bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of CD4 T-cell immunity by eukaryotic Elongation Factor 2 Kinase
Authors: Peng Hao-Yun , Song Jianxun
Published: August 2023

CD4 T cells are a major source of inflammatory cytokines to manage the immune responses, and there is evidence showing that IL-17-secreting helper CD4 T cells (Th17 cells), play a critical role in autoimmune diseases. Eukaryotic Elongation Factor 2 Kinase (eEF2K) is a protein kinase that negatively regulates the elongation step of protein synthesis. eEF2K regulates vascular inflammatory responses and hypertension development, yet a complete immunologic characterization of eEF2K, particularly in CD4 T cells and autoimmune diseases, remains obscure. Defining eEF2K-mediated CD4 T cell activities, including CD4 T cell inflammation and metabolism, will significantly enhance our knowledge toward the goal of improving health span. Here, we reported that eEF2K is essential for the survival and proliferative capacity of CD4 T cells. eEF2K deficient mice displayed a higher metabolic profile and increased production of inflammatory cytokines. By analyzing CD4 T cells from the wild type (WT) and eEF2K deficient mice, we revealed that eEF2K regulates mitochondrial stress capacity and the production of reactive oxygen species (ROS) through the master transcription factor, STAT3. Depletion of eEF2K agitated Th17-related autoimmune diseases, including arthritis and colitis. A STAT3 inhibitor, C188–9, attenuated Th17 cell-mediated autoimmune colitis by reducing inflammation in the murine colitis model that received eEF2K deficient CD4 T cells. Our findings provide insights into the regulation of CD4 T cells and autoimmunity by eEF2K, and further investigations would help design novel strategies by targeting eEF2K-based immunotherapy in autoimmune diseases.
Obesity Modulated Gut Microbiota Exacerbate Disease Severity in HLA-Class II Transgenic Mouse Model of Multiple Sclerosis
Authors: Shahi Shailesh K , Ghimire Sudeep , Lehman Peter , Rux Allison G , Mangalam Ashutosh K
Published: August 2023

Multiple sclerosis (MS) is an inflammatory and demyelinating disease of the CNS where interaction among multiple environmental and genetic factors influence disease outcome. HLA class II alleles such as HLA-DR2 and –DR3 are considered strongest genetic factors linked with MS, whereas recently gut microbiota have emerged as an important environmental factor linked to MS. Additionally, obesity has been shown to promote MS disease severity. However, the mechanistic link between gut microbiota and obesity in pathobiology of MS is unknown. HLA-DR3 transgenic mice on a high-fat diet (HFD) to induce obesity, resulted in severe EAE compared to normal chow diet (NCD). Antibiotic-mediated gut microbiota depletion and fecal transplant studies established that the microbiota is necessary and sufficient for obesity to potentiate EAE severity. Obesity-associated microbiota alterations included increases in bacterial genes linked with hydrogen sulfide (H2S)-metabolism, with concordant increases in fecal H2S. Further, mice on HFD showed increased gut permeability and systemic inflammation compared with NCD group. Altogether, this study provides evidence for involvement of the gut microbiome and associated H2S-metabolism microbial genes in obesity induced modulation of EAE severity. Dissection of these pathways will help to identify novel therapeutic targets to reduce disease severity in obese patients.

The Author acknowledge funding from the National Multiple Sclerosis Society grant (RG 5138A1/1T), National Institutes of Health/NIAID (1R01AI137075), the University of Iowa Environmental Health Sciences Research Center, NIEHS/NIH (P30 ES005605), and a gift from P. Heppelmann and M. Wacek to AM.
Development of monoclonal antibodies and antigens as liquid biopsy reagents for IgA nephropathy
Authors: Ka Shuk-Man , Chen Yi-Liang , Chen Ann
Published: August 2023

IgA nephropathy (IgAN) is the most common primary autoimmune glomerular disorder across the world. A renal biopsy examination is absolutely required for final diagnosis. In particular, IgAN has been categorized as an autoimmune disease that features IgA-ICs formed by galactose-deficient (Gd)-IgA1 (autoantigen) and corresponding IgG anti-Gd-IgA1 (autoantibody). Therefore, it is clinically warranted to develop a liquid kidney biopsy for non-invasive or only minimally invasive approaches for early diagnosis of IgAN and to ease monitoring the evolution of the disease. We have recently developed three monoclonal antibodies namely EASON1, EASON2, and EASON3, and each of them shows high discriminative rate in differentiating IgAN patients and healthy control subjects by doing ELISA-based analyses to determine serum Gd-IgA1 levels (Provisional patent - T2392-US, USA entitled “Production of IgG monoclonal antibody series anti-galactose deficient IgA1 and IgA1 bearing O-linked glycans; Provisional patent - 63/119,215, USA entitled “Development of a series of IgM monoclonal antibodies as non-invasive diagnostic reagents for IgA nephropathy; a regular patent application under review). Moreover, we have successfully developed 62 human hybridoma cell lines that produce Gd-IgA1, each of which will be useful in designing tests for the detection of serum IgG autoantibodies against Gd-IgA1 levels (Provisional patent - T2392-US, USA). In conclusion, both the 3 mouse monoclonal antibodies and 62 human Gd-IgA1-producing hybridoma cell lines may be further developed as promising diagnostic reagents for a liquid kidney biopsy for IgAN, allowing for early, rapid, and non-invasive tests for the renal disease.

Supported by grants from Ministry of Science and Technology, Taiwan (MOST 108-2321-B-016-002, 109-2314-B-016 -034 -MY3)
Distinct roles of kindlin-3 and talin-1 in neutrophil beta2 integrin clustering
Authors: Wu Yuanyuan , Wen Lai , Ley Klaus , Fan Zhichao
Published: August 2023

Neutrophils are the most abundant leukocytes in human blood. Neutrophil malfunction may cause various immunological diseases. Beta2 integrins are critical for neutrophil recruitment. Kindlin-3 and talin-1 are important regulators for beta2 integrin activation. We have confirmed this using conformation-specific antibodies KIM127 and mAb24 that recognize activation epitopes in beta2 integrin. Besides activation, clustering is also critical for beta2 integrin avidity and adhesion of neutrophils. Whether kindlin-3 and talin-1 are involved in beta2 integrin clustering remains unclear. Here we used super-resolution stochastic optical reconstruction microscopy (STORM) to study their roles in beta2 integrin clustering on human neutrophil-like HL60 cells. We find that lymphocyte function-associated antigen 1 (LFA-1) and macrophage-1 antigen (Mac-1) are significantly clustered in wild-type cells after IL-8 or fMLP stimulation. Using CRISPR-Cas9-mediated kindlin-3 and talin-1 knockout HL60 cells, we found that kindlin-3 but not talin-1 are responsible for beta2 integrin clustering. Our study provides new insights into the regulation of beta2 integrin clustering and leukocyte recruitment.

Supported by grants from the National Institutes of Health, National Heart, Lung, and Blood Institute, USA (R01HL145454), and a startup fund from UConn Health.
In vivo cell tracking reveals the pattern of neutrophil tissue distribution at baseline and in response to glucocorticoid treatment
Authors: Bohner Ariel M , Gadkari Manasi , Makiya Michelle , Hong So Gun , Cao Qilin , Potter E Lake , Xing Fan , Calvo Katherine , Klion Amy , Howe Katherine N , Kaplan Mariana J , Roederer Mario , Sato Noriko , Dunbar Cynthia , Franco Luis M
Published: August 2023

Neutrophils are the most abundant circulating immune cell in humans and primates, and they play a central role in inflammation and innate immunity. Glucocorticoids (GCs) are the cornerstone of anti-inflammatory and immunosuppressive therapies. While GCs are known to induce neutrophilia, little is known about the underlying kinetics or molecular mechanisms. To better define the kinetics of GC-induced neutrophilia, we studied healthy human volunteers and rhesus macaques, and found that the neutrophilia peaks within 4 hours of GC infusion and lasts beyond 72 hours. Flow cytometry, nuclear morphology, and single-cell RNA-seq confirmed that GC-mobilized neutrophils are mature and do not have the CXCR1lo/ICAM1hi reverse-transmigration phenotype. To study the pattern of neutrophil tissue distribution in vivo, we are performing live tracking of neutrophils by PET/CT and serial intravascular staining (SIVS), before and after GC in a rhesus model. Initial Zr-89 oxine PET/CT results indicate that re-injected radiolabeled neutrophils transit the lungs quickly and within 24 hours localize to the liver, bone marrow, and spleen, where the signal remains stable for at least 72 hours. GC treatment does not appear to lead to significant re-distribution of tissue neutrophils. Initial SIVS results indicate that most GC-mobilized neutrophils had been in circulation for less than a day and came from the intravascular compartment. In summary, our findings to date indicate that neutrophils have stable and prolonged tissue localization, and that GCs rapidly mobilize a small proportion of the total neutrophil pool, composed mainly of mature cells that were present in the intravascular compartment at the time of treatment.

supported by grants from NIH (Intramural Research Program – NIAMS/NIH)
Dendritic cells regulate innate cell trafficking and lymph node organization during inflammation
Authors: Huang Jessica , Wu Yixuan , Gerner Michael
Published: August 2023

Organization of immune cells within lymph nodes (LNs) is critical for effective host defense. During vaccination or infection, layers of innate cell activity in the outer periphery of the LN provide immediate protection, while antigen-presenting dendritic cells (DCs) migrate into the T cell zone (TZ), located deeper in the LN, to initiate later-stage adaptive responses. Using mouse models of vaccination and infection, we now find that DC activation and repositioning into the TZ is tightly associated with localized recruitment of multiple innate effector cells, including neutrophils, monocytes, and NK cells, which also rapidly infiltrate the TZ. As activated DCs migrated towards the TZ, they passed by the high endothelial venules (HEVs), where they expressed multiple inflammatory chemokines and high levels of the integrin ligand, ICAM-1, generating a chemokine- and adhesion molecule-rich matrix ripe for innate cell migration. Depletion of DCs, or conditional ablation of ICAM-1, significantly impaired innate cell trafficking into LNs, and cells which did enter remained largely trapped near the HEVs and were less able to infiltrate the TZ. While essential for innate defense, neutrophil influx also led to large-scale disruption of the TZ and impaired early T cell activation. However, over time this was controlled by DCs and DC-recruited monocytes, which efferocytosed the infiltrating neutrophils and cleared the TZ to allow productive adaptive responses to occur. Thus, in addition to their classical roles in antigen presentation, DCs orchestrate innate cell recruitment and activation across the entire LN, to promote both effective innate defense as well as rapid restoration of LN architecture for the generation of adaptive immunity.

Supported by grant from NIH (1F31AI161316-01).
Diabetic wound macrophage IL6 drives fibroblast dysfunction via the histone methyltransferase, SETDB2
Authors: Joshi Amrita D , Barrett Emily C , Mangum Kevin D , Mazumder Purba , Rocco Sabrina , Erturk Asuman , Kai Rohan , Moon Jadie Yoonjoo , Wolf Sonya , Gallagher Katherine
Published: August 2023

Interactions between innate immune cells and structural cells are vital to proper tissue repair and regeneration after injury. Under pathologic conditions such as type 2 diabetes (T2D), Mϕs exist in a chronic inflammatory state that drives dysregulated wound repair, in part by altering the fibroblast response to injury. We have previously shown that wound Mϕs from a murine model that mimics T2D (diet-induced obese mice; DIO) physiology, display increased levels of inflammatory cytokines, including IL6. Here, we found that rIL6 drives CD202-, CD45-, CD31-, Epcam-, TER119-fibroblasts isolated from wounds to produce decreased levels of col1A and periostin, two mediators that are crucial for fibroblast function in wound repair. Although the underlying mechanisms remain unclear, we and others have shown that both immune and structural cell functions in tissues can be controlled by epigenetic modifications that alter gene expression. Using our human single cell sequencing data from T2D wounds we identified that the histone methyltransferase, SETDB2, was increased in T2D fibroblasts, compared to non-wounded controls. We then isolated fibroblasts from wounds and stimulated them with rIL6 and identified an increase in H3K9me3, the histone methylation mark specific for SETDB2, at the promoters of the col1A and periostin genes. Increase in this repressive mark serves to decrease gene expression. Our findings reveal a previously unreported role for SETDB2 in regulating fibroblast function in tissue repair. Moreover, our data suggest that the diabetic milieu promotes increased expression of SETDB2 which, in turn, may contribute to maintaining the impaired fibroblast function in diabetic wounds that hinders tissue repair.
Pulmonary Mycobacterium tuberculosis infection alters the hepatic immunometabolic landscape and compromises insulin responsiveness early after infection
Authors: Cooper Andrea May , Das Mrinal , Pearl John
Published: August 2023

Immunity against Mycobacterium tuberculosis (Mtb) is associated with both regional and systemic inflammation as well as with metabolic disruption in tuberculosis (TB) patients. The metabolic and immunological responses are intertwined but the specific impact of localized disease on systemic metabolism is not well defined. We investigated the impact of low dose aerosol infection of the lung with Mtb on the systemic metabolic regulatory organ – the liver. Using an unbiased approach we defined the mRNA and protein dysregulation occurring in the early stage of infection. We found that infection leads to immune pathway activation and reduced metabolic activity in liver. Comparison of the gene expression patterns between of the lung, liver and blood revealed organ specific metabolic gene dysregulation in the context of non-organ specific activation of interferon response pathways. We have also found that infection leads to suppression of carbohydrate and lipid metabolism, without alteration of fasting or random blood glucose levels. Infected mice show reduced sensitivity to exogenous insulin but the glucose tolerance remains intact indicating insulin resistance. Analysis of metabolomics data from public repository reveals a metabolic signature of insulin resistance correlates with TB progression. Taken together, our finding shows that aerosol Mtb infection influences hepatic metabolism very soon after infection and may contribute to early metabolic disease. Defining the early interplay between localised infection, inflammation and metabolic regulation is key to understanding the impact of latent and subclinical TB on the large proportion of the World’s population who do not exhibit TB symptoms but who are infected with Mtb.

Supported by Grants from Royal Society Wolfson Merit Award (AMC) Newton Fellowship (MKD), Wellcome Trust Institutional Support Fund Fellowship (MKD), MRC award MR/P011136/1 (AMC).
Investigating the Role of Bacterial Long-Chain Polyphosphates in Macrophages during Legionella pneumophila Infection
Authors: Farquharson Kara L , Platten Johannes , Brueck Kevin , Sharma Arjun , Bosmann Markus
Published: August 2023

Up to 10% of community-acquired pneumonia cases are identified as Legionnaires’ Disease, caused by the pathogen Legionella pneumophila. L. pneumophila naturally resides in freshwater systems and becomes pathogenic when man-made plumbing systems grow biofilm and amoeba if not maintained. Hosts are accidentally infected by L. pneumophilavia aerosol inhalation into the respiratory system. L. pneumophila primarily reside in human alveolar macrophages, entering and replicating via the Legionella-containing vacuole. This vacuole will also release and recruit hundreds of effector molecules to prevent itself from being phagocytosed and disguise itself as a host cellular component. Polyphosphates are highly conserved molecules found in essentially every cell performing essential functions. Long-chain polyphosphates, containing hundreds of phosphate units, are produced by bacteria to perform functions critical to their survival in hosts. We have previously found that long-chain polyphosphates produced by E. coli interfere with the host innate response against infection. Here, we show that the infection of L. pneumophila and synthetic long-chain bacterial polyphosphates interfere with the inflammatory response in macrophages. We utilized a mouse model for in vitro infection of L. pneumophila in bone-marrow-derived macrophages, showing a modulated inflammatory cytokine response and cell death. Additionally, the addition of S. cerevisiae derived exopolyphosphatase, which degrades long-chain polyphosphates, decreases inflammation in vitro.
Sensitivity and specificity of an indirect ELISA using Hawaii 31 kDa Angiostrongylus cantonensis antigen in experimentally-infected rats (Rattus norvegicus)
Authors: Jarvi Susan Irene , Steel Argon , Jacob John
Published: August 2023

The parasitic nematode Angiostrongylus cantonensis is the leading cause of neuroangiostrongyliasis, is an emerging tropical disease, and is the leading cause of eosinophilic meningitis worldwide. Diagnosis is generally presumptive and can be easily confused with other central nervous system disorders. Several studies have demonstrated the promise of immunodiagnosis, most frequently in the form of ELISA assays. Taking advantage of plasma samples from previous rat studies, we developed an indirect ELISA assay based on the 31 kDa antigen isolated from A. cantonensis collected in Hawai’i. Using IgG, our assay showed 100% sensitivity, and specificity in diagnosing A. cantonensis infection in lab-reared rats fed 50 live larvae six weeks previously. We saw diminishing sensitivity to IgM, IgA, and IgE, respectively, and no antibody response in rats fed killed A. cantonensis larvae. Our experiments validate the use of indirect ELISA with 31 kDa antigen as an effective immunodiagnostic assay for rats, and our use of different antibody isotypes provided an opportunity to investigate in more detail the humoral immune response in lab-reared rats to A. cantonensis infection.

Daniel K. Inouye College of Pharmacy, Hawaii State Legislature
Fasciola hepatica Fh15 promote survival in a mouse septic shock model and downregulates inflammatory cytokines
Authors: Armina-Rodriguez Albersy , Ocasio-Malavé Carlimar , Méndez-Torres Loyda B , Valdés-Fernández Bianca , Espino Ana M
Published: August 2023

Parasitic helminths and helminth-derived molecules, possess an inherent anti-inflammatory capacity. Fasciola hepatica has been reported to decrease in vivo particular Th1 immune responses caused by concurrent bacterial infections. Studies have shown that some Fasciola hepatica’s excretory-secretory products has the capacity to reduce phagocytic activity, affect cell recruitment, and induce activation of alternative activated macrophages. We hypothesized that recombinant F. hepatica FABP (Fh15) may be an excellent target for the development of new anti-inflammatory drugs against sepsis. For this, we developed a mouse model of septic shock to determine the capacity of Fh15 to suppress the cytokine storm and prevent the mortality rate of mice exposed to lethal doses of LPS. BALB/c mice were allotted into two experimental groups of 5 animals each, which received an IP injection with 12mg/kg body wt. of LPS from E. coli 0111: B4. One hour after LPS injection one group received a single IP injection with 100 μg Fh15 and the other group received two injections of 100 μg Fh15 two hours apart. Control groups received IP injection with LPS, Fh15 or PBS alone. Animals were monitored for mortality for 72 hrs. Blood samples were collected from all animals at baseline and by the time they were about to die or survived the LPS lethal challenge. As expected, all animals from LPS-control group succumbed between 12 to 24h following the LPS challenge. In contrast, between 40 to 100% of animals that received the Fh15 treatment survived to the LPS challenge. This notable increase in the survival rate was found associated to significant suppression in the production of pro-inflammatory cytokines such as IL-12, TNF-a and IFN-g, which were measured in serum by BioPlex.
Sialophorin is an essential host element for vaccine immunity against pulmonary fungal infections
Authors: Nanjappa Som G , Sharma Jaishree , Deepe George S , Mudalagiriyappa Srinivasu
Published: August 2023

The global incidence of fungal infections is alarming due to the increasing immunocompromised population. Vaccine measures are necessary to prevent fungal diseases. Identifying a protective host element as a functional phenotypic marker is immensely valuable as a target to enhance vaccine responses, measure vaccine efficacy, and assess disease outcomes. We identified a host element, O-glycosylated sialophorin, preferentially associated with antifungal memory Tc17 (IL-17A+) cells. Using a mouse model of fungal vaccine immunity during CD4+ T-cell deficiency, we found that sialophorin was essential to bolster vaccine-induced CD8+ T-cell responses and immunity to pulmonary infection. Sialophorin-sufficient CD8+ T cells were superior in responses than the deficient cells under a similar inflammatory micromilieu. Supplementation of sialophorin mAb that binds to O-glycosylated form potentiated the CD8+ T cell responses to fungal antigens, and an inhibitory mAb that binds to non-O-glycosylated form selectively reduced Tc1 (IFNγ+) but not Tc17 cell responses. Sialophorin-mediated immunity was independent of T cell trafficking into the lung. Finally, using fungal antigens vaccination, we show that O-glycosylated sialophorin is a marker of vaccine-potency and immunity. Our study revealed that sialophorin is an essential host element to bolster vaccine responses and serves as a potential biomarker of fungal immunity.

NIAID-NIH 5R01AI153522
Updating the definition of V(D)J recombination signal sequences with high-throughput recombination assays
Authors: Hoolehan Walker Aloysius , Rodgers Karla K
Published: August 2023

V(D)J recombination initiates the production of a diverse antigen receptor repertoire in developing B and T cells. Antigen receptor diversity is required for adaptive immunity in all jawed vertebrates. Recombination activating proteins, RAG1 and RAG2 (RAG1/2), catalyze V(D)J recombination by cleaving adjacent to recombination signal sequences (RSSs) that flank antigen receptor gene segments. Previous studies defined the consensus RSS as containing conserved heptamer and nonamer sequences separated by a less conserved 12 or 23 base-pair spacer sequence. However, many RSSs deviate from the consensus sequence, and the molecular mechanism for semi-selective V(D)J recombination specificity is unknown. Here, we developed a cell-based, high-throughput V(D)J recombination assay to evaluate RAG1/2 activity on thousands of RSSs in parallel. Specifically, we focused our study on the RSS heptamer and adjoining spacer region because it undergoes extensive conformational changes during RAG-mediated DNA cleavage. While the consensus heptamer sequence (CACAGTG) was marginally preferred, RAG1/2 was highly active on a wide range of non-consensus sequences. RAG1/2 generally selected purine/pyrimidine motifs instead of specific bases. Molecular dynamics simulations of RSSs showed that DNA sequences preferred by RAG1/2 had dynamic structural features that may accommodate heptamer unwinding in the RAG1/2 active site. Our results suggest RAG1/2 specificity for RSS heptamers is primarily dictated by dynamic DNA properties dependent on purine/pyrimidine pattern. Further investigation of RAG1/2 specificity will help elucidate the genetic instructions guiding V(D)J recombination to antigen receptor gene segments.

National Institutes of Health (AI128137, AI15635); Oklahoma Center for Advancement in Science and Technology (OCAST) [HR21-142]; Presbyterian Health Foundation of Oklahoma City; National Institute of Allergy and Infectious Diseases Immunology training grant [T32 AI7633-16]
IL-9 responsive macrophages utilize Arginase 1 to enhance lung tumor growth
Authors: Cannon Anthony Michael , Kaplan Mark H
Published: August 2023

In cancer, the immunosuppressive function of myeloid cells that support tumor progression is controlled by secreted factors in the tumor microenvironment. In the tumor microenvironment, arginine and arginine-derived metabolites have been demonstrated to be crucial factors in tumor development. Within macrophages, arginine metabolism influences the polarization of macrophages and therefore, tumor growth by eliciting an anti-tumoral or pro-tumoral phenotype. Interleukin 9 (IL-9) is a pleiotropic cytokine that signals through the IL-9 receptor (IL-9R) and can function as a positive or negative regulator in tumor immunity. Recently, our lab has demonstrated that IL-9 signaling promotes tumor progression in the lung by expanding pulmonary interstitial macrophage populations and inducing Arginase 1 (ARG1), an enzyme associated with pro-tumoral macrophage function, activity. However, the mechanism by which IL-9R/ARG1+ interstitial macrophages promote tumor development remains unknown. Here, using a B16F10 model of metastatic lung cancer, we demonstrate that knockdown of ARG1 using macrophage targeting Arg1 siRNA containing nanoparticles results in altered pulmonary T-cell and macrophage proportions. Moreover, attenuation of IL-9 signaling and ARG1 expression in macrophages impacts arginine and arginine-derived metabolite concentration in lung tissue and BAL fluid which correlates with decreases in CD4+ T Cell exhaustion marker expression and a concomitant reduction in PDL1 expression on pulmonary interstitial macrophages in tumor-bearing mice. Thus, our work suggests that the IL-9R/ARG1 axis in macrophages is sufficient to alter arginine in the tumor microenvironment and promote tumor growth.
Stem-like CD4 T differentiation controls CD8 T cell immunity or tolerance in cancer
Authors: Cardenas Maria A , Prokhnevska Nataliya , Jansen Caroline S , Sobierajska Ewelina , Master Viraj , Kissick Haydn T
Published: August 2023

In this work we wanted to understand how CD4 T cell differentiation shapes the CD8 T cell response in cancer. We identified a population of tumor specific CD4 T cells that maintain an activated, undifferentiated phenotype throughout the cancer response. These CD4 T cells were the dominant population within tumor draining lymph nodes (TDLNs), characterized by high expression of TCF1 and little to no expression of other lineage defining transcription factors. Tumor specific TCF1+ CD4s exhibited functional stem-like properties, as they self-renewed and differentiated almost exclusively into Tregs in tumor bearing mice. These cells, however, were not limited to Treg differentiation, as sorted TCF1+ CD4s from a 5-week tumor response expanded and gave rise to TH1 and TFH effectors in response to LCMV Armstrong infection. Interestingly, we also found a similar population of activated PD1+ TCF1+ CD4s with stem-like capabilities infiltrating various human tumors, indicating this phenotype of CD4s is comparable in human cancer and mouse models. Importantly, the choice of lineage differentiation of TCF1+ CD4s regulated the quality of the CD8 T cell anti-tumor response. When Tregs were selectively depleted, TCF1+ CD4s differentiated into TH1s, which enhanced tumor specific CD8 T cell effector differentiation in both tumor and TDLNs, resulting in reduced tumor burden. Using a bone marrow chimeric CRISPR-Cas9 approach, we found that IFNg from TH1s was intrinsically required for CD8 stem to effector differentiation within TDLNs, promoting an improved anti-tumor response. Overall, this work shows that CD4 differentiation dictates tolerance or immunity in cancer by regulating the phenotype and magnitude of the effector CD8 T cell response.
Varied T cell repertoire bearing intact TCR/CD3 complexes required to observe maximal therapeutic anti-tumor effects of CD3 Fab fragments when binding to T cells
Authors: Mahadevappa Chandrashekara Puthanapura , Huynh Hien , Okpasuo Onekachi , Radhakrishnan Vijay , Cheatham Christa , Vu Nhan , Schrum Adam G , Gil Diana
Published: August 2023

A cognate antigen engagement of TCR induces a conformation change in the CD3 complex (CD3Δc) that is required for productive T cell activation. CD3Δc fails when TCRs interact with poorly immunogenic antigens, like many tumor-associated-antigens (TAAs), which do not active T cells. Anti-CD3 monovalent Fab fragments (Mono-Fabs) bound to the CD3 complex mimic CD3Δc, producing T cell “co-potentiation” when associated with TCRs bound to weak antigens that enhances T cell activation, neither stimulating non-engaged T cells, nor interrupting T cell responses to strong antigens. Previously, we showed T cell co-potentiation can be exploited as a novel immunotherapeutic principle by reducing melanoma burden in B6 mice treated with anti-CD3 Mono-Fabs. TCR signaling initiation requires CD3Δc for the exposure of a conserved sequence in the cytosolic domain of CD3ɛ rich in prolines (PRS) that enables the recruitment of adaptor protein Nck. The mutation of cysteines in the CXXC motif of CD3ɛ extracellular domain inhibits the transmission of TCR signaling across transmembrane that defects CD3Δc and PRS accessibility. Here, using the B6 syngeneic melanoma cell lines Yumm 1.7 and Yummer1.7, which differ in the number of somatic mutations they carry, in B6 mouse strains either wild or mutated in either the CXXC or the PRS motifs in CD3ɛ, we have established the need of tumor cells to carry potential tumor neoantigens, and a more varied repertoire of T cells responding to the tumor with intact TCR/CD3 complexes to observe full anti-tumor effects by anti-CD3 Mono-Fab treatments.

MU Start-up funds

NIH, NCI, U01 CA244314

NIH, NIAID, R01 AI097187
CNPY2-orchestrated unfolded protein responses play redundant roles in regulating anti-tumor immunity of CD8+ T cells
Authors: Wang Yi , Li Anqi , Song No-joon , Shannon Ariana E , Hong Feng , Searle Brian C , Li Zihai
Published: August 2023

Tumors evade immune surveillance by creating a hostile environment that impairs the functions of immune cells. Persistent perturbations including chronic antigen stimulation and metabolic challenges cause endoplasmic reticulum (ER) stress and unfolded protein responses (UPR) in T cells. CNPY2 is one of the key initiators of UPR in the ER, but its roles in T cell activation and persistence have not been reported. Herein, we found that the expressions of CNPY2 and other molecules in UPR pathways were upregulated in both activated and exhausted T cells, indicating UPR activation. However, genetic deletion of Cnpy2 in CD8+T cells did not result in changes in viability, proliferation, mitochondrial functions, and protein translation rate under acute or chronic antigen stimulation in vitro. Furthermore, no differences were seen between wild-type and CD8-specific Cnpy2 knockout mice in protection against multiple syngeneic tumor models including colon (MC38 and CT26), bladder (MB49), melanoma (YUMM1.7 and B16-F10) and breast tumors (4T1). Although CD8+ T cells from tumors were moderately more activated in the absence of CNPY2, no apparent impact was observed on CD8+ infiltration and effector differentiation. Finally, by mRNA profiling and proteomic analysis, we found that the downstream UPR signaling pathways remained intact in T cells despite loss of CNPY2. In summary, we concluded that persistent TCR stimulation and the tumor microenvironment induce CNPY2 expression and UPR in CD8+ T cells. However, ablating CNPY2 alone failed to overcome UPR-mediated T cell dysfunction, suggesting that targeting multiple initiators of UPRs in CD8+ T cells need to be considered to improve cancer immunotherapy.
Impaired IL-17 and cytotolytic responses to influenza vaccine antigens in aging nonhuman primates
Authors: Rout Namita , Walker Edith , Bodogai Monica , Mattison Julie , Jazwinski Stanislaw Michal , Biragyn Arya
Published: August 2023

Despite widespread flu vaccination, influenza remains a major cause of morbidity and mortality in the elderly. The early immune response, including CD4+ helper T cells, and CD8+ CTLs, plays a critical role in efficient control of virus and initiation of adaptive immunity. However, the correlates of protection in the elderly are not well understood. Here, we sought to investigate early cellular responses to re-exposure with seasonal flu vaccine antigens in a cohort of pre-vaccinated young and old rhesus macaques (human age equivalent of 30–45 yrs, and 55–85 yrs). In vitro T cell responses were examined following overnight stimulation of PBMCs with flu vaccine antigens by flow cytometry for Th1-type and Th17-type cytokine responses, and cytolytic functions. Notably, older macaques showed significantly impaired IL-17 production and CD107a upregulation in response to vaccine antigens, along with greater HLA-DR+/PD-1+CD8 T cells. Further, they produced significantly higher TNF-α than young macaques. These results suggest that circulating flu vaccine-specific T cells in older macaques are deficient in mucosal barrier-protective and cytotolytic functions and possess a dominant proinflammatory function. These findings suggest that re-exposure to flu vaccine antigens in older individuals likely induces a proinflammatory response that is impaired in cytolytic capacity and the ability to repair lung mucosal damage during influenza infection, thereby resulting in more severe disease outcomes. Further studies on in vivo modulation of proinflammatory T cells while enhancing IL-17 functions during vaccination could support the development of more effective influenza vaccine strategies for older adults.
The adaptation model of immunity: tumor cell adaptation to tumor-reactive immune responses
Authors: Manjili Masoud H
Published: August 2023

Tumor cells are genetically unstable carrying continuously changing mutations which are the basis for neoantigen vaccines. Pre-existing immune responses against tumor-associated self-antigens as well as against semi-nonself neoantigens have been reported in cancer patients. These data are against the self-nonself (SNS) model of immunity, proposing that tumor cells expressing self-antigens induce tolerance. The adaptation model proposes that expression of many adaptation receptors (AdRs) linked to anti-apoptotic pathways in tumor cells, one of which is reported to be B7-H1, is responsible for the survival of tumor cells in the presence of anti-tumor immune responses. To this end, anti-tumor efficacy of immune checkpoint inhibitors is mainly because of the blockade of survival pathway in tumor cells downstream of B7-H1. Limited efficacy of these therapies is because several AdRs may be expressed by tumor cells which compensate for the blockade of B7-H1. In fact, B7-H1 is mischaracterized as PD-L1 ligand while it is a bi-directional receptor that relays survival signal both as trans, when engaged with PD-1 on T cells, and as cis, when both B7-H1 and PD-1 or B7.1 are expressed on DCs or tumor cells. AdRs could also exist as a dual receptor system consisting of those linked to anti-apoptotic pathways, i.e., TNFR2, and countered by those linked to pro-apoptotic pathways, i.e. TNFR1. The outcomes of tumor-reactive T cell responses would depend on the presence/absence of AdRs or balance between an AdR and its counter-receptor on tumor cells. Therefore, current immunotherapies should focus on the discovery and inhibition of tumor-associated AdRs, or knocking out the nominal AdLs in tumor-specific T cells.

This work was supported by the Office of the Assistant Secretary of Defense for Health Affairs through the Breast Cancer Research Program under Award No. W81XWH2210793. Opinions, interpretations, conclusions, and recommendations are those of the authors and are not necessarily endorsed by the U.S. Department of Defense. This work was also supported by multi-investigator pilot funding from the VCU Massey Cancer Centre, supported in part with funding from NIH/NCI Cancer Centre support grants P30 CA016059.
Development of an Ultrahigh-plex Antibody Panel for Spatial Phenotyping of Murine Cancer Models
Authors: Nikulina Nadezhda , Fisher Jeremy , Loebelenz Jean , Jensen Samuel , LeungLee Paulina , Braubach Oliver
Published: August 2023

Single cell spatial phenotyping of protein biomarkers is an established tool for cancer researchers aiming to understand the biology of the tumor microenvironment. This technology has been developed around human tissues and there remains a paucity of applications available to study murine cancer models. Yet, mouse models are widely used for immuno-oncology (IO) research, justifying the development of spatial phenotyping applications for use in mice. We developed a >30-plex mouse IO antibody panel by selecting biomarkers for tumor, immune cell lineages, immune activation and checkpoints, as well as proliferation and structural markers. Purified antibodies, previously IHC validated in mouse models using rigorous protocols developed by Cell Signaling Technology (CST), were then conjugated to PhenoCycler barcodes, using a standardized conjugation protocol, and confirmed to show positive staining. We then performed ultrahigh-plex imaging using this IO antibody panel on the PhenoCycler-Fusion Platform, using murine lymphoid organs and/or tumor tissues. The resulting data were analyzed for cellular phenotypic compositions as well as the presence of cellular neighborhoods. The work presented here demonstrates the feasibility of conducting ultrahigh-plex spatial phenotyping in FFPE mouse tissues. The tools that were developed as part of this study can readily be deployed for discovery and translational research and will engender future comparative mouse and human spatial phenotyping studies that can further our understanding of cancer biology.
Indolent B-cell splenic lymphomas have T cells with an exhausted phenotype
Authors: Herne Christine Ann , Bruno Jennifer E , Baran Andrea M , Peterson Derick R , Mosmann Tim R , Quataert Sally A , Evans Andrew G , Zent Clive S , Chu Charles C
Published: August 2023

T cell dysregulation has been observed in many tumors, but has not been well-characterized in rare B-cell splenic lymphomas. We began defining the T cell populations in the tumor microenvironments of four human indolent B cell splenic lymphomas: splenic marginal zone lymphoma (SMZL), hairy cell leukemia (HCL), hairy cell leukemia variant (HCLv), and splenic diffuse red pulp small B-cell lymphoma (SDRPL). After Institutional Research Subjects Review Board approval, we studied de-identified cryopreserved bulk splenic tumor suspension cell isolates from fresh (less than 4 h) splenectomy tissue and matched intact formalin-fixed paraffin embedded (FFPE) tissue sections from 17 splenic lymphoma patients and 4 trauma patients (controls). Initial immunohistochemical staining analysis of FFPE tissue sections suggests that the distribution of intratumoral T cells is markedly different among B cell splenic lymphomas. In HCL, residual T cell zones appear retained, albeit diminished, while HCLv and SDRPL have markedly reduced T cells throughout with non-distinct zonation, and SMZL has prominent peritumoral collections of T cells at the interface of the neoplastic white pulp. High-parameter (32-color) fluorescence spectral flow cytometry (Cytek Aurora) analysis suggests that splenic lymphomas exhibit CD4+ and CD8+ T cells with higher proportions of transitional memory, regulatory, and exhausted phenotypes than controls. T cell activation may be enhanced in HCLv. HCLv and SMZL may have decreased naïve T cells. Finally, splenic lymphomas had higher proportions of T cells with an exhausted phenotype compared to trauma samples. In summary, our data suggests T cells are dysregulated in B-cell splenic lymphomas.

American Association of Immunologists, Hairy Cell Leukemia Foundation / Sass Foundation for Medical Research, and generous donations by Elizabeth Aaron.
Precise activation of antiviral memory T cells in solid tumors triggers a multi-cytokine response that ultimately drives tumor clearance
Authors: Gavil Noah Veis , Smith Olivia , Weyu Eyob , Joag Vineet , Vezys Vaiva , Masopust David
Published: August 2023

The reactivation of CD8+ tissue-resident memory T cells (TRM) triggers a broad, yet local response that mobilizes adaptive and innate immune cells. This immunostimulatory process can be initiated in solid tumors when infiltrating antiviral memory T cells are reactivated. The intratumoral delivery of viral peptides is sufficient to clear poorly immunogenic tumors in mice and synergizes with immune checkpoint therapy. This study investigated the mechanisms required for this anti-tumor response and novel “Peptide Alarm Therapy” that is currently being tested in a Phase I clinical trial. In contrast to expectations, viral peptide presentation by cancer cells was not required for treatment efficacy, and while NK and CD4 T cells are activated and/or recruited to the tumor microenvironment (TME) following antiviral memory T cell activation, depletion of either population did not limit tumor clearance. Interestingly, inflammatory monocytes were greatly increased in both the TME and tumor-draining lymph node (tdLN) following therapy. Moreover, antigen presenting cells (APCs) containing tumor-derived antigen accumulated within the tdLN. TNFα, IFNγ, and IL-2 are cytokines that can signal to APC populations and are produced by activated TRM. When these cytokines were blocked collectively, but not independently, the treatment efficacy of Peptide Alarm Therapy was greatly reduced. Further, when these cytokines were intratumorally injected, they recapitulated some phenomena achieved by antiviral T cell activation. Thus, local reactivation of antiviral memory T cells, which infiltrate tumors and possess known specificities, drives a multi-cytokine response that promotes the immune-stimulation that effectuates tumor clearance.

1R01CA238439-01A1, 1F30CA253992-01
Tumor cell secretome induces the expression of the immune checkpoint NKG2A on CD8+ T cells
Authors: Rodriguez-Garcia Gabriel J , Schares Henry A , Bachmann Brian O , Kim Young J , Korrer Michael J , Rathmell Jeffrey C
Published: August 2023

PD-1 blockade has been approved for head and neck squamous cell carcinoma (HNSCC) patients. However, only about 20% of HNSCC patients respond to PD-1 blockade. We hypothesize that other inhibitory receptors play a role as a resistance mechanism to PD-1 blockade. Our laboratory and others found that the NKG2A/HLA-E pathway is overexpressed in HNSCC patients. NKG2A is an inhibitory receptor expressed by NK cells and CD8+ T cells. How NKG2A expression is regulated on human T cells is not clear. Additionally, we previously identified a significant increase in NKG2A+ T cells in the tumors when compared to T cells in matching PBMC samples. Therefore, we hypothesize that the tumor microenvironment is inducing NKG2A’s expression. We have identified that tumor conditioned media (TCM) of HNSCC cell lines induce the expression of NKG2A on activated CD8+ T cells isolated from PBMCs of healthy donors. In our efforts to identify what factor(s) in the TCM are inducing NKG2A, we boiled the TCM to denature proteins or used filters to exclude molecules larger than 3kDa. However, both boiled and filtered TCM show similar induction of NKG2A. In addition, we further divided the media through hydrophobic fractionation of the filtered TCM and showed NKG2A is induced by the most hydrophobic fraction. Altogether, these findings suggests that NKG2A is being induced by a heat stable small hydrophobic molecule. This is a novel mechanism for tumor immune evasion, in which tumor cells induce NKG2A expression through secretion of small molecules. Our findings can lead to targets for novel cancer therapies or biomarkers for NKG2A blockade response.

Supported by grant from NIH/NCI (R01 CA217987) and the T32 AI138932 “Immunological Mechanisms of Disease Training Program”.
Discovery of Human Papilloma Virus and patient-specific neoantigens targeted by CD8+ T cells in HNSCC
Authors: Makani Venkata Krishna Kanth , Li Housaiyin , Kulkarni Aditi , Arshad Sanya , Vujanovic Lazar , Ferris Robert L , Joglekar Alok V
Published: August 2023

Head and neck squamous cell carcinoma (HNSCC) is amongst the rapidly growing form of cancer associated with high mutation burden and Human Papilloma virus (HPV) infection presenting approximately 800,000 new cases and 400,000 deaths annually. HPV-positive (HPV) HNSCC has been strongly associated with better prognosis than HPV-negative HNSCC due to the immune activation in the tumor microenvironment including cytotoxic CD8+ T cells. One of the key missing links is the relative contribution of HPV-reactive and neoantigen-reactive CD8+ T cells in anti-tumor response. To address this, we utilized epitope libraries presented in Signaling and Antigen-presenting Bifunctional Receptors (SABRs), which were previously described by our lab. As a proof-of-concept, we generated SABR libraries presenting 8392 epitopes derived from HPV or from personalized neoantigens from five HNSCC patients. In parallel, we used single cell RNA sequencing to identify 25 clonally expanded TCRs from tumor-infiltrating lymphocytes of the patients. Using SABR screens, we have identified several putative HPV-derived and neoantigen-derived epitopes targeted by CD8+ T cells in HNSCC patients. We have established a robust pipeline for identification of patient-specific epitope specificities from tumor-infiltrating CD8+ T cells. The goal is to determine whether and how effective anti-HPV or anti-neoantigen T cell responses may contribute to therapeutic efficacy using human samples from clinical trials and murine HNSCC models. Our study will aid in the discovery of novel tumor-associated antigens in HPV+ HNSCC patients, which can be successfully employed for precise adoptive T cell transfer therapies.
Generation of repetitive element-specific T cells to target ovarian cancer
Authors: Grundy Erin E , Cox Olivia , Lee Abigail , Omar Khadra , Chiappinelli Katherine Bakshian
Published: August 2023

The immunosuppressive tumor microenvironment in ovarian cancer (OC) enables these tumors to evade the immune system, resulting in late diagnosis and poor therapeutic outcomes for patients. This immunosuppression can be partially reversed with epigenetic inhibitors, which stimulate an immunogenic interferon response in OC cells by inducing the transcription of repetitive elements (REs). However, epigenetic therapy alone is not curative in clinical trials and new treatments that further augment the immune response to OC are needed. While REs are epigenetically silent in terminally differentiated cells, their increased expression in OC cells implicate these genomic elements as an unexplored pool of tumor antigens. As epigenetic therapy also upregulates antigen processing and presentation in OC cells, the generation of T cells specific for over-expressed RE-derived antigens may boost the immune recognition of OC cells.

Transducing healthy donor T cells with a T cell receptor construct that recognizes the RE ERV-K-Env resulted in antigen specificity only when co-cultured with B lymphoblastoid cells derived from the same donor that the T cell receptor was isolated from. Transduced T cells were not specific for ERV-K-Env in autologous systems. As previous reports have shown that ERV-K-Env-specific T cells can only be expanded from patients with OC, current studies are underway to expand T cells from OC patients. Upon successful expansion of ERV-K-Env-specific T cells from multiple OC patients, we will assess if specific recognition is enhanced by treating autologous tumor cells with epigenetic therapy. These efforts serve as a novel approach to enhance the immune response to OC and improve the overall OC patient survival rate.

NIH F31 CA271788-01 DOD W81XWH2010273