mouse colony we address the immunological underpinnings of asymptomatic malaria. This model shows that asymptomatic Plasmodium carriage is genetically controlled. Furthermore, wild-derived mice have varying levels of anemia associated with non-lethal Plasmodium yoelii XNL infection; some mice exhibit an asymptomatic phenotype with mild anemia independently of parasitemia. Anemia levels correlate with the production of TNF-a and IL-10, which is consistent with patterns seen in asymptomatic children in Cameroon. CD4 T cells producing IL-10 and IFN-g were also correlated with the severity of anemia, with few T regulatory cells that expanded during infection, consistent with the pattern of circulating CD4 T cells responses seen in pediatric asymptomatic malaria. Previously unknown genes are also found in GWAS analysis in these mice. Our results highlight that this novel model can be used to determine the immune mechanisms that underpin the asymptomatic carriage of Plasmodium parasites.

Supported by a grant from NIH (R01AI167422)
Distinct 3D remodeling of Il4-Il13-Il5 loci mediates differential type 2 responses in innate versus adaptive lymphocytes
Authors: Nagashima Hiroyuki , Shayne Justin , Kanno Yuka , O’Shea John J
Published: August 2023

The type 2 cytokines, interleukin (IL)-4, IL-5 and IL-13 reside within a tandem multi-gene cluster in mammals. These cytokines represent the hallmark of type 2 immune responses controlling parasites, promoting tissue repair as well as causing allergic diseases. Both innate and adaptive lymphocytes secrete type 2 cytokines with discordant production spectra. However, how those two related but distinct type 2 lymphocytes configure the extended type 2 cytokine loci and elicit selective output of this cassette genes is not well understood. Here, we took a holistic structural and functional view of the type 2 cytokine locus before and after activation, comparing innate (ILC2) and adaptive (Th2) lymphocytes to understand mechanisms underlying their distinctive programs. Rapid induction of IL-5 dominates in ILC2, whereas IL-4 does so in Th2 cells. Using high-resolution chromatin conformation capture we found that global cellular chromatin architecture remained constant, whereas the type 2 cytokine locus rapidly remodeled. In ILC2, Il13 and Il5 loci were aligned in proximity whereas Il4 locus was insulated. In Th2 cells, Il4 and Il13 positioned in proximity while the Il5 locus remained distal. Select REs were separately deleted in mice to confirm cell-type specific and activation-dependent roles in type 2 responses in vivo. Thus, contrary to the premise that chromatin architecture plays a minimal role in steady-state gene induction, signal-dependent remodeling of 3D configuration underlies the discordant cytokine outputs in ILC2s versus Th2 cells.
Bottoms up!: Inferring relevant immune phenotypes from bulk cytokine kinetics via semi-supervised machine learning
Authors: Krishnan Anagha Yogam , Wahlsten Madison , Lau Vivian , Gerard Audrey , Altan-Bonnet Grégoire
Published: August 2023

Immunologists frequently collect data on continuous multi-dimensional single-cell characteristics then classify cells based on these characteristics. Often, relevant cellular phenotypes (clusters) are determined by an iterative, intuition-driven process that requires significant biological validation. This begs the question: can we efficiently predict immunologically-relevant phenotypes by directly linking them to the biological question we are asking?

To that end, we developed a semi-supervised machine learning algorithm that directly links multi-parametric single cell data to bulk cytokine kinetics. Using T cells primed by antigen-presenting tumor cells as a biological model system, we measured bulk cytokine levels and single-cell surface marker expression every 6 hours for 72 hours. To aid with experiments, we developed a novel protocol for automated sample collection and processing. From these samples, we constructed detailed fitted time kinetics of three cytokines: interferon-gamma (IFNg), interleukin-2 (IL-2), and tumor necrosis factor alpha (TNFa). By training a semi-supervised algorithm on these kinetics, we determined an optimal set of phenotypes that explained cytokine production by T cells. When we applied our model to novel experimental systems (varying the effector:target ratio of cells, changing the peptide quality, etc.), we predicted the resultant cytokine levels and found phenotypes relevant to early shutdown of cytokine production in large, immunosuppressive tumors.

Our algorithm serves as a broadly applicable method to directly link single-cell phenotypes to bulk biological data. We aim to use this method to quickly identify phenotypes of interest for a variety of immunological questions.

Supported by the NIH Oxford-Cambridge Doctoral Fellowship and the Cambridge Trust Fellowship.
Tissue-specific determinants of the adaptive NK cell response
Authors: Mujal Adriana M , Owyong Mark , Santosa Endi K , Wingert Claire , Grassmann Simon , Lau Colleen M , Sun Joseph Chai-Yuen
Published: August 2023

Natural killer (NK) cells are circulating innate lymphocytes that are poised to respond rapidly upon encounter with cancer or virally-infected cells as well as proinflammatory cytokines. In addition to their early role in immune responses, it is now appreciated that NK cells can also take on features of adaptive lymphocytes such as antigen-specific clonal expansion and long-lived memory responses, with these processes best characterized following cytomegalovirus (CMV) infection in mouse and human. NK cell receptor recognition of virally-encoded proteins drives CMV-specific NK cells, but the signals that collectively give rise to these adaptive NK cells are still not fully understood. Mouse cytomegalovirus (MCMV) infection is systemic and while NK cells exhibit cytokine-induced activation across multiple tissue sites of infection, we were surprised to find that MCMV-specific NK cell activation and expansion occurs predominantly in the spleen. Transcriptional analysis indicated that canonical and non-canonical NF-kb components may be engaged to a greater extent in NK cells primed in the spleen, and we further identified TNFa signaling as a critical regulator of both innate and adaptive NK cell responses through engagement of distinct downstream signaling arms. Notably, TNFR2-deficient NK cells exhibited hyper-activation and ultimately failed to undergo the strong proliferative burst we observed in the spleen. These findings highlight the central role of the spleen in facilitating the innate-to-adaptive transition NK cells undergo and provide insight into how we can better generate adaptive NK cell immunity across diverse settings.

Adriana M. Mujal is supported by the Cancer Research Institute as a Cancer Research Institute/Amgen Fellow.
Synergy of heterodimeric IL-15 immunotherapy and a Fatty Acid Metabolism Modulator enhances intratumoral lymphocyte metabolism and eradicates EO771 murine breast tumors
Authors: Karaliota Sevasti , Stellas Dimitris , Stravokefalou Vasiliki , Myers Breana , Felber Barbara K , Pavlakis George N
Published: August 2023

Tumor-infiltrating cytotoxic CD8+ T cells frequently acquire an altered state of differentiation referred to as “exhaustion”, which prevents control of tumor growth. We study hetIL-15, a cytokine that stimulates the generation, proliferation and cytotoxic function of CD8+ T and NK cells. The objectives of this study was to assess the effects of hetIL-15 immunotherapy in TNBC tumors, to evaluate the metabolic profile of the tumor-infiltrating T cells and to study any potential synergy with a Fatty Acid Metabolism Modulator (FAMM). Using the EO771 orthotopic breast cancer model we monitored the efficacy of locoregional administration of hetIL-15 in combination with a FAMM. We monitored the effect of treatment on tumor growth, survival of mice and the status of tumor-infiltrating immune cells using Seahorse and flow cytometry. hetIL-15 locoregional administration resulted in complete tumor regression in 40% of the treated animals and increased survival. The infiltrating cytotoxic CD8+ T and NK cells were increased in hetIL-15 treated tumors and showed enhanced activation and proliferation. Their metabolic flux analysis confirmed a robust rise in Oxygen Consumption Rate (OCR) with substantial increase of spare respiratory capacity. Combined treatment of hetIL-15 immunotherapy and FAMM resulted in increased mitochondrial function, FA uptake and OCR. In addition, combination therapy resulted in statistically significant tumor growth delay and complete eradication of the tumors in 85% of mice. Our results indicate that metabolic reprogramming of tumor-specific CD8+ T cells might represent a strategy to promote survival in the metabolically hostile Tumor Microenvironment (TME), enhancing the clinical efficacy of immunotherapy.
Circulating cell-free DNA directly inhibits T cell function and contributes to peripheral immunosuppression in experimental GBM
Authors: Ayasoufi Katayoun , Wolf Delaney M , Reesman Rachael A , Rangel Frances A , Gulbicki Lauren R , Tritz Zachariah , Jin Fang , Hansen Michael , Fain Cori E , Pfaller Christian K , Johnson Aaron J
Published: August 2023

Glioblastoma (GBM) is an incurable brain cancer that is associated with severe peripheral immunosuppression. This immunosuppression is a critical barrier to patient survival and the success of immunotherapies. C57BL/6 mice harboring GL261 gliomas present with the hallmark features of peripheral immunosuppression observed in GBM patients, including T cell lymphopenia. We determined that peripheral immunosuppression involves release of non-steroidal soluble factors with large molecular weight that are found in sera of glioma-bearing mice. Sera of glioma-bearing mice inhibits T cell proliferation ex vivo. To determine the identity of the immunosuppressive molecules in serum, we used two parallel approaches: 1.) evaluation of abundant macromolecules and 2.) proteomics analysis using tandem mass tag mass-spectrometry (TMT-MS). We determined that serum of glioma-bearing mice contains significantly higher levels of cell free DNA (cfDNA) compared to controls. TMT-MS identified histones as a highly abundant protein in serum, further implicating DNA-histone complexes as top candidates. DNAse treatment of sera isolated from GL261 glioma-bearing mice reduced the suppressive effect on T cell proliferation. We next performed T cell proliferation assays in the presence of pure cfDNA isolated from sera of glioma-bearing mice while genomic DNA was used as control. cfDNA from serum potently inhibited T cell proliferation while control genomic DNA did not. These results were AIM2 independent implicating a novel pathway of DNA sensing in T cells. We contend that cfDNA induces immunosuppressive effects in experimental GBM. Devising strategies to reverse immunosuppression through targeting cfDNA could improve outcomes in GBM patients.

Supported by grants from NIH (K99NS117799-01A1 (KA), R01NS103212 (AJJ), and RF1NS122174(AJJ) and Brains Together for a Cure Foundation (KA).
Detection of PD-1 for flow cytometry immunophenotyping using commercial antibodies in clinical studies with check point inhibitors
Authors: Bisconte Angelina R , Mohanram Venkat , Yang Ming , Phippard Deborah
Published: August 2023

Precision for Medicine (Precision) has optimized the use of commercial anti-PD-1 and anti-IgG4 reagents to detect both free PD-1 and/or Pembro-bound PD-1 in pharmacodynamic studies when Pembrolizumab, a concomitant check point inhibitor, is used in combination with a new therapy. Precision has developed and qualified flow immunophenotyping assays to quantify PD-1 on CD4+ and CD8+ T cells using a commercial PD-1 clone (PD1.3.1.3) and an anti-IgG4. This enabled detection of both free and drug bound PD-1 receptor. We compared detection of PD-1 in PHA stimulated healthy donors treated with saturating Pembrolizumab or no drug treatment. There was not a significant difference observed in PD-1 quantification in the presence or absence of Pembrolizumab, which demonstrated that our approach was able to accurately measure total PD-1 whether free or bound. As Pembrolizumab was titrated from the saturating concentration there was a dose-dependent decrease in detection of PD-1. Here we will demonstrate the detection of PD-1 using the above methodology compared to other commercial PD-1 antibody clones. In clinical trials which incorporate checkpoint inhibitors like either PD-1 or PD-L1, the selection of commercial reagents are key considerations to detect these receptors when designing a sensitive, selective, and accurate biomarker assay.
Obesity-induced dysregulation of a unique subset of Tregs promotes skin inflammation
Authors: Li Chaoran , Sivasami Pulavendran , Elkins Cody , Goss Kyndal , Diaz-Saldana Pamela P , Peng Amy , Hamersky Michael , Bae Jennifer , Pollack Brian P , Horwitz Edwin M , Scharer Christopher D , Seldin Lindsey
Published: August 2023

Obesity is associated with increased skin inflammation and is a major risk factor for psoriasis, raising the question of how obesity disrupts the regulatory mechanisms that keep skin inflammation in check at steady state. We found that skin was enriched with a unique subset of CD4+ Foxp3+ regulatory T cells (Tregs), which is critical to limit IL-17A-mediated psoriatic inflammation. Diet-induced obesity, however, resulted in a significant reduction of this subset of skin Tregs and a corresponding loss of control over IL-17A-mediated inflammation. Mechanistically, this specific skin Treg population preferentially took up elevated levels of long-chain saturated fatty acids during obesity, which led to cellular lipotoxicity and mitochondrial dysfunction. Harnessing the anti-inflammatory property of this unique subset of skin Tregs could have therapeutic potential for obesity-associated inflammatory skin diseases.

Supported from grants from NIH/NIDDK (1R01DK128061), National Psoriasis Foundation, and Emory University Research Committee (URC).
Lung injury induces preferential expansion of Foxp3+ regulatory T cells over conventional T cells in self-antigen specific CD4+ T cell repertoire
Authors: Shin Daniel Seong-Joo , Moon James J
Published: August 2023

Self antigen-specific T cells are prevalent in the mature adaptive immune repertoire. At steady state, these T cells are regulated by various mechanisms of peripheral tolerance. Characterizing the activation of these self antigen-specific T cells during inflammation may shed crucial insights into the pathogenesis of autoimmunity. Utilizing an experimental animal model where mice express three model T cell epitopes (2W, gp66, and OVA) in a tissue restricted manner, we detect the exclusive expansion of autoreactive regulatory T cells (Tregs) following acute lung injury that is dependent on recognition of their cognate self antigen and IL-2 signaling. Conversely, conventional T cells of the same self antigen-specificity did not expand during inflammation, even following the depletion of endogenous Tregs. Therefore, our findings suggest that the self-antigen specific T cell repertoire may serve a regulatory function during acute tissue damage to regulate the ongoing immune response and limit the possibility of autoimmunity.
IEDB-3D 2.0: Enabling visualization of epitopes on the 3D structure of the corresponding antigens
Authors: De Almeida Mendes Marcus F , Mahita Jarjapu , Blazeska Nina , Wheeler Daniel , Ha Brendan , Greenbaum Jason , Wang Jiyao , Shackelford Deborah , Sette Alessandro , Peters Bjoern
Published: August 2023

The Immune Epitope Database and Analysis Resource (IEDB) contains information related to antibodies, T cells, and MHC across a broad spectrum of research fields (infectious diseases, cancer, allergy, autoimmunity, and transplantation). A critical component of this information is three-dimensional structural data on T cell receptors (TCRs), antibodies, and pairwise residue interactions between immune receptors and antigens, which we refer to as IEDB-3D. Analysis using atomic models helps to understand the intricate interactions between immune receptor and epitopes. Here, we present IEDB-3D 2.0, which incorporates a complete overhaul of how we obtain and present 3D structural data from PDB and AlphaFold database. We implemented a new molecule visualizer, iCn3D, to replace outdated java-based technology. In addition, we have designed a new epitope mapping system that matches each epitope available in the IEDB with its antigen structural data. Lastly, we integrated our epitope mapping system with immunogenic data retrieved from the IEDB’s ImmunomeBrowser, which now can be used to highlight immunogenic regions of an antigen directly in iCn3D. Overall, the IEDB-3D 2.0 provides an updated tool platform to visualize epitope data cataloged in the IEDB.

Research reported in this publication was supported by the National Institutes of Health contract 75N93019C00001 and grant U24CA248138.
An integrated reagent and multimodal analysis workflow to enrich and characterize peptide-specific CD8+T cells
Authors: Adams Bruce A , Shahi Payam , Reyes Daniel , Maheshwari Shamoni , Mousavi Nima , Krishnan Sreenath , Ramen Nandhini , Tsai FuNien , Kumar Poornasree , Finnegan Peter , Fernandes Sunjay , Bali Gunmeet Kaur , Terry Jessica
Published: August 2023

Immune responses to disease are complex and the only way to understand such a response is to consider the behavior of multiple different cell types across both the innate and adaptive branches of the immune system. We developed Barcode Enabled Antigen Mapping (BEAM) technology to enable easy enrichment of antigen-specific cells. This technology is fully integrated with the 10x Genomics Chromium Single Cell Immune Profiling Solution to allow multimodal evaluation of immune cells including full-length, paired sequences of T and B cell receptor genes while simultaneously screening their specificity for a set of genes. Our antigen-specific TCR workflow (BEAM-T) identifies antigen-specific CD8+T cells. We demonstrate the assay’s performance with spike-in experiments using various percentages of HLA-A*02:01-restricted peptide-stimulated T cells (targeted mixture being 40% T cells with specificity for cytomegalovirus peptide, 10% T cells with specificity for flu peptide and 0–5% anti-SARS-CoV-2 T cells) into a background of human PBMCs. Cells were labeled with a panel of five viral pathogen peptide-loaded multimers, plus a HLA-A*02:01 negative control peptide to assess antigen specificity and were then sorted for CD8+PE+ T cells. We demonstrated that the major VDJ-T clonotypes identified for the SARS-CoV2 cells were retained, albeit with roughly proportionally fewer cells, as the percentage of spiked cells decreased. We further feature the robustness of the protocol with various sample types and models including H2Kb-restricted OT-1 transgenic mouse splenocytes, as well as healthy human donor PBMCs and dissociated tumor cells to demonstrate the utility of an integrated reagent-to-data analysis offering across a range of samples.