bluuebunny/crossref_metadata_2025 huggingface dataset:
Distinct B cell signatures and tertiary lymphoid structures are driven by two etiol-ogies in head and neck cancer
Authors: Ruffin Ayana T , Cillo Anthony R , Tabib Tracey , Liu Angen , Onkar Sayali , Kunning Sheryl , Lampfield Caleb , Abecassis Irina , Qi Zengbiao , Soose Ryan , Duvvuri Umamaheswar , Kim Seungwon , Lafyatis Robert , Ferris Robert L , Vignali Dario , Bruno Tullia C
Published: January 2023

B cells can regulate immune responses by presenting antigen, producing antigen-specific antibodies and immunomodulatory cytokines. Their role in the anti-tumor immune response is poorly understood. However, in many cancers including head and neck squamous cell carcinoma (HNSCC), intratumoral B cells correlate with better survival. HNSCC has two distinct etiologies (HPV−) and (HPV+) where patients who are HPV+ have increased B cell infiltration and respond better to therapy. We hypothesized that (1) intratumoral B cell phenotype is different between HPV+ and HPV− HNSCC (2) location within the tumor microenvironment (TME) is distinct and (3) antibodies produced by intratumoral B cell in HPV+ HNSCC are specific for viral antigens.

Using single-cell RNA sequencing and spectral flow cytometry, we observed that B cell signatures in HPV− HNSCC patients were predominantly memory B cells and plasma cells, while the signatures in HPV+ HNSCC were naïve and germinal center (GC) B cells. Further, we quantified B cells in tertiary lymphoid structures (TLS) using multispectral immunofluorescence, and the presence of GC-rich TLS were increased in HPV+ patients. In fact, GC-rich TLS within the tumor of HPV+ patients correlated with increased overall survival. Overall, high enrichment for GC B cells were positively associated with longer progression-free survival. Antibodies produced by intratumoral B cells from HPV+ patients were positive for HPV viral antigens. Ultimately, characterization of B cell phenotype and function in HNSCC is important for devising new therapeutic options for cancer patients. Specifically, therapeutics to enhance B cell responses in the TME should be prioritized as a compliment to T-cell mediated therapies.
Polyfunctional endogenous EBV-specific T cell response in nasopharyngeal carcinoma
Authors: Chen I-Yu , Lay Fang-Yun , Chia Jean-San , Liao Li-Jen , Chiu Yen-Ling
Published: January 2023

Nasopharyngeal carcinoma (NPC) is associated with Epstein-Barr virus (EBV) infection and can be treated by adoptive transfer of EBV-specific T cells. Although EBV-specific T cell immune response has been identified in NPC, the phenotype and functionality of EBV-specific, tissue-resident memory T cells has not been explored. Tumor-infiltrating lymphocytes (TILs) were isolated from newly-diagnosed NPC and two types of EBV-unrelated malignancies; oral squamous cell carcinoma (OSCC) and renal cell carcinoma (RCC) tissue samples. We found that endogenous EBV-specific T cell response can be detected in the all the tissue types, including NPC, OSCC and RCC, but were rare in peripheral blood. In all tissues, EBV-specific T cells in tumor tissues exhibit resident memory phenotype characterized by high CD69 and CD103 expression and also an exhaustion phenotype characterized by high CD39 and PD-1 expression. Unexpectedly, EBV-specific T cell frequency as well as response magnitude were not enhanced in NPC when compared to other tissue types. The lack of an enhanced EBV-specific T cell response magnitude in NPC tissue confirms the concept that enhancement of EBV-specific immunity by adoptive T cell therapy may improve the outcome of NPC.
A Novel Bifunctional Fusion Protein Comprising of TGF-βRII trap and IL15/IL-15Rα as an Immunotherapeutic against Cancer
Authors: Liu Bai , Chaturvedi Pallavi , Wang Meng , Zhu Xiaoyun , George Varghese , Revelo Kristine , Spanoudis Catherine , Kong Lin , Xing Jilan , Prendes Caitlin , You Lijing , Rhode Peter , Wong Hing C
Published: January 2023

Interleukin-15 (IL-15) is a promising cytokine for cancer therapy. However, it has shown limited antitumor efficacy as monotherapy in clinical trials to date. Removal of immunosuppression of the tumor microenvironment is considered a promising approach to enhance IL-15-mediated immune responses. TGF-β is a key component for creating an immunosuppressive tumor microenvironment. Therefore, we constructed a novel bifunctional fusion protein complex, designated HCW9218, comprising a soluble fusion of two TGFβRII, human tissue factor, and human IL-15, and a second soluble fusion of two TGFβRII and a sushi domain of IL-15Rα. HCW9218 activates IL-15R signaling and the dimeric TGFβRII functions as “trap” for all the three TGF-β isoforms. In healthy and tumor-bearing C57/BL6 mice, subcutaneously administrated HCW9218 was well tolerated with a long serum half-life and was able to stimulate and induce proliferation of CD8+ T cells and NK cells. Splenocytes from HCW9218-treated mice showed enhanced metabolic activity and cytotoxicity against Yac-1 target cells compared to untreated mouse splenocytes. In the syngeneic B16F10 melanoma mouse model, a single-dose of HCW9218 exhibited strong antitumor activity in combination with chemotherapy and TA99 antibody. HCW9218 also significantly increased immune cell infiltration into tumors. Mutagenesis studies demonstrated that both the TGFβRII and IL-15/IL-15Rα domains are essential for antitumor efficacy of HCW9218. Collectively, our preclinical data demonstrate that HCW9218 elicits potent immune responses with a remarkable safety profile and serves as a novel immunotherapeutic against cancer alone or in combination with therapeutic antibodies.
Antibiotic-mediated gut dysbiosis alters innate-like T cell frequency and phenotype in tumor-bearing mice
Authors: Wong Stephanie Wing-Yan , Ohashi Pamela S. , Sacher Adrian G.
Published: January 2023

Innate-like T cells bind to non-peptide antigens in a TCR-dependent manner and are not MHC-restricted. This subpopulation of T cells consists of mucosal associated invariant T cells (MAIT), natural killer T cells (NKT), and gamma delta (γδ) T cells. Innate-like T cells can recognize microbially-derived antigens and interact with gut commensals to help maintain intestinal homeostasis. Additionally, they are implicated in tumour immunity as producers of cytokines and cytotoxic molecules. Thus, innate-like T cells may serve as an intermediary between the gut microbiota and tumor development. Using syngeneic mouse tumour models and flow cytometric analysis, we aim to evaluate the relationships between gut microbiota modulation, tumour growth, checkpoint inhibitor response, and innate-like T cell phenotype. Gut dysbiosis was induced using broad spectrum oral antibiotics two weeks prior to subcutaneous injection of MC38 colon adenocarcinoma cells. Mice with palpable tumours were treated twice with anti-PD-L1 during the experiment. Compared to vehicle control, tumor size reduction in response to anti-PD-L1 was not significantly altered with antibiotic treatment. The following observations were noted in antibiotic-treated mice. The frequency of γδ T cells was decreased in the tumour, blood, and spleen. MAIT, NKT, and γδ T cells in the spleen had lower expression of tissue localization-associated markers, CD103 and CXCR6. MAIT cell activation state, characterized by CD25 and CD69 expression, was also elevated in the spleen. These findings suggest that changes to the immune landscape of tumor-bearing mice due to antibiotic-driven dysbiosis is characterized by alterations in innate-like T cell abundance and phenotype.
MHC genotype, immune repertoire, and tumor immunity all influence spontaneous cancer development and metastasis in spontaneous breast cancer models
Authors: Castro Sarah A , Pena Alvaro , Khazaie Khashayarsha , Pease Larry R
Published: January 2023

20 to 30% of breast cancers overexpress ERRB2, encoding the human epidermal growth factor-2 receptor (HER2). Overexpression of HER2 is associated with poorer prognosis, particularly in metastatic disease. Although HER2 therapeutics exists many patients eventually develop uncontrolled tumor growth. The BALB/c-neuT mouse has been a popular model for investigating HER2-positive breast cancer. We have found that immune responsiveness and MHC genotype are important factors in spontaneous tumor development and tumor rejection in the neuT model. To assess the influence of immune response potential on tumor biology we introduced the skg ZAP-70 mutation or the H-2b MHC haplotype into the neuT model. Spontaneous tumors appeared with decreased frequency and metastasis or increased frequency and metastasis, respectively, in comparison to the BALB/c model. H-2b tumors were readily transplantable into the breast fat pads of syngeneic BALB.B-neuT hosts which developed systemic immune abnormalities very similar to those observed in BALB/c - neuT or BALB/c WT mice bearing neuT tumors. However, BALB.B WT hosts initially accepted the grafts, but subsequently rejected them after they were established. Adoptive transfer of splenocytes from BALB.B WT mice which had rejected tumor conferred resistance to the tumor grafts, as well as to spontaneous tumors developing in BALB.B neuT littermates. The strong H-2b-mediated antitumor response in WT mice indicates efficient antigen presentation, and presumably leads to strong tolerance in neuT+ animals. The importance of an intact T cell signaling and MHC genotype indicate a critical role for T cell-mediated immunity and tolerance in the biology of the spontaneously developing tumor and metastasis.
Reactive oxygen species and L-type calcium channels mediates hypoxia-dependent CCL2 secretion in mast cells
Authors: Castillo-Arellano Jorge Ivan , Ramírez-Moreno Itzel Guadalupe , Ibarra-Sánchez Alfredo , Jiménez-Estrada Manuel , Blank Ulrich , Gonzalez-Espinosa Claudia
Published: January 2023

Mast cells (MCs) are well-known cells for their important role in initiating allergic response through of cross-linking of FcɛRI receptor with IgE-Ag complexes. As participants on inflammatory reactions, they are exposed to environmental conditions such as hypoxia, which on MC-dependent cytokine secretion has not been described.

Here, we show that bone marrow derived MCs (BMMCs) exposed to hypoxia, produce important amounts of CCL2 mRNA and protein, that is maximal secreted between 12 and 24 hours. CCL2 production induced by hypoxia was sensitive to the antioxidant trolox and L-type Ca2+ channel blocker nifedipine.

In order to analyze the possible mechanism involved on hypoxia-induced CCL2 production, we found that this conditions caused an increase on reactive oxygen species (ROS) that was maximal after one hour. We also observed an increase on [Ca2+]i two hours after hypoxia, which was sensitive to trolox. With the aim to confirm this mechanism, BMMCs were treated with H2O2 and as expected, H2O2 caused an increase on ROS and [Ca2+]i in BMMCs. Ca2+ increase produced by H2O2 was sensitive to nifedipine. CCL2 mRNA accumulation and CCL2 protein secretion induced by H2O2 was sensitive to trolox and nifedipine too.

When trolox and nifedipine were used in the B16-F1 murine model of melanoma, a close relationship between ROS, Ca2+ rise and CCL2 production was found, since those compounds were able to prevent pimonidazole-positive areas and CCL2 accumulation inside tumors. Our data suggest that conditions prevalent in tumor microenvironment contribute to the generation of ROS and L-type Ca2+ channel-dependent Ca2+ entry in MCs, causing the secretion of the potent immunosuppressor chemokine CCL2.
Metastatic ovarian cancer shapes the abdominal immune microenvironment by manipulating IL20-IL20RA signaling pathway
Authors: Xiang Rong , Li Jia , Wang Longong , Shi Yi
Published: January 2023

The disseminated of Ovarian cancer (OC) cells shape the tumor abdominal immune environment (TAIE) for survival. However, the key genes and mechanisms mediating the reprogramming of TAIE by OC remain largely unexplored. Here, through a genome-wide in vivo knockout screening by CRISPR/Cas9, we identified IL-20-IL20RA as a key checkpoint preventing the abdominal metastasis of OC cells. In OC patients, the level of IL20RA dramatically decreases in metastatic OC cells when compared with paired OC specimen in the primary sites. Ectopic expression of IL20RA in highly metastatic OC cells greatly suppressed the abdominal metastasis and reduced the formation of ascites. In contrast, silencing IL20RA could increase the metastasis of OC cells. We further examined IL02RA mediated crosstalk between the disseminated cancer cells and the TAIE in both in vivo and in vitro. Silencing IL20RA in OC cells dramatically decreased the M1 macrophages and increased the M2 subtype, while the populations of T and B lymphocytes in ascites were not affected. Further mechanistic studies revealed that the OC cells greatly induced the mesothelial cells in peritoneum to produce IL20RA ligands, i.e., IL-20 and IL-24, which in turn activated the IL20RA downstream signaling in OC cells to trigger the production and maturation of IL-18. IL-18, when secreted into the microenvironment, was able to promote the polarization of macrophages into M1-subtype to kill the cancer cells. Thus, we identified a novel immune checkpoint IL-20/IL20RA that had to be overcome during the metastasis of OC cells to avert an anti-tumor TAIE, which has the potential to be developed as a novel immunotherapy method for OC.
IL-15 deficient colon carcinomas have decreased cytolytic lymphocytes and skewed myeloid cell populations
Authors: Schluns Kimberly S , Chou Chih-Chien , Carrero Rosa M Santana , Davis Richard Eric , Hegde Shweta M
Published: January 2023

Loss of IL-15 expression by colon tumors strongly correlates with classification as microsatellite stable, decreased tumor infiltrating lymphocytes (TILs), increased metastasis, and poor responses to immunotherapy. To better understand how IL-15 expression regulates immune cells in the tumor microenvironment (TME) and identify the importance of IL-15 expressed by tumor cells, we developed a mouse model system to examine this by generating MC-38 tumor cells deficient in IL-15 using CRISPR/Cas9 system. After one round of IL-15 deletion, MC-38 cells that lack one IL-15 allele (i.e IL-15+/− MC-38 cells) were identified and expressed decreased levels of soluble IL-15 complexes. Surprisingly, deletion of one IL-15 allele was sufficient to affect tumor growth as IL-15+/− MC-38 tumors grew faster than Wt MC-38 tumors. IL-15+/− MC-38 tumors had decreased CD8 and NK TILs and increased CD11b+Ly6G+ and CD11b+Ly6ChiLy6G− myeloid cells than Wt tumors. Gene expression analysis of myeloid cells showed that blocking IL-15 in tumors increased expression of CD206, ARG1, and TDO2 and decreased IL-12β and IL-6. A second round of deletion led to complete deletion of IL-15 in MC-38 cells (i.e. IL-15−/− MC-38) and upon implantation developed tumors with further decreases in CD8 and NK TILs. In mice lacking IL-15Rα in the intestinal epithelium, AOM/DSS-induced colon tumors also harbored decreased numbers of CD8 TILs. These results demonstrate that even partially decreasing levels of IL-15 in the TME negatively impacts the numbers of CD8 and NK TILs and skews myeloid cells towards a pro-tumor landscape. Overall, these models that can be used to identify the mechanisms contributing to the poor therapeutic responses observed in human colon carcinomas.
An effective peptide vaccine strategy circumventing heterogeneity of murine myeloid leukemia
Authors: Cho Hyun-Il , Shin A-Ri , Lee Sang-Eun , Choi Haeyoun , Sohn Hyun-Jung , Kim Tai-Gyu
Published: January 2023

Therapeutic cancer vaccines are an attractive approach for treating malignant tumors, and successful tumor eradication depends primarily on controlling tumor immunosuppression status as well as heterogeneity of tumor cells driven by epigenetic alterations. Here we describe an effective peptide vaccination approach capable of circumventing antigenic heterogeneity by targeting multiple shared tumor antigens simultaneously in murine myeloid leukemia. Peptide-loaded dendritic cell (DC) prime and noninfectious peptide booster heterologous immunizations were assessed for the immunogenicity of polo-like kinase-1 (PLK1)-derived peptides, and targeting multiple shared tumor antigens simultaneously with PD-L1 blockade was evaluated for antitumor effects against murine myeloid leukemia. A synthetic PLK1122 (DSDFVFVVL)-based heterologous vaccination generated large numbers of long-lasting antigen-specific CD8 T-cells eliciting therapeutic effects against various established tumors. The therapeutic efficacy of single antigen-targeting PLK1122-based vaccine with sufficient endurance of PD-L1 blockade toward C1498 leukemia relied on the heterogeneous clonal levels of MHC-I and PD-L1 expression. A novel multi-peptide-based vaccination targeting PLK1 and survivin simultaneously along with PD1 blockade led to complete tumor eradication and long-term survival in mice with clonally heterologous C1498 myeloid leukemia. Our findings suggest that PLK1 could be an attractive immunotherapeutic target antigen for cancer immunotherapy, and that similar strategies would be applicable for the optimization of cancer vaccines for the treatment of numerous viral diseases and malignant tumors.
Exploiting metabolism of vaccine-elicited T cells to enhance tumor immunotherapy
Authors: Willett Benjamin A. S. , Klarquist Jared , D’Alessandro Angelo , Kedl Ross M.
Published: January 2023

Given the highly glycolytic nature of most cancers, glycolysis inhibition remains a strong potential treatment strategy. However, effective glycolysis blockade may also inhibit the function and survival of infiltrating T cells. Unlike T cells activated by infections and cancer, T cells activated by subunit vaccination do not rely on aerobic glycolysis, either during their initial proliferative burst or thereafter. Instead, these vaccine-elicited T cells primarily rely on mitochondrial metabolism of fatty acids and OXPHOS. Thus, we hypothesize that vaccine-elicited T cells will maintain anti-cancer therapeutic efficacy even under conditions of glycolytic inhibition. Additionally, vaccine-elicited T cells are characterized by robust expression of the transcription factor Tcf1, which has been shown to identify a stem-like pool of T cells capable of sustaining an anti-cancer response. We show that a therapeutic regimen combining subunit vaccination with glycolysis inhibition significantly impairs tumor growth in mice. These effects are further enhanced by the addition of clinically-relevant αPD-1 inhibitors. Collectively, these data show the potential of controlling tumor progression by exploiting the distinct metabolic and transcriptional program of vaccine-elicited T cells through a novel combination immuno/metabolomic therapeutic regimen.
Development of AAV6-based vaccine with prime-boost capacity by using exosome-protected viral particles
Authors: Aslanidi George , Krotova Karina
Published: January 2023

We recently showed that the vaccine based on optimized AAV6 vector generates high level of antigen (Ag)-specific immune response. However, certain limitations in use of viral vectors as vaccine carriers need to be overcome to ensure its wide use for clinical application. Particularly, the primary injection of AAV lead to generation of neutralizing antibodies (NAB) against the virus capsid, hampering AAV re-administration. To address this limitation, we exploited the ability of AAV to accumulate in exosomes. AAV vectors associated with exosomes (exoAAV) were purified from conditioned media of HEK 293 cells, along with the standard AAV production (stAAV) from the same lysed cells. Here we report several significant observations that highlight the advantages of using exoAAV for development in a prime-boost protocol. First, we confirmed comparable infectivity between stAAV and exoAAV using YFP and luciferase reporter genes. Second, we showed that exoAAV is highly resistant to NAB compared to stAAV. Third, we developed a prime-boost protocol with injections of stAAV followed by exoAAV expressing OVA Ag as a model system. This protocol led to significantly higher levels of OVA-specific CD8+ T cells compared to the single injection method. The therapeutic potential of prime-boost was evaluated in a melanoma B16F10 tumor prevention model in comparison to single injection vaccination. While a single injection was able to significantly delay tumor growth, the prime-boost protocol completely prevented tumor development. In summary, we use exosome-protected AAV to allow for repeated vaccination, and this method produced a superior protective immune response compared to single vaccination.
A novel TLR adjuvant increases the efficacy of vaccines against fentanyl-induced toxicity and overdose
Authors: Miller Shannon M , Robinson Christine , Whitacre Margaret , Buhl Cassandra , Crouse Bethany , Pravetoni Marco , Evans Jay T
Published: January 2023

The widespread illicit use of fentanyl and its analogs is associated with the increased incidence of fatal drug overdoses in the United States in recent years. Pre-clinical studies with vaccines targeting opioids show selectivity and efficacy but clinical data suggests vaccine efficacy against drugs of abuse may be limited by individual variability and a requirement for high and sustained antibody levels. New strategies are needed to enhance vaccine efficacy and increase the quantity and quality of the anti-drug antibody response. We investigated the ability of novel synthetic TLR adjuvants to increase anti-fentanyl antibody titers and protect from fentanyl challenge in mice. Fentanyl-based haptens were conjugated to CRM and combined with alum, novel synthetic TLR adjuvants, or combinations thereof. The most promising TLR adjuvants and combination adjuvants increased anti-fentanyl hapten total IgG, IgG1, and IgG2a antibody concentrations. Splenocyte restimulation with CRM protein showed adjuvant combinations significantly increased secretion of Th1-type cytokines compared to antigen only or single adjuvants alone. Likewise, in a fentanyl challenge model, combination adjuvanted vaccines increased protection from respiratory depression compared to mice vaccinated with antigen alone or single adjuvant controls. These data demonstrate that TLR adjuvants improve the anti-fentanyl adaptive immune response and increase protection against fentanyl challenge in mice, and may enhance vaccine-induced protection against fentanyl overdose in humans.
LAG-3: a potentially important checkpoint of the humoral immune response
Authors: Haun Brien K , To Albert , Wong Teri Ann , Nakano Eileen , Ndhlovu Lishomwa , Lehrer Axel T
Published: January 2023

Emerging infectious viruses such as Zika virus (ZIKV) pose serious threats to human health. Currently, there are no FDA-approved therapeutics or vaccines available for ZIKV. Our laboratory has developed a vaccine candidate using recombinant envelope protein (E) of the ZIKV. Our previous work has shown that neutralizing antibody responses are the protective factor upon viral challenge in BALB/c mice and non-human primates. Since lymphocyte-activation gene 3 (LAG-3) is an immune checkpoint receptor that directly interacts with MHC-II during antigen presentation, we decided to use the mouse model to investigate the effects of administering a LAG-3 blockade prior to immunization. We hypothesized that this pre-treatment may increase the number of CD4+ T helper (Th) cells available to interact with B cells in germinal centers, thus increasing the antibody response. We observed an increase in CD4+ T cells among the treatment group, changes in overall Th phenotypes, and a reduction of phenotypically exhausted CD8+ T cells. This alteration in cell populations was accompanied by an increase in vaccine potency based on ZIKV neutralizing titers. Based on preliminary observations, we believe that pre-treatment with LAG-3 blockade prior to immunization may modulate the magnitude of humoral responses by increasing Th cell activity in germinal centers. Overall, we think that LAG-3 may be an important controller of the quality of adaptive immune responses and work to further investigate these phenomena is ongoing.
The synergy of TLR and STING in cancer immunity
Authors: Taylor David R , Young Kim , Korrer Michael J
Published: January 2023

Adjuvants are essential components of cancer vaccine formulations to promote effective immune responses. We initially screened multiple immune-activating targets, including TLR and STING adjuvants using in-vitro assays to test their ability to activate bone marrow DC (BMDC) and showed STING had the most potent activation (i.e., MHC and co-stimulatory molecules) on DCs. Since STING and TLR signaling is non-redundant, TLR and STING adjuvants were combined to enhance DC activation. We utilized the multidimensional synergy of combinations (MuSyc), a novel synergy algorithm, to assess molecular synergy in combining STING and TLR adjuvants, and we noted that the combination of R848 (TLR7-8) plus STING agonists provided a synergistic efficacious and potent response on BMDCs. From this data-set, we generated a MuSYC synergy strategy where we will utilize max signal dose for STING adjuvant and ten times less of max signal dose for R848 (TLR7-8) to determine synergy for other assays. We tested this same MuSYC strategy for activation of human monocytic cell line THP-1 and determined that the synergy strategy induced similar or better activation effects compared to the max signal dose for both adjuvants. Finally, we tested the in-vivo anti-tumor immune response for the B16 melanoma and MOC2 head/neck tumor-bearing vaccination models that resulted in an improved anti-tumor response for the combinatorial STING-TLR adjuvanted vaccines compared to the single agents.
Porcine messenger RNA and microRNA pathway analysis of Porcine Reproductive and Respiratory Syndrome Virus infection
Authors: Miller Laura C , Fleming Damarius
Published: January 2023

OBJECTIVE

Studies of the host-pathogen interaction between pigs and porcine respiratory and reproductive syndrome virus (PRRSV) have tried to uncover the process that allows the virus to evade the immune response to its benefit. This study examined the effects messenger RNA (mRNA) and microRNA (miRNA) expression has on host biological networks.

METHODS

Analysis was carried using whole blood from both mock infected and PRRSV infected pigs. Transcriptomic techniques were then applied to examine the differential expression of miRNAs using Hisat2 for alignment to the Sus scrofa 10.2 genome and changes in expression analyzed using DeSeq2. Statistical significance for pathway analysis was set at FDR ≤ 0.5. The resulting list of miRNAs were examined for their effects on porcine biological networks using the mirPATH software to generate KEGG pathways affected by changes in miRNA differential expression.

RESULTS

The results of the statistically significant (FDR ≤ 0.15) miRNAs were defined by days post inoculation (dpi) to examine biological networks inhibited or activated by the identified miRNAs. Post-transcriptional control of host gene expression appeared to take place over several host biological pathway groupings which effect homeostasis that included immune, metabolic, and structural pathways. This included pathways such as the proteoglycan in cancer pathways that showed multiple genes targeted for inhibition that would cause host dysregulation and impair the ability to properly maintain homeostasis during infection.

CONCLUSION

The analyses suggests inhibition and activation of networks involved in viral entry, proliferation, and pro-inflammatory signaling may underlie the ability of PRRSV to hinder homeostasis.
Characterization of systemic, mucosal and lymphoid granulocyte subsets in rhesus macaques
Authors: Manickam Cordelia , Jones Rhianna , Ram Daniel , Kroll Kyle , Hueber Brady , Varner Valerie , Reeves R. Keith
Published: January 2023

Granulocytes mediate immune protection by phagocytosis, NETs, degranulation, antibody mediated effector functions and recruitment of innate and adaptive cells against pathogens. However, limited research describing granulocytes in HIV infection and in mucosal tissues exists. In particular, the role of granulocytes in the rhesus macaque (RM) model of HIV infection and vaccine/treatment studies have been understudied. Our goal was to characterize granulocyte subsets in systemic, mucosal and lymphoid tissues of naïve and infected RM. Mononuclear cells from jejunum, colon, cervix, vagina, lymph nodes, spleen, liver, and whole blood of naïve, SIV+, and SHIV+ RM were characterized using flow cytometry and Imagestream. Phenotype of granulocyte subsets were identified as- CD45+CD66+CD49d+ eosinophils, CD45+CD66+CD14+ neutrophils, and CD45+CD123+FcRE+ basophils. Polynuclear and mononuclear visualization with DAPI staining and surface marker images by Imagestream analysis further confirmed granulocytic phenotypes. Flow cytometry data showed that differential expression of FcRgs (CD64, CD32 and CD16) can be helpful in distinguishing granulocyte subsets while FcRE was only expressed by basophils. Interestingly, frequencies of granulocyte populations were generally higher in mucosal compared to lymphoid tissues in both naïve and infected animals. Eosinophils were particularly enriched within jejunal tissue, with a median of 45% of CD45+ cells. High frequencies of granulocytes in mucosal tissues indicate their importance in gut and reproductive tract immunity. Overall, further investigation is warranted to understand the significance of granulocytes in tissue homeostasis and infection.
Oclacitinib induces Foxp3 expression in CD4+ T cells under activation conditions
Authors: Jasiecka-Mikolajczyk Agnieszka , Socha Piotr , Jaroszewski Jerzy Jan , Maslanka Tomasz
Published: January 2023

Oclacitinib (OCL) is a novel immunosuppressive agent approved for dogs that controls inflammation in allergic disease via the inhibition of the JAK/STAT pathway. The present study attempts to shed light on the involvement of Foxp3-expressing regulatory CD4+ T (Treg) cells and inducible type 1 regulatory T (Tr1) cells in the anti-inflammatory and anti-allergic actions of OCL. Our previous study demonstrated that exposure of unstimulated canine lymphocytes to OCL did not affect Foxp3 expression in T cells. This study investigates the effect of OCL on Foxp3 expression by canine CD4+ T cells in the course of in vitro activation. Effect of OCL on generation of Tr1 (i.e. CD223+CD49b+CD4+) cells was studied using murine lymphocytes due to the lack of appropriate antibodies for the induction and detection of these cells in dogs. The exposure of canine lymphocytes to both concentration of OCL, with a concomitant stimulation, led to a significant increase in the percentage of Foxp3-expressing cells within the CD4+ T cell subset. In the light of our previous results, it can be suggested that the effect of OCL on Foxp3 expression in CD4+ T cells may depend on T cell activation status. The mean percentage of CD3/CD28-induced CD223+CD49b+CD4+ T cells was approximately 6- and 2-fold lower in the cultures exposed to OCL 10−6 and 10−7M, respectively, compared to untreated cells. Thus, the results strongly suggest that the generation of inducible Foxp3-expressing CD4+ T cells may constitute one of mechanisms responsible for the production of anti-inflammatory and anti-allergic effects of OCL. These effects are not mediated by generation of Tr1 cells. What is more, the present results indicate that OCL may counteract the induction of these cells.
Code Blue: Nurse Cells Can Resuscitate Lymphocyte Receptors in Nurse Shark Thymus by Secondary Rearrangement
Authors: Mitchell Christian Danielle , Criscitiello Michael F
Published: January 2023

The thymus is the most conserved primary lymphoid organ present, in some form, in mammals to all the way back in lamprey. It is composed of a densely-packed outer cortex and an exiguous inner medulla. The thymus functions to provide a microenvironment conducive to T lymphocyte maturation and receptor repertoire formation. Sharks are situated at a pivotal location in evolutionary immunology. They are the oldest vertebrates to have similar adaptive immune characteristics to mammals. Shark lymphocyte antigen receptors are diversified by V(D)J recombination via RAG and heavy chain class switch recombination catalyzed by AID. Somatic hypermutation occurs in both B and T cell antigen receptors of shark, mediated by AID as well. Shark T cell receptors also employ antibody-like NARTCR and IgHV components. Looking at the oldest lymphoid tissue in the oldest vertebrate ancestor with adaptive characteristics similar to mammals will yield important information on the evolution of the adaptive immune response as it relates to T lymphocytes. Nurse cells are cortical thymic epithelial cells that play an important role in selection of functional T cells during the maturation process. Nurse cells have been shown, in mice, to surround non-functional T cells in order to allow them to reconfigure their receptors and attempt selection another time via RAG and/or AID. Through the use of immunohistochemistry and in situ hybridization, we will investigate whether nurse cells provide a microenvironment for shark thymocyte receptor revision in sharks as in mammals.
Transcriptomic profiling of Strongylocentrotus purpuratus coelomocyte populations
Authors: Fugmann Sebastian , Cheng Jun Fang , Huang Hsiao Han , Buckley Katherine M
Published: January 2023

Coelomocytes are important cellular mediators of immunity in echinoderms. In the purple sea urchin (Strongylocentrotus purpuratus) these cells can readily be divided into phagocytes, vibratile cells, red spherule cells, and colorless spherule cells based on their morphology. While some information has been reported about genes that are expressed in specific types of coelomocytes, a comprehensive analysis of the gene expression pattern in each cell type was not available. Using a density gradient coelomocytes from individual adult urchins were separated into three populations (phagocytes, a mixture of vibratile and colorless spherule cells, and red spherule cells), RNA was isolated, and next generation sequencing was employed to determine the transcriptome of each cell population. All three cell populations showed clear differences in their gene expression patterns, and the differences in transcript levels for selected genes that were found to be uniquely expressed in one cell type were confirmed by qRT-PCR using cDNA obtained from additional sea urchins. We now complemented this bulk population data with single-cell RNAseq to define coelomocyte identity and function based on their gene expression profiles. The new findings from this study will be discussed.

This work was supported by the Chang Gung Memorial Hospital (CMRPD1C0242, CMRPD1F0421, and CORPD1F0051) and the Ministry of Science and Technology Taiwan (MOST 107-2320-B-182-002 and 108-2320-B-182 -018 -MY3)
Development of a multiplex assay for the bovine inflammatory cytokines IL-1β, IL-6, and TNF-α
Authors: Chitko-McKown Carol G , Bierman Stacy L.
Published: January 2023

Commercially available bovine-specific assays are limited in number, and multiplex assays for this species are rare. Our objective was to develop a multiplex assay for the bovine inflammatory cytokines IL-1β, IL-6, and TNF-α using the Meso Scale Discovery U-PLEX platform.

"Do-It-Yourself" ELISA kits that contained polyclonal antibodies, both unlabeled and biotinylated, and the specific recombinant bovine cytokine, were purchased for each of these three cytokines. The biotinylated antibodies that would normally be used as reporters in a traditional ELISA were coupled to linkers that bind to specific locations within each well of the U-PLEX plate. Unique linkers were used for each of the cytokines. The unlabeled antibodies were conjugated with electrochemiluminescent labels to serve as detection antibodies. Each cytokine assay was optimized individually prior to performing an optimization on the multiplex assay containing reagents for all three cytokines. To calculate cytokine concentrations, standard curves were developed using the recombinant cytokines and were run concurrently on each plate.

Standard curves for IL-1β and TNF-α were run at concentrations ranging from 0 – 50,000pg/ml, and for IL-6 from 0 – 10,000pg/ml. The minimum average concentrations measured by the standard curves were 5.3pg/ml, 0.92pg/ml, and 22.34pg/ml for IL-1β, IL-6, and TNF-α respectively as determined by data from seven plates. Only 37 out of 280 unique samples assayed had IL-β or TNF-α concentrations above the upper limits of these standard curves.

In our hands the U-PLEX platform was a viable means to develop analyte- and species-specific multiplex assays using privately developed or purchased sets of commercially available reagents.
Mitigation of Equine Recurrent Uveitis through topical Suppressor of Cytokine signaling-1 (SOCS1) mimetic peptide: Open Label Safety and Efficacy Pilot study
Authors: Larkin Joseph , Polk Timothy , Sharma Jatin , Bae Sarah , Barr Olivia , Jones Amari , Kitchen Holly , Wihelmy Michelle , Devine Katherine , Plummer Caryn
Published: January 2023

Recurrent Uveitis (RU), is a relapsing/remitting disease that accounts for more than 10% of severe visual handicaps in US patients per year, despite treatments that often include steroids. Even when effective, prolonged steroid use is accompanied by significant negative side effects. As such, there is a critical need for the development of novel therapeutic strategies to treat RU. Notably, equine recurrent uveitis (ERU) is the only spontaneous model of human recurrent uveitis, and is also a leading cause of equine blindness. RU is driven by aberrant inflammation from the immune system. Notably, the body naturally generates a family of intracellular proteins, known as suppressors of cytokine signaling (SOCS), which prevent excessive inflammatory responses. We have previously shown that topical application of an eye drop, containing a cell-penetrating peptide that mimics SOCS1 (SOCS1-KIR), could mitigate experimental uveitis in mice. In order to evaluate the ability to translate our preclinical findings we conducted a first in equine, open label study in which we evaluated the safety and efficacy of topical SOCS1 peptide administration in ERU patients. We found that topical SOCS1-KIR administration was well tolerated in equine patients. Moreover, topical administration of SOCS1-KIR peptide twice a day was associated with reductions in ocular discomfort, hyperemia, and aqueous flare. Additionally, biochemical analyses of SOCS1-KIR revealed an enrichment strategy that could potentially be utilized to optimize ocular peptide dosing. Together, these results prove justification for conducting larger additional treatment studies in horses, and exploring SOCS1-KIR use in humans.
LAIV vaccination prior to pregnancy protects against lethal influenza infection and prevents infection-associated outcomes in offspring
Authors: Strutt Tara M , Satchmei Ali R.
Published: January 2023

Influenza is a highly contagious respiratory infection of public health concern. Pregnant women are among those at greater risk of suffering from serious influenza A virus (IAV) infection. Compromised cell-mediated immune responses, low inactivated vaccine efficacy, and poor vaccine uptake all increase the likelihood that exposure to the virus during pregnancy will result in infection-associated maternal and gestational complications such as stillbirths, preterm delivery, and low birthweights. The protective potential of IAV-specific immunological T cell memory induced by vaccination with a cold adapted live attenuated influenza vaccine (LAIV) during pregnancy has not been described; this immunity has the potential to mediate universal protection against influenza. Here, we investigated how LAIV priming before pregnancy and subsequent heterosubtypic IAV challenge during pregnancy impacts maternal and fetal outcomes in mice. LAIV priming prior to pregnancy completely protects pregnant dams against the morbidity and mortality of infection that is lethal to unprimed mice and primed dams go on to drop average sized litters. Pups born to unprimed dams are significantly smaller in terms of weight on days 3, 7, 14, and 21 when compared to pups born to LAIV primed dams. In contrast, morphological development determined by crown-rump length and skeletal measurements, while initially hindered, does not significantly differ between pups born to unprimed and primed dams after the first week following birth. LAIV priming prior to pregnancy thus appears to prevent adverse maternal and fetal IAV-associated outcomes. These data support that LAIV has potential for use as a universal vaccine to protect pregnant females against pandemic IAV.
Respiratory Syncytial Virus Induces a Functional tRNA-derived Fragment to Promote Infection by Targeting SYNE2
Authors: Chen Yu , Bao Xiaoyong
Published: January 2023

RSV is the most common cause of lower respiratory tract infection, such as pneumonia and bronchiolitis, in infants. RSV also leads to significant outpatient visits. However the host-virus interaction is still poorly understood. The tRNA-derived RNA fragments (tRFs) is a recently discovered family of small non-coding RNAs. They are significantly induced by RSV and some have been demonstrated to play a role in mediating virus replication and host responses. However, the mechanisms used by most tRFs for the regulation are not clear. Previously, we have shown that an RSV-induced tRF derived from the 5′-end of mature tRNAs decoding GlyCCC (named as tRF5-GlyCCC) promotes RSV replication and has a regulatory function in gene expression. To identify target(s) of tRF5-GlyGCC, we first investigated tRF5-GlyCC-associated mRNAs by HIgh-Throughput Sequencing of RNAs Associated with Biotinylated tRF (HITS-ABt). The targets were prioritized by identifying common mRNAs whose expression is suppressed by RSV and predicted free energy for the interaction of tRF5-GlyCCC and targets. SYNE-2 is one of the most likely target genes for tRF5-GlyCCC. Our experiments confirmed that SYNE-2 was downregulated by RSV infection. In addition, we found that antisense oligonucleotides of tRF5-GlyCCC reversed the SYNE-2 downregulation by RSV. We also found that SYNE2 is not involved in the replication of RSV, but important in RSV-regulated innate cellular response. Two immune mediators MIP-1β and IP-10 are selectively regulated by tRF5-GlyGCC, possibly through controlling the activation of transcription factor p50. In summary, this study provides accumulative evidence that tRFs induced by RSV are not degraded byproducts, but functional molecules.
Sodium pyruvate reduces immune signaling during influenza A virus infection in macrophages
Authors: Reel Jessica Morgan , Abysalamah Hazzar M. , Lupfer Christopher R
Published: January 2023

Pyruvate is a key metabolite for energy synthesis. After the production of pyruvate through glycolysis, the molecule is shuttled into the mitochondria. Once there, it is modified for use in the TCA cycle and the energy derived from pyruvate is eventually converted into ATP. While pyruvate is a key metabolite, it also appears to have anti-inflammatory properties. In models of sterile inflammation, like ischemia, pyruvate limits inflammation. We observed that infecting murine bone marrow derived macrophages (BMDM) with influenza A virus (IAV) and treating those macrophages with sodium pyruvate results in lower inflammasome activation and reactive oxygen species (ROS) production. However, this was specific to IAV infection as inflammasome activation and ROS were not affected by sodium pyruvate during E. coli infection or lipopolysaccharide (LPS) and ATP treatment of BMDMs. Our results show that IAV induces a potent and unique metabolic reprograming of infected cells. The addition of sodium pyruvate facilitates ATP production, which correlates with less mitochondrial damage and reduced inflammasome activation. Thus, pyruvate deserves additional examination as an anti-inflammatory treatment in diseases where mitochondrial metabolic stress is a factor.
MLKL-independent RIPK3 signaling alters adaptive immune responses to IAV pneumonia
Authors: Riegler Ashleigh Nichole , Jenkins Meagan M , Ballesteros-Tato Andre , Orihuela Carlos J
Published: January 2023

Necroptosis, a programmed form of lytic cell death, is initiated by various viral and bacterial pathogens as result of irreparable ion dysregulation and energy depletion. This cellular damage results in the activation of the kinases RIPK1 and RIPK3, then the activation and membrane targeting of MLKL, resulting in cell lysis. We demonstrated that necroptosis of airway epithelial cells (AECs) is key in the development of the adaptive immune response to the bacterial pathogen, Streptococcus pneumoniae. Here we examined the role of necroptosis in the generation of an adaptive immune response to influenza A virus (IAV), a pathogen also shown to induce necroptosis of AECs. Wildtype (WT), RIPK3 KO, and MLKL KO mice were challenged intranasally with IAV strain A/Puerto Rico/8/1934 (i.e. PR8). Weight loss was monitored as a measure of disease severity. Compared to WT and MLKL KO, RIPK3 KO mice experienced the most severe weight loss, independent of viral titers, as they were equivalent. The lungs of RIPK3 KO mice showed a significant increase in IAV-specific CD8+ T cells compared to those of WT and MLKL KO. In vitro we observed increased IL-8 production by IAV-infected RIPK3 deficient AECs. IAV-specific germinal center B cells in RIPK3 KO and MLKL KO mice were decreased compared to WT. No differences were observed in IAV specific lymph node and lung regulatory, follicular helper, central, effector, or tissue resident memory T cells. These results suggest fundamental differences in the roles of RIPK3 and MLKL in the development of adaptive immunity against IAV infection. Ongoing studies are focused on determining the contribution of MLKL-independent RIPK3 responses in AECs and immune cells in IAV infection and pathology.
NMP4 regulates innate immune responses to influenza A virus infection
Authors: Yang Shuangshuang , Adaway Michele , Sun Jie , Huang Shengping , Bidwell Joseph , Zhou Baohua
Published: January 2023

Severe influenza A virus infection typically triggers excessive and detrimental lung inflammation with massive cell infiltration and hyper-production of cytokines and chemokines. Defining research models to alleviate lung damage without compromising viral control is critical. We identified a novel function for nuclear matrix protein 4 (NMP4), a zinc-finger-containing transcription factor in regulating lung inflammation and antiviral innate immune responses. Nmp4-deficient mice are protected from H1N1 influenza infection, losing less body weight compared to the wild-type mice. Nmp4-deficiency does not affect viral clearance or adaptive immune responses, but significantly reduces the accumulation of neutrophils and monocytes in the lungs and the early inflammation. Consistent with fewer innate cells in the airways, infected Nmp4-deficient mice show decreased expression of chemokine genes Ccl2 and Cxcl1 as well as pro-inflammatory cytokine genes Il1b and Il6. We further reveal that NMP4 binds to the promoters and/or conserved non-coding sequences (CNS) of Ccl2 and Cxcl1 in mouse lung epithelial cells. Besides, the knockdown of NMP4 by CRISPR/Cas9 or siRNA significantly lessens the luciferase activities of Ccl2 and Cxcl1 promoters and/or CNS. Taken together, our data suggest that NMP4 regulates monocyte- and neutrophil-attracting chemokine expression in airway epithelial cells upon influenza infection, resulting in exaggerated innate inflammation and lung tissue damage.