bluuebunny/crossref_metadata_2025 huggingface dataset:
Multi-peptide cancer immunotherapy in combination with sorafenib
Authors: Marquez-Manriquez Juan P. , MONTANO GEOBANNI TORRES , VERBURG MARK L. , CAMACHO-HERNANDEZ ALEJANDRO , LUCERO-DIAZ PEDRO-ALEJANDRO , MONTIJO-FERNANDEZ LORENA , VILLALOBOS-MOLINA PERLA-GABRIELA , Hinojosa-Martinez Simon , VERDUGO-VILLASENOR ALEXIS , MATUTE-BRISENO JOSE-ANTONIO
Published: January 2023

Background

Little is known why most of the cancer immunotherapies failed despite good results in pre-clinical studies. We believe that if we focus on clinically relevant multi-proteins to immune-target simultaneously in combination with drugs with potential immune properties we may improve the clinical outcomes.

Methods

12 proteins overexpressed in several human tumors and associated with prognosis were identified. Indirect ELISA (IgA+ IgM + IgG) and ELISPOT was used to evaluate immunity proteins in 50 cancer patients and 50 controls. Immunohistochemistry (IHC) staining was done for proteins and CD8 T-cell quantitation. Peptides that were highly immunogenic by ELISA and ELISPOT were formulated into multi-peptide immunotherapy and with low dose of oral sorafenib. Patients treated with active progressive disease 6 months, 12 times were monitored biweekly to evaluate the immune response.

Results

Serum responses for all positive antigens were significantly elevated in advanced cancer patient sera when compared to donor controls (Bcl-2 p=0.001, survivin p=0.0001, EGFR P=0.005, Fascin-1 p=0.0001, Sox2 p<0.0002), RCAS1 p= 0.004, VCP = 0.05 and Ape-1 (p=0.0001). ELISPOT screening yielded epitopes that were immunogenic in advanced cancer patients. Metastatic areas of liver and lungs in advanced cancer patients were significantly inhibited after treatment with the multi-peptide immunotherapy and sorafenib.

Discussion

Advanced cancer is still immunogenic and overexpressed proteins involved in clinical relevant pathways can serve as specific active immunogens. These data could be the proof of principle for the development of multi-peptide immunotherapy in combo with drugs with immune properties such as sorafenib.
A B Cell Lymphoma-Associated LncRNA Modulates BCR-Mediated Calcium Signaling
Authors: Payton Jacqueline , Pyfrom Sarah , Luo Hong , Oltz Eugene M.
Published: January 2023

Long non-coding RNAs (lncRNA) act as transcriptional regulators, scaffolds, and signaling modulators, but their role in normal or malignant lymphocytes is unknown. We identified lncRNAs with altered expression in >100 human Non-Hodgkin Lymphoma (NHL) and normal B cell samples. One of these lncRNA genes is upstream of PLCG2 and parallels its expression, but the transcript is localized in the cytoplasm, making transcriptional regulation of PLCG2 unlikely. As expected, knock-out (KO), knock-down (KD), or over-expression of lncRNA-PLCG2 did not affect PLCG2 levels. PLCG2 is a B-cell specific phospholipase C enzyme that stimulates Ca2+ signaling after BCR activation. We assessed Ca2+ signaling via IgM stimulation of the BCR in multiple lncRNA-PLCG2 KO/KD NHL cell lines and observed defects in Ca2+ efflux from the ER and influx through Orai channels. To identify lncRNA-PLCG2 interacting proteins, we performed RNA pull-down experiments using sense (S) and anti-sense (AS, control) lncRNA-PLCG2 as bait incubated with B cell lysates. Mass spectrometry of eluted proteins showed several proteins significantly enriched in S versus AS, including PLD1, DICER1 and DHX9; we confirmed these by Western blot. We next performed RNA-binding protein Immunoprecipitation (RIP) using antibodies for PLD1, DHX9 and DICER1, which showed 2–10-fold enrichment of lncRNA-PLCG2 compared to IgG isotype control. Ongoing studies are mapping lncRNA-PLCG2-protein interactions and evaluating the impact of lncRNA-PLCG2 on PLD1 activity. Taken together, these results suggest that lncRNA-PLCG2 modulates BCR-mediated Ca2+ signaling via interaction with BCR downstream signaling proteins, a previously unrecognized mechanism that may promote lymphomagenesis.
Mapping the transcriptome and epigenomic landscape of HDAC-inhibitor resistant CTCL
Authors: Andrews Jared , Schmidt Jennifer , Payton Jacqueline
Published: January 2023

Epigenetic alterations deregulate gene expression and are a significant contributor to cancer pathogenesis. Unlike gene mutations, epigenetic changes are reversible, and this distinction has been exploited therapeutically for Cutaneous T-cell Lymphomas (CTCL). Histone deacetylase inhibitors (HDACi) are FDA-approved; however, two-thirds of patients do not respond to HDACi. Therefore, we are addressing this gap by 1) establishing new techniques to identify HDACi response elements from all stages of CTCL and 2) developing targeted epigenetic modifiers (TEM) as a novel precision therapy.

Our key innovations in this study are the generation of targeted epigenetic modifiers (TEM) as potential therapeutics and the development of a technique called Ultra-Low Input Chromatin Immunoprecipitation (ULI-ChIP) that enables epigenome-wide profiling of histone modifications in 10,000–30,000 cells. We are utilizing this method to identify recurrently altered regulatory elements in HDACi-resistant CTCL patients, which, in conjunction with RNA-seq data, allow us to determine epigenomic and transcriptomic states that predispose patients for resistance to these treatments.

We have identified several regulatory elements upstream of CXCR4, a chemokine receptor with known roles in cell survival and metastasis in B cell lymphomas and T cell leukemias, that are dysregulated in HDACi resistant patients. Similar loci were also found near key transcription factor (FOXP1) and anti-apoptotic genes (BCL2). The most promising HDACi response elements will be targeted in CTCL cells using locus-specific epigenetic modifiers to validate and evaluate the potential of each locus to be further developed for TEM protein therapeutics.
Unique molecular identifiers of indolent versus aggressive phenotyping in non-Hodgkin lymphoma: spotlight on SMZL.
Authors: Robinson Jacob E. , Cutucache Christine E.
Published: January 2023

Splenic marginal zone lymphoma (SMZL) is an indolent, non-Hodgkin’s lymphoma that lacks clear genotypic classification. While most cases progress slowly, approximately 30% of cases will become aggressive or undergo transformation to a more lethal lymphoma. The purpose of this study was to characterize the molecular profiles of SMZL cases compared with healthy B-cells and other B-cell lymphomas (BCL) in an effort to delineate indolent versus aggressive presentations. We conducted a meta-analysis of previously published gene expression data in order to identify genes most dysregulated in SMZL. We compared gene expression datasets among SMZL, similar B-cell neoplasms (all B-cell lymphomas), and healthy B-cell controls in order to extract a SMZL-specific, indolent phenotype profile. As a result, we observed a unique profile of genes upregulated in SMZL that are involved in immune defense (inclusive of leukocyte chemotaxis), motility (inclusive of lamellipodium assembly), cell proliferation, as well as other mechanisms. Of these, the most upregulated genes in SMZL were CXCL6, KAZN, NTN4, CD5L, AIF1L, and CD36. Additionally, 20 key markers of potential indolence were identified, with the largest expression differences seen in ABI3BP, GPNMB, CCL19, and ZC3H7B. Further investigation of these genes and their resultant impacted pathways will be evaluated for diagnostic and prognostic significance using immunohistochemistry, and hypothesized targets evaluated in vitro. Ultimately, we expect that the translation of these results will assist with patient staging in SMZL.
Glycolipid stimulation of invariant NKT cells mobilizes precursors of mature NK cells and potentiates their participation in immune surveillance against metastatic cancer.
Authors: Choi Joshua , Chan Cindy , Lesage Sylvie , Haeryfar S.M. Mansour
Published: January 2023

Precursors of mature natural killer (pre-mNK) cells, initially called IFN-producing killer dendritic cells, have been recently characterized as a novel intermediate in NK cell differentiation. Typified by a unique B220+NK1.1+CD11c+MHCII+ phenotype, pre-mNK cells exhibit prolific anti-tumor cytotoxicity while retaining the ability to present antigens thereby activating neighbouring T cells. Invariant NKT (iNKT) cells are another population highlighted for their potential in anti-cancer immunity. They are activated very early and play an important role in transactivating downstream effectors, such as NK cells. The extent to which iNKT cells can activate pre-mNK cells is unknown. We hypothesized that iNKT cells are able to activate pre-mNK cells and potentiate their anti-tumor properties. Wildtype C57BL/6 mice were injected i.p. with the iNKT cell superagonist, α-galactosylceramide (αGC), and various lymphoid tissues were harvested after 5 days for phenotyping or cytotoxicity assays. We have shown, for the first time that iNKT cell activation via αGC results in robust expansion of pre-mNK cells in the spleen, lung and most notably, in the liver. These cells exhibited cytotoxic activity against YAC-1 thymoma and B16 melanoma cancer cells via the granule exocytosis pathway and significantly contributed to overall NK cytotoxic activity in vivo. The anti-cancer responses due to αGC activation have been well documented; however, the contribution of pre-mNK cells in these responses have never been shown. Our findings demonstrate that pre-mNK cells rapidly expand due to iNKT activation while retaining their capacity to kill tumor targets. This may suggest a novel mechanism of targeting pre-mNK cells in anti-cancer therapies.
A distinct human NKp30+FcɛRIγ+CD8+ T cell population exhibiting high NK-like anti-tumor potential
Authors: Correia Margareta P. , Stojanovic Ana , Bauer Katharina , Juraeva Dilafruz , Tykocinski Lars , Lorenz Hanns-Martin , Brors Benedikt , Cerwenka Adelheid
Published: January 2023

CD8+ T cells are considered prototypical cells of adaptive immunity. Here, we uncovered a distinct CD8+ T cell population, expressing the activating NK receptor, NKp30, in peripheral blood of healthy individuals. We revealed that IL-15 could de novo induce NKp30 expression in a population of CD8+ T cells, and further drive their differentiation towards a broad innate transcriptional landscape. The adapter FcɛRIγ was concomitantly induced, and shown to be crucial to enable NKp30 cell surface expression and function in CD8+ T cells. FcɛRIγ de novo expression required promoter demethylation and was accompanied by the acquisition of the signaling molecule Syk and the “innate” transcription factor PLZF. IL-15-induced NKp30+CD8+ T cells exhibited high NK-like anti-tumor activity in vitro, able to synergize with TCR-signaling. Importantly, this population potently controlled tumor growth in a preclinical xenograft mouse model. Our study, while blurring borders between innate and adaptive, reveals a unique NKp30+FcɛRIγ+CD8+ T cell population with high anti-tumor therapeutic potential.
Differential T follicular helper cell responses induced by candidate HIV vaccine strategies utilizing ALVAC versus Ad5 vectors
Authors: Auclair Sarah , Soong Lynn , Hu Haitao
Published: January 2023

The development of a safe and effective vaccine against Human Immunodeficiency Virus (HIV) remains a major priority in global health research. Several candidate HIV vaccines have undergone late-stage clinical trials in recent years, several of which utilized non-replicating viral vectors based on canarypox (ALVAC) or human adenovirus (Ad5.) Thus far, the only trial to show moderate (31.2%) efficacy in preventing HIV infection is the RV144 trial, which utilized an ALVAC prime/protein boost strategy. Subsequent analyses showed that protection from HIV infection correlated with the production of IgG3 antibodies directed against the highly conserved V1V2 loop of the HIV envelope (Env) protein, and was abrogated by the production of Env-specific serum IgA antibodies. Although the ineffective Ad5-based vaccines also induced strong Env-specific antibody responses, they failed to induce protective IgG3 and/or V1V2-specific antibodies. To better understand the mechanisms underlying the differential antibody profiles generated by these two vectors, we measured the frequency and functional phenotype of follicular T helper-like cells found in the peripheral blood of vaccine recipients (pTfh). We found that subjects who received an ALVAC prime/protein boost vaccine regimen had higher levels of Env-specific pTfh cells than subjects who received a DNA prime/Ad5 boost regimen, and that these pTfh cells have enhanced ability to induce B cell activation and class switching compared to those found in Ad5-based vaccine recipients. We hypothesize that the higher frequency and functionality of these ALVAC-induced pTfh cells contributes to the uniquely protective antibody response generated by ALVAC prime/protein boost vaccination.
Induction and relationship of HIV Envelope-specific B cells in the peripheral blood and bone marrow following vaccination in humans.
Authors: Kobie James , Basu Madhubanti , Piepenbrink Michael , Francois Czestochowa , Roche Fritzlaine , Fucile Christopher , Rosenberg Alexander , Liesveld Jane , Keefer Michael
Published: January 2023

Induction of long-lived plasma cells producing protective HIV-1 Envelope (Env)-specific antibody (Ab) is a primary goal of HIV vaccine strategies. However, vaccine-induced Env-specific human plasma cells have not yet been identified. In an effort to do so, samples were obtained from HVTN 105, a phase I trial in which participants were immunized with the same bivalent gp120 protein, AIDSVAX B/E, as used in RV144, although combined with a DNA immunogen (instead of ALVAC-HIV) in various prime/boost strategies. Participants who received a boosting regimen that included AIDSVAX B/E had robust peripheral blood plasmablast responses as measured by Env-specific ELISpot (n=21) and flow cytometry (n=20). The Env-specific immunoglobulin repertoire of the plasmablasts, assessed by monoclonal Ab generation (>110 mAbs total from 15 participants), was dominated by VH1 gene usage (~60%, VH1-2>VH1-46>VH1-24), and had modest mutation from germline (~5%). Ongoing epitope mapping has indicated V3 is a dominant target; however other regions including V1/V2 and C1/C5 were also targeted. The majority of the mAbs are high avidity, and a subset can mediate Ab-dependent cellular phagocytosis; additional functional analysis is ongoing. Bone marrow was evaluated ~7 months after immunization (n=3), and although ELISpot of CD138+ plasma cells could readily detect influenza specific IgG Ab secreting cells (ASCs), Env-specific IgG ASCs were not convincingly detected by ELISpot. However, when using VH deep sequencing of the bone marrow (n=4), multiple members of plasmablast-derived mAb clonal lineages were detected. Our results indicate that HIV-1 Env-specific B cells are present in human bone marrow after vaccination, but may persist at very low frequency.
Rescue of vaccine immunity with nicotine and isoproterenol after total body irradiation
Authors: Gandhi Deep , Lehtimaki Mari K. , Surujdin Ryan , Aryankalyil Joseph , McFarland Hugh I. , Rosenberg Amy S.
Published: January 2023

Radiation exposure results in DNA damage and apoptosis of lymphocytes, including memory T cells, leading to the loss of vaccine immunity and resulting in a potentially lethal susceptibility to infection. We have shown that memory CD8+ T cells are preserved by revaccination within 3–4 days after exposure to sub-lethal γ radiation. This study explores therapeutics selected to replicate the effects of revaccination. These therapeutics may promote memory T cell survival by acting on survival pathways stimulated by T cell activation. Using a Listeria monocytogenes (LM) model in mice, nicotine tartrate and isoproterenol were chosen from a screen of 22 therapeutic candidates. Both drugs have been shown to act on the AKT and ERK1/2 survival pathways to prevent apoptosis and promote cell survival and DNA repair. Thirty days after vaccination against LM, mice were irradiated and treated 1 day later with one of the therapeutics. Thirty days later, mice were challenged with wild-type LM, followed by harvest and plating of the spleen. Colonies were counted as a measure of vaccine immunity to LM. Nicotine significantly decreased the number of LM colonies when administered subcutaneously in a TiterMax adjuvant emulsion, as well as transdermally in the form of a patch. The decrease in LM was comparable to revaccination. Similarly, pilot experiments using isoproterenol show decreased colony counts, indicating potential preservation of vaccine immunity. Further studies will include varying doses of isoproterenol and varying routes of administration, as well as combinations of treatments. These therapeutics offer a practical effective means of preserving vaccine memory as a potential new medical countermeasure for Acute Radiation Syndrome.
Activated macrophages as pathogenesis factors in Ebola virus disease in humans
Authors: McElroy Anita K. , Shrivastava-Ranjan Punya , Harmon Jessica R. , Martines Roosecelis , Silva-Flannery Luciana , Flietstra Timothy D. , Kraft Colleen S. , Mehta Aneesh K. , Lyon G. Marshall , Varkey Jay , Ribner Bruce , Nichol Stuart T. , Zaki Sherif , Spiropoulou Christina
Published: January 2023

Ebola virus disease (EVD) is associated with elevated cytokine levels, that are more pronounced in fatal cases. This type of hyperinflammatory state is reminiscent of other inflammatory disorders, such as macrophage activation syndrome (MAS) or hemophagocytic lymphohistiocytosis (HLH). These are both part of a spectrum of rheumatologic phenomena characterized by both macrophage and T cell activation. These disorders can be secondary to infection, malignancy, underlying rheumatologic disorder, or, paradoxically, immune deficiency. An evaluation of two cohorts of patients with EVD revealed that several of the common laboratory features of MAS and HLH are shared by EVD. Neither fibrinogen nor soluble IL-2 receptor were significantly different between fatal and non-fatal cases. However, elevated levels of triglycerides, ferritin and sCD163, a marker of macrophage activation were noted in patients with EVD and they correlated with disease severity and a fatal outcome. Furthermore, significant immunoreactivity for CD163 + cells in host tissues was observed in fatal cases, predominantly in areas of extensive immunostaining for EBOV antigens. These data suggest that host macrophage activation contributes to EVD pathogenesis and that directed anti-inflammatory therapies could be beneficial in the treatment of EVD.
-Microglia mount a robust immune response to Rift Valley Fever virus that is dependent on MAVS and independent of TLR7
Authors: Weilhammer Dina R. , Bourguet Feliza , Rasley Amy
Published: January 2023

The mosquito-borne Rift Valley Fever virus (RVFV) is a category A biodefense pathogen due to the potential for intentional spread by aerosol and the lack of any licensed vaccine or antiviral therapeutics. Animal studies and limited data from human infection have demonstrated that RVFV pathogenesis following aerosol exposure differs from subcutaneous exposure, and is characterized by severe encephalitis, high viral titers in the brains, and long lasting neural pathology in human survivors of infection. Despite the brain being a major target of viral replication and tissue damage, little is known about innate immune responses to RVFV in the brain following aerosol infection. Thus, we are investigating the responses of the resident innate immune cells of the brain microglia and astrocytes to RVFV infection. Microglia mount a robust response to RVFV infection evidenced by upregulation of immunoregulatory genes, secretion of cytokines and upregulation of surface markers of activation. The response of microglia to RVFV is dependent upon MAVS and independent of TLR7, with cells derived from MAVS knockout animals unresponsive to infection while cells derived from TLR7 knock out animals responded similarly to infection as wild type cells. Experiments to determine the in vivo innate immune response to RVFV in the brain are ongoing. Understanding the in vivo innate immune response in the brain will be will be critical to the development of vaccines and antiviral therapeutics that are successful in preventing viral encephalitis caused by RVFV.
HPV18 E1-antigen specific CD8+ T cells show cross-reactivity against cells expressing E1 from HPV16, 31, 33 and 53.
Authors: Amador Alfredo , Amador Juan Carlos , Lizano Marcela
Published: January 2023

CD8+ mediated immune response plays a major role in the clearance of Human Papillomavirus (HPV) infected cells. Although HPV vaccines prevent HPV infections through humoral response, they do not protect against established infections and its cross protection is limited. In the HPV infection, the expression of E1 helicase during viral replication makes it a potential immunological target. The E1 protein is one of the most conserved proteins among papillomaviruses. We demonstrate in previous work, that immunization of HPV type 18 E1 protein plus α-GalactosylCeramide (α-GalCer) elicited CD8 T cell-immune response, able to eliminate tumor cells expressing HPV18 E1 protein. For this study, we analyzed the amino acids sequence of E1carboxi-terminal (CT) domains from HPV18, 16, 31, 33 and 53, using Rankpep and Clustal software to determine the number of conserved peptides among E1 proteins. These sequences share 20 predicted peptides. We cloned E1-CT domains of HPV16, 31, 33 and 53, and its expression in B16-F0 cells was determine. We show that CD8 T cells from mice immunized with HPV18 E1 protein plus α-GalCer, are actively cytotoxic in co-culture with B16-F0 cells expressing different E1-CT proteins in comparison with unimmunized mice, showing a cross-reactivity. Cytotoxicity activity was evaluated by detection of CD107a and CD107b on the CD8 T cells surface as part of cytotoxic granule exocytosis, through flow cytometry. Ongoing experiments will examine if mice immunized with HPV18 E1 protein plus α-GalCer are able to eliminate tumors cells expressing different E1-CT proteins in a mouse model. The characterization of new immunological targets against HPV, could help to develop new strategies to clearance HPV established infections.
Anti-inflammatory effects of PUFA’s on T cells reduce TCR affinity for antigen
Authors: Kolawole Elizabeth Motunrayo , Evavold Brian D.
Published: January 2023

Polyunsaturated fatty acids (PUFAs) have been shown to modulate inflammation yet little is known on their specific effects on recognition of antigen by T cells despite. Since the T cell receptor (TCR) and its peptide-MHC (pMHC) antigenic ligand are embedded in the membrane, incorporation of omega-3 fatty acids into lipid membranes could likely change the 2D kinetics of TCR:pMHC engagement and the membrane environment. We used the 2D micropipette adhesion frequency assay to define the proximal kinetics of antigen recognition by T cells on a single cell basis. We found that dietary fish oil modifies the structural lipid content in plasma membranes as measured by mass spectrometry, resulting in reduced TCR affinity for antigen at the clonal and polyclonal level. The reduced affinity led to deceased frequency and effector functions from both CD4 and CD8 T cells at the peak immune response to viral (LCMV) infection. Thus, our work provides mechanistic insight for the anti-inflammatory effects of PUFAs by altering plasma membrane composition which leads to decreased 2D kinetics of antigen recognition in T cells and dampened response.
Z-DNA binding protein 1 (ZBP1) is an innate sensor of Influenza vRNPs and activates programmed cell death and assembly of NLRP3 inflammasome
Authors: Sannula Kesavardana , Kuriakose Teneema , Samir Parimal , Malireddi RK Subbarao , Kanneganti Thirumala-Devi
Published: January 2023

Influenza A virus (IAV) infection triggers pulmonary epithelial cell death and inflammation which promote bronchioalveolar tissue damage and failure of lung function. However, the mechanism of IAV induced cell death and inflammation remains unclear. RIP homotypic interaction motif (RHIM) has evolved to play critical role in cell death and inflammation. Z-DNA binding protein 1 (ZBP1/DAI) is unique among RHIM-containing proteins since it has additional Za nucleic acid binding domains. Although ZBP1 domain architecture suggests its function as nucleic acid sensor, its precise physiological role is not established.

We discovered that ZBP1 is a central activator of IAV induced programmed cell death and NLRP3 inflammasome activation in both immune and non-immune cells. Mechanistically, ZBP1 senses ribonucleoprotein complexes (vRNPs) of IAV and engage RIPK3-caspase-8 dependent apoptosis, necroptosis and NLRP3 inflammasome assembly. ZBP1 also drives RIPK1 dependent secretion of TNF and IL-6 cytokines after IAV infection. We also discovered that apical activation of RIG-I/MAVS/IFNAR signaling licenses ZBP1 upregulation and activation. In contrast to other RNA viruses, IAV replicates inside the nucleus. Accordingly, we observed localization of ZBP1 in the nucleus suggesting its role in sensing IAV inside the nucleus. Strikingly, we also found that ZBP1 undergoes ubiquitination in response to IAV infection to assemble cell death signaling complexes. In IAV infected mice, ZBP1 induces cell death in lung epithelial cells and its absence conferred better survival advantage, despite high viral titers in the lung. Overall, our work demonstrates the central role of ZBP1 in sensing IAV infection to trigger cell death and inflammation.
Natural killer cell regulation of HIV vaccine responses
Authors: Rydyznski Carolyn E. , McNally Jonathan P. , Gyurova Ivayla E. , Cranert Stacey A. , Waggoner Stephen N.
Published: January 2023

Understanding mechanisms that regulate humoral responses are crucial for the design of more efficacious vaccines. We have previously shown that natural killer (NK) cells suppress antiviral T-cell function and antibody production during acute virus infection in mice. NK cells also inhibited follicular helper (TFH) T-cell and germinal center (GC) B-cell responses following immunization with an alum-adjuvanted, non-replicating hapten-protein, NP-KLH. In addition, the frequency and type of mutations observed during SHM in NP-specific GC B cells is linked to NK cells, demonstrating that NK cells are important modulators of not just the quantity, but also the quality of the humoral response. To test the impact of NK cells on clinically-relevant HIV vaccine regimens, mice were depleted of NK cells prior to administration of an HIV envelope-expressing vaccinia virus vector. When NK cells were absent during priming, vaccine-induced TFH and GC B cell responses were increased along with production of HIV-specific immunoglobulin. Using a prime-boost vaccination scheme, we observed that NK cells also suppressed recall responses of B cells, such that combined depletion during both prime and boost immunizations had an enhanced effect on humoral responses. These results demonstrate that NK cells are important inhibitory regulators of affinity maturation and HIV-specific humoral responses, such that NK cells may limit vaccine-mediated generation of broadly neutralizing antibodies by curtailing GC responses. These data demonstrate the exciting potential to manipulate NK cells in order to promote effective vaccine-elicited protection against HIV infection.
Dock8-Positive CD4 T cell as Autoantibody-Inducing CD4 T (aiCD4 T) Cell That Causes Systemic Lupus Erythematosus (SLE): Proof of Concept of Self-Organized Criticality Theory as the Cause of SLE
Authors: Shiozawa Shunichi , Tsumiyama Ken , Miyazaki Yumi , Sakurai Keiichi , Miyazawa Masaaki
Published: January 2023

INTRODUCTION

We found in reproducible experiments in which the mice not prone to autoimmune disease were immunized repeatedly with antigen that overstimulation of CD4 T cells led to the development of aiCD4 T cell which had undergone TCR revision capable of inducing varieties of autoantibody and antigen-specific CTL via antigen cross-presentation, after which they caused SLE (Tsumiyama K et al., 2009). Here we identify aiCD4 T cell as DOCK8+CD4 T cell.

METHODS

aiCD4 T cell was searched by transferring different fractions of CD4 T cells of x12 OVA-immunized BALB/c female mice into naïve mice and tested if autoantibodies were raised, mass spectrometry, and FACS.

RESULTS

We focused CD45RBlo CD122lo PD1+CD4 T cell as aiCD4 T candidate, and its membrane uniquely expressed DOCK8. DOCK8+CD4 T cell was a large effector CD4 T cell with abundant ER and mitochondria, ICOS+ CXCR5-PD1+ Ly6C+ LFA1hi, produced increased IFNγ, IL4, IL6, IL17 and IL21, and its TCR repertoire was deviated. Upon transfer to naïve mice or to the x8 OVA-pre-immunized and CD4 T-depleted mice in which CTL was yet immature, DOCK8+CD4 T cells induced SLE, where anti-dsDNA/Sm Ab, glomerulonephritis of WHO IV/V types, skin liquefaction degeneration, splenic periarteriolar fibrosis with amyloid-like deposits classical of Onion-skin lesion, pericholangitis, pneumonitis, thyroiditis, perineuritis and panniculitis developed. Manifestations were cured by anti-DOCK8 Ab treatment.

CONCLUSION

We prove the self-organized criticality theory explaining that autoimmunity arises as a natural consequence of routine but exaggerated immune response against antigen when stimulated maximally beyond immune system’s self-organized criticality, and show that DOCK8+CD4 T cell causes SLE.
TFH-like cells promote TH17-induced neuro-inflammation
Authors: Quinn James L. , Kumar Gaurav , Agasing Agnieshka , Ko Rose M. , Axtell Robert C.
Published: January 2023

The cooperative role of T cells and B cells is a topical research area in multiple sclerosis. Previous studies, using experimental autoimmune encephalomyelitis (EAE), have shown that TH17 responses are correlated with ectopic B cell follicles in the CNS. As follicular T helper (TFH) cells control B cell responses, this study examines the role TFH cells play TH17-induced EAE. As previously reported, we found that B cells are a major cell type infiltrating the CNS of mice with TH17-EAE. We now show that the severity TH17-EAE was diminished in B cell deficient mice demonstrating that B cells contribute to the severity of disease in these mice. We next focused on the role TFH cells play in TH17-EAE. We found substantial numbers of TFH cells in the CNS of TH17-EAE mice and these cells are correlated with infiltrating B cells. Using transfer experiments, we found that TFH cells alone do not infiltrate the CNS but require the infiltration of TH17 cells to gain entry to the CNS. We next tested the effects of blocking TFH trafficking in TH17-EAE using an antagonistic antibody against CXCL13, the chemokine that binds to CXCR5 on TFH cells. We found that anti-CXCL13 treatment significantly reduced TH17-EAE disease. This treatment blocked T cells from entering the CNS but had no effect on B cells. Surprisingly, we observed that anti-CXCL13 treatment reduced disease in B cell-deficient mice. Collectively, these data demonstrate that B cells and TFH cells contribute to the severity of TH17-induced EAE. Furthermore, this report describes an important role for TFH cells that is independent of their support for B cell function and begins to answer important questions regarding their contribution in neuro-inflammatory diseases.
Neuroantigen-specific CD8 T cells inhibit ongoing demyelinating disease by autoregulating CD4 T cell responses using temporally distinct IFNγ- and perforin-dependent mechanisms.
Authors: Boyden Alexander , Brate Ashley A. , Karandikar Nitin
Published: January 2023

Pathogenesis of immune-mediated demyelinating diseases like multiple sclerosis (MS) is thought to be governed by a complex cellular interplay between immunopathological and immunoregulatory responses. We have previously shown that neuroantigen-specific CD8 T cells have an unexpected protective role in the mouse model of MS, experimental autoimmune encephalomyelitis (EAE). In this study, we interrogated the immunotherapeutic potential of PLP178–191-specific CD8 T cells. Here, we show that PLP178–191-specific CD8 T cells, when administered post-disease onset, swiftly ameliorated EAE progression and suppressed PLP178–191-specific CD4 T cell responses as measured by delayed-type hypersensitivity (DTH). To accomplish DTH suppression, PLP178–191-specific CD8 T cells required differential production of perforin and IFNγ. Perforin was ancillary for the rapid suppressive action of PLP178–191-specific CD8 T cells, but was necessary for maintenance of optimal longer term DTH suppression. Conversely, IFNγ production by PLP178–191-specific CD8 T cells was necessary for swift DTH suppression, but was less significant for maintenance of longer term suppression. These data indicate that neuroantigen-specific CD8 T cells employ an ordered regulatory mechanism program over a number of days in vivo during demyelinating disease and have mechanistic implications for this immunotherapeutic approach.
NKG2D signaling within the pancreas enhances CD8+ central memory T cell formation and decreases non-obese diabetic (NOD) diabetes development
Authors: Trembath Andrew P. , Sharma Neekun , Markiewicz Mary A.
Published: January 2023

The immune receptor NKG2D is implicated in autoimmune diabetes. However, the mechanism by which NKG2D signaling affects diabetes is unclear. Among the aspects not known are 1) the relevant anatomical site(s) of NKG2D signaling, 2) the cells in which NKG2D signaling occurs and 3) whether NKG2D signaling enhances or inhibits diabetogenesis. These are among the critical questions that must be answered to determine whether modulation of NKG2D signaling could be used in intervention strategies in type 1 diabetes. To determine whether NKG2D signaling within the pancreatic islets modulates autoimmune diabetes, we generated a novel NOD mouse strain in which the mouse NKG2D ligand RAE1-e is constitutively expressed in the beta-islet cells (RIP-RAE1e mice). Insulitis progressed faster in RIP-RAE1e mice compared with control NOD mice. Despite this, there was a significant decrease in spontaneous diabetes in RIP-RAE1e mice compared with control mice. Using flow cytometry, we compared the immune cells present in the pancreas and spleen of pre-diabetic RIP-RAE1e and control NOD mice. Similar percentages and numbers of CD4+ T cells, CD8+ T cells and NK cells were detected. Similarly, equivalent numbers of CD69+ CD4+T and CD69+CD8+T cells were present. However, RIP-RAE1e mice contained increased numbers of CD44hiCD62Lhi (central memory T cell, Tcm, phenotype) CD8+ T cells and decreased numbers of CD44hiCD62Llo (effector/effector memory, Tem, phenotype) CD8+ T cells in both the pancreas and spleen. This increased Tcm:Teff/em ratio was not observed in the CD4+ T cell compartment. Together, these data demonstrate that NKG2D signaling within the pancreas drives an increased ratio of CD8+ Tcm:Teff/em cells and is protective against NOD diabetes.
Autoreactive T cells use IFNγ and Fas Ligand to clear melanocytes in vitiligo
Authors: Strassner James P. , Azzolino Vincent N. , Richmond Jillian M. , Harris John E.
Published: January 2023

Vitiligo is an autoimmune disease of the skin in which melanocytes are selectively destroyed, leading to a disfiguring depigmentation. Studies by our group and others demonstrated that IFNγ-induced chemokines drive pathogenesis through the recruitment of CD8+ T cells that kill melanocytes. We examined potential mechanisms of melanocyte killing by T cells in our mouse model by performing adoptive transfer of autoreactive cells lacking IFNγ, perforin, or Fas ligand. We found that disease severity and T cell accumulation in the skin was reduced in mice that received either IFNγ KO or Fas ligand KO melanocyte-specific T cells; however, perforin was not required. We partially rescued the disease phenotype by performing 50:50 co-injections of effector molecule KO T cells with WT T cells, however KO T cells outcompeted WT cells in recruitment to the epidermis, suggesting IFNγ and Fas Ligand are needed in situ to cause disease. To determine if IFNγ signaling was directly cytotoxic to melanocytes, we used a Cre-Lox system in which the IFNγR was specifically deleted in melanocytes. Floxed/Floxed Cre+ littermates were not protected from disease, suggesting IFNγ signaling in melanocytes is dispensable for melanocyte clearance. In summary, IFNγ has pleiotropic effects in vitiligo, required both for T cell recruitment and functional killing of melanocytes. This further supports targeting IFNγ signaling for the treatment of vitiligo. In addition to IFNγ, Fas ligand is a new potential target for therapy that warrants further investigation.
Type-1 interferon receptor knockout mice generate high titers of ADCC antibodies in response to vaccination with ΔgD-2 but display defects in executing ADCC
Authors: Dardick Joseph , Weiss Kayla , Jacobs William R.
Published: January 2023

ΔgD-2 is a live-attenuated Herpes Simplex Virus type-2 (HSV-2) strain genetically deleted for glycoprotein D (gD) that elicits complete protection against HSV-1 and HSV-2 through antibody-dependent cell-mediated cytotoxicity and phagocytosis (ADCC and ADCP). The mechanisms by which immunogens elicit ADCC/ADCP activating antibodies are not well understood but type-1 interferons (IFNα/β) have been shown to play a role in activating humoral immunity and FcγR-mediated immune modulation. To investigate the role of IFNα/β in generating ADCC/ADCP antibodies, we vaccinated IFNα/β receptor knockout mice (IFNAR−/−−) with ΔgD-2 and compared the resulting immunity to that of WT mice. IFNAR−/− mice vaccinated with ΔgD-2 generated similar overall and isotype specific anti-HSV IgG titers to WT mice. Serum from vaccinated IFNAR−/− mice also induced significantly more ADCC/ADCP killing than serum from vaccinated WT mice (p<0.05). To determine whether this serum initiated ADCC/ADCP in vivo, serum from vaccinated IFNAR−/− mice was passively transferred into naive WT and FcγRIV−/− mice. WT mice that received the vaccinated serum were partially protected from 10x LD90 challenge with HSV-2 but FcγRIV−/− mice were not (p<0.05). Additionally, serum from vaccinated WT mice did not confer any protection to naive IFNAR−/− mice, indicating difficulty in mobilizing ADCC/ADCP (p<0.01). However, IFNAR−/− mice that were vaccinated with ΔgD-2 were completely protected from morbidity and mortality following a 10x LD90 challenge with HSV-2 (p<0.0001). Together, our data show that the production of ADCC/ADCP antibodies in response to ΔgD-2 is independent of IFNα/β, but IFNAR signaling is important for executing ADCC/ADCP protection.
Quantitative Bead-Based Multiplex Immunoassays for Mouse Anti-Virus Immune Response
Authors: Zhao Amy , Sun Binggang , Lehmann Jason , Ji Shaoquan , Jiang Weiping
Published: January 2023

In response to pathogens, especially viruses, cells release interferons and other cytokines to combat infection. Interferons are typically divided into three types: 1 (e.g., IFN-α, IFN-β), 2 (e.g., IFN-γ), and 3 (e.g., IFN-λ1, IFN-λ2). All inter-ferons are important for fighting viral infections and for regulating the immune system. In addition, interferons are critically involved in cancer and autoim-mune diseases such as psoriasis, systemic lupus erythematosus, and multiple sclerosis. Studying the expression profile of interferons and other related cyto-kines is essential to understanding the immune response against viral pathogens and other related disease processes.

We have developed a multiplex panel, using fluorescence-encoded beads, which is suitable for use on commonly available flow cytometers. The panel simultaneously quantifies 13 mouse anti-viral immune proteins, including IFN-α, IFN-β, IFN-γ, CXCL1 (KC), TNF-α, CCL2 (MCP-1), IL-12p70, CCL5 (RANTES), IL-1β, CXCL10 (IP-10), GM-CSF, IL-10, and IL-6. The assay was validated for specificity, linearity, cross-reactivity, and inter and intra-assay precision. The assay was further validated using biological samples to quantify analyte concentration changes in response to poly (I:C) stimulation compared to untreated controls.

This multiplex panel is a robust tool for measuring the concentration of anti-viral inflammatory mediators in murine biological samples, and offers greater efficiency and broader dynamic ranges compared to conventional ELISA assays.
Hypoxia and monomeric IgE activate common signaling pathways leading to chemokine CCL2 production in mast cells
Authors: RAMIREZ-MORENO ITZEL GUADALUPE , Ibarra-Sánchez Alfredo , Gonzalez-Espinosa Claudia
Published: January 2023

Although presence of mast cells (MCs) to sites with low oxygen concentration (such as inflamed tissues with compromised blood flux and solid tumors) has been reported, the effects of hypoxia on the cytokine profile produced by MCs under those conditions has not been fully explored. We used the murine B16 melanoma model to quantify the presence of MCs in normoxic or hypoxic zones of the tumor and, bone marrow-derived mast cells (BMMCs) subjected to a 1% O2 atmosphere to characterize the production of specific cytokines and chemokines by this cell type in hypoxic conditions. Hypoxic areas of melanoma tumors were identified with hypoxyprobe and the number of MCs was determined by confocal analysis of tryptase positive cells. Using this methodology, important MCs presence in hypoxic zones of the tumor was detected. In vitro, BMMCs subjected to hypoxia were able to produce VEGF and the potent chemokine CCL2. Chemokine production in hypoxia was sensitive to the antioxidant Trolox and the IKK inhibitor BAY117085. CCL2 production was also induce by monomeric IgE by a mechanism also sensitive to Trolox and BAY117085. CCL2 production triggered by hypoxia or monomeric IgE was sensitive to calcium chelator BAPTA and the L-type calcium channel blocker Nifedipine. Our results show that MCs are importantly recruited to hypoxic sites of melanoma tumors and that hypoxia induces the production of the chemokine CCL2 by a mechanism that require the production of reactive oxygen species (ROS) and intracellular calcium rise through the activation of L-type calcium channels. Also, data indicate that monomeric IgE triggers a very similar response than hypoxia does to lead to the production of CCL2 in mast cells.
Prenatal smoke exposure exacerbates lung inflammatory response by modulating neutrophil infiltration to the lungs after RSV infection
Authors: Cheemarla Nagarjuna R. , Naidu Shan , Guerrero-Plata Maria Antonieta
Published: January 2023

Exposure to second hand tobacco smoke (SHS) is associated with an increased frequency and severity of viral respiratory tract infections. The mechanisms of recurrence and susceptibility to viral pathogens after passive smoke exposure are multifactorial and include alteration of the structural and immunologic host defenses. Maternal smoking during pregnancy is associated with a number of adverse health effects in infants, including higher susceptibility to respiratory syncytial virus (RSV) infection and associated bronchiolitis. Given the extensive impact on human health, further studies are needed that identify the mechanisms responsible for the increased severity of respiratory viral infections in individuals exposed to cigarette smoke. In this work, we used a well-established mouse model of in-utero smoke exposure. Offspring (5-day old) were infected intranasally with RSV and sacrificed at different times post infection. Sample analysis indicated that in-utero SHS exposure led to increased lung inflammation and higher viral lung titers, as shown by lung histopathology and plaque assay, respectively. In further understanding the mechanism of inflammation, cytokine profile revealed a significant increase in CXCL1 (KC), a potent neutrophil chemoattractant. Thus, we investigated the infiltration of neutrophils in lungs of SHS and air-exposed mice. FACS analysis revealed not only that RSV-infected neonates exposed to in-utero SHS had a significant increase in neutrophil influx into airways compared to air-exposed mice, but also that their neutrophils were more activated and expressed higher levels of CXCR2. In conclusion, our results help elucidate the mechanisms underlying harmful effect of smoking during pregnancy.
Zika, dengue and yellow fever viruses induce differential anti-viral immune responses in human monocytic and first trimester trophoblast cells
Authors: Luo Huanle , Winkelmann Evandro , Fernandez-Salas Ildefonso , Li Li , Mayer Sandra V. , Daniz-Lozano Rogelio , Sanchez-Casas Rosa Ma , Vasilakis Nikos , Tesh Robert , Barrett Alan D. , Weaver Scott C. , Wang Tian
Published: January 2023

Zika virus (ZIKV) is a mosquito-borne flavivirus associated with severe neonatal birth defects, but the causative mechanism is incompletely understood. ZIKV shares sequence homology and early clinical manifestations with yellow fever virus (YFV) and dengue virus (DENV) and are all transmitted in urban cycles by the same species of mosquitoes. However, YFV and DENV have been rarely reported to cause congenital diseases. Here, we compared infection with a contemporary ZIKV strain (FSS13025) to YFV17D and DENV-4 in human monocytic cells (THP-1) and first-trimester trophoblasts (HTR-8). Our results suggest that all three viruses have similar tropisms for both cells. Nevertheless, ZIKV induced strong type 1 IFN and inflammatory cytokine and chemokine production in monocytes and peripheral blood mononuclear cells. Furthermore, ZIKV infection in trophoblasts induced lower IFN and higher inflammatory immune responses. Placental inflammation is known to contribute to the risk of brain damage in preterm newborns. Inhibition of toll-like receptor (TLR)3 and TLR8 each abrogated the inflammatory cytokine responses in ZIKV-infected trophoblasts. Our findings identify a potential link between maternal immune activation and ZIKV-induced congenital diseases, and a potential therapeutic strategy that targets TLR-mediated inflammatory responses in the placenta.
TRIM29 suppresses the innate immune response to RNA virus
Authors: Xing Junji , Zhang Ao , Minze Laurie J. , Li Xian Chang , Zhang Zhiqiang
Published: January 2023

The innate immunity is critically important in protection against virus infections, and in the case of RNA viral infections, the signaling mechanisms that initiate robust protective innate immunity without triggering autoimmune inflammation remain incompletely defined. We have previously found that TRIM29 is constitutively expressed in alveolar macrophages and plays a critical role in the control of innate immunity in respiratory tract. Also, we have shown that TRIM29 is highly expressed in human airway epithelial cells and promotes DNA virus infections by inhibiting innate immune response. Here, we found the E3 ligase TRIM29 was specifically expressed in poly I:C stimulated human myeloid dendritic cells (mDCs). The induced TRIM29 played a negative role in type I interferon (IFN) production in response to poly I:C or dsRNA virus reovirus infection. Importantly, the challenge of wildtype mice with reovirus led to lethal infection. In contrast, deletion of TRIM29 protected the mice from this developing lethality. Additionally, TRIM29−/− mice have lower titers of reovirus in the heart, intestine, spleen, liver, and brain due to elevated production of type I IFN. Mechanistically, TRIM29 was shown to interact with MAVS and subsequently induce its K11-linked ubiquitination and degradation. Taken together, TRIM29 regulates negatively the host innate immune response to RNA virus, which could be employed by RNA viruses for immune evasion.