bluuebunny/crossref_metadata_2025 huggingface dataset:
TLR/BCR co-engagement induces mature and protective T-cell independent antibody responses through MyD88 and PI(3)K signalings
Authors: Rivera Carlos Eduardo , Xu Zhenming , Casali Paolo
Published: January 2023

B cells are central to immune responses to infections and vaccines by producing high-affinity and class-switched protective antibodies, often in a T cell-dependent manner. Prompted by our findings that dual TLR/BCR engagement induces efficient CSR in vitro, to a high level comparable to that induced by CD40 engagement, here we have analyzed induction of T cell-independent CSR/SHM and antibody responses by TLR-BCR co-engagement. Immunization of wildtype C57 mice with NP hapten conjugated to TLR4-engaging LPS (NP-LPS), but not NP-Ficoll admixed with LPS, elicits high-affinity and class-switched IgGs (IgG1, IgG2a, IgG2b, IgG3), at levels comparable to those elicited by a T cell-dependent antigen NP-CGG. NP-LPS also induces sustained and high levels of IgG2a, IgG2b and IgG3, but not IgG1, in Tcrβ−/−Tcrδ−/− mice, which are devoid of T cells and, as expected, fail to mount any response to NP-CGG. Immunization of both wildtype and Tcrβ−/−Tcrδ−/− mice with highly purified Salmonella typhimurium (S. typhimurium) flagellin, which has dual BCR and TLR5 engaging activities, elicits flagellin-specific antibodies that can opsonize S. typhimurium. T cell-independent immune responses, which are impaired in mice lacking Myd88 or Pik3r1 in B cells, are associated with high mutation loads in VH genes, generation of switched B cells, and B cell differentiation into long-lived plasma cells and memory B cells, which can be re-activated for anamnestic antibody responses. Thus, SHM/CSR and B cell differentiation into plasma cells and memory B cells can occur independently of T cells, leading to protective mature antibody responses.
Toll-like receptor 9 signaling antagonizes antigen-specific B cell-helper T cell interactions
Authors: Akkaya Munir , Akkaya Billur , Kim Ann S , Miozzo Pietro , Sohn Haewon , Pena Mirna , Skinner Jeff , Kabat Juraj , Pierce Susan K
Published: January 2023

Key events in T cell-dependent antibody responses are dependent on the ability of B cells to capture antigen and process and present it to follicular helper T cells. Despite its importance, little is known about the regulation of this critical B cell function. Here we focus on the impact of signaling through the innate immune receptor, Toll like receptor 9 (TLR9), on the outcome of a B cell’s encounter with antigen. We show that although TLR9 signaling induced B cell proliferation, and secretion of cytokines and IgM, it blocked the ability of antigen-specific B cells to capture, process and present antigen. In the presence of the TLR9 agonist, CpG, B cells were less able to form synapses with antigen-containing membranes and to pull antigen from them. CpG also diminished the ability of the BCR to traffick antigen within the cell to antigen processing compartments resulting in fewer MHC-peptide complexes on the B cell surface. TLR signaling also antagonized the BCR-induced increases in the expression of both MHC class II and important co-receptors, including CD86, resulting in a decrease in the duration of antigen-specific B cell-T helper cell interaction and the inability of B cells to activate antigen-specific T cells. Chimeric mice that contained either wild type B cells or B cells deficient in TLR signaling when immunized with antigen adjuvanted with CpG showed that the loss of the B cells’ ability to respond to CpG resulted in lower antigen-specific IgM responses but enhanced high affinity antigen-specific IgG responses. Thus, B cell intrinsic TLR9 signaling may ensure rapid B cell expansion and antibody secretion at the expense of the ability of B cells to engage in germinal center events that are highly dependent antigen capture and presentation.
Dynamic regulation of T Follicular Regulatory (Tfr) cell responses by Interleukin-2 (IL-2) during influenza infection
Authors: Ballesteros-Tato Andre , Botta Davide , Fuller Michael J , Bachus Holly , Bradley John E , Zajac Allan J , Randall Troy D , Lund Frances E , Ruiz Beatriz León
Published: January 2023

IL-2 promotes FoxP3+ regulatory T (Treg) cell responses, but inhibits the differentiation of T follicular helper (Tfh) cells. However, it is not clear how IL-2 affects T follicular regulatory (Tfr) cells, a cell type with properties of both Treg and Tfh cells. Here we show that IL-2 prevents, rather than promotes, the development of Tfr cells. Using an influenza infection model, we found that Tfr cells were barely detectable at the peak of the infection, but accumulated as the immune response resolved. High levels of IL-2 at the peak of the infection promoted the expression of Blimp-1 in Treg cells, which suppressed Bcl6 expression and thereby precluded Tfr cell development. However, as IL-2 levels declined some CD25+ Treg cells down-regulated CD25, up-regulated Bcl6 and differentiated into Tfr cells, which prevented the accumulation of self-reactive antibody-secreting cells. Thus, unlike its effects on conventional Treg cells, IL-2 inhibits Tfr cell responses.
The RNA transcriptome of in vivo influenza virus infection reveals unique gene expression profiles in type 2 alveolar epithelial cells.
Authors: Stairiker Christopher , Hancock Aidan S , Boesteanu Alina C , Monzon-Casanova Elisa , Lukasiak Sebastian , Stubbs Andrew , Turner Martin John Charles , Katsikis Peter D
Published: January 2023

Globally, influenza virus outbreaks represent a serious threat to public health. Influenza virus infects type 2 alveolar epithelial cells (AEC) and utilizes cellular machinery in order to produce new infectious virions. During infection, multiple cell types release cytokines and other soluble factors that influence gene expression in both infected and uninfected cells. Here, the unique in vivo gene expression profiles between infected and uninfected bystander AEC from influenza virus infected mice are compared via next generation RNA-sequencing analysis. Mice were infected with PR8-expressing GFP virus (PR8-GFP). On day 3 post infection, GFP positive and negative AEC were FACS sorted from influenza virus infected mouse lungs. Transcriptome next generation RNA-sequencing analysis was performed comparing influenza-infected GFP-positive cells versus uninfected bystander GFP-negative cells from influenza virus-infected mouse lungs. Results from the sequencing data suggest a number of genes and associated pathways that are significantly differentially expressed in GFP-positive versus GFP-negative AEC. Pathways such as interferon signaling and cell death were found to be increased while Wnt signaling was decreased specifically within GFP-positive infected cells. RNA sequencing also identifies unique genes that are upregulated during virus infection, such as heatr9, that have yet to characterized during the response to influenza.
Sterile-activated cDC1 cells in neonatal lung induce T cell IL-4 production and lymph node maturation
Authors: Silva-Sanchez Aaron , Meza-Perez Selene , Stone Sara L , Randall Troy
Published: January 2023

Neonates are susceptible to respiratory tract infections, possibly due to immature pulmonary dendritic cells (cDCs). Here we assessed the development and function of conventional DCs in the lung and the lung-draining mediastinal lymph node (MedLN). We found that neonatal lungs, from days 3 to 10 of life, contained a higher proportion of CD103+ cDC1 cells than adult lungs and that these cells could be divided into a small population of CD103hi cDC1 and a larger population of CD103int cDC1, which were not found in adult lungs. A similar expansion of cDC1 cells was observed in the MedLN migratory cDCs during the first 2 weeks after birth. Transcriptional and functional assays showed that neonatal CD103int cDC1 cells had an activated phenotype and efficiently migrated to the MedLN, their formation was dependent on the transcription factor Batf3 but CD103int cDC1 cells developed independently of the microbiota or MyD88 signaling. Neonatal cDC1 cells express high levels of OX-40L and produce IL-12p40, but not IL-12p70, and stimulated the differentiation of IL-4-producing OT-II cells. Importantly, the MedLNs of neonatal Batf3−/− had poorly developed HEVs and FRCs, which were restored by transfer of neonatal lung CD103int into Batf3−/− mice. These changes were not observed following transfer of CD103hi or CCR7−/− CD103int DCs. Overall our results indicate that neonatal lung CD103int cDCs are activated as part of a developmental program that promotes MedLN maturation and biases CD4 T cell activation towards IL-4 production.
Gp120 transgenic mice infected with LP-BM5 virus show exacerbated symptoms compared to LP-BM5 murine AIDS alone
Authors: Arabatzis Taxiarhia J , Wakley Alexa A , McLane Virginia D , Rau Sanjay , Cao Ling
Published: January 2023

It is estimated that 36.7million people live with HIV/AIDS globally, and nearly half of them suffer from HIV associated neurocognitive disorder (HAND). The LP-BM5 virus murine AIDS (MAIDs) model and HIV gp120 transgenic (tg) mice are two distinct and well characterized models for investigating the pathophysiology of HIV/AIDS in mice. The LP-BM5 virus does not contain any HIV viral components, while gp120tg mice develop only CNS-related pathology without brain region differences. To generate a more clinically relevant murine HAND model, we examined the progression of LP-BM5 infection in gp120tg mice. Gp120tg (+) mice showed an exacerbation of the disease following infection when compared to their gp120tg (−) littermate controls and wild type (WT) C57BL/6 controls. At 12 wks post infection, all infected mice showed splenomegaly, and infected gp120tg (+) mice had higher IgG2a serum levels and an increased viral burden in hippocampi compared to the gp120tg (−) and WT controls. CCL5 expression in the hippocampus and striatum was also increased in all infected mice, with gp120 (+) mice showing the greatest elevation of hippocampal CCL5 expression. Interestingly, LP-BM5 appeared to induce brain region-dependent changes in type I interferons in gp120tg (+) mice. In addition, infected gp120tg (+) mice trended towards more errors in the spontaneous alternation T-maze test suggesting an association between a higher viral load in the hippocampus and greater memory and learning deficits. These results suggest that gp120tg mice can be productively infected with the LP-BM5 virus and display more severe MAIDS and HAND associated changes, representing a clinically relevant HAND model.
Isolation and characterization of a novel bacteriophage SH1 and optimizing PFGE to determine the molecular weight of SH1 bacteriophage’s DNA
Authors: Hoda Smriti , Greguske Elizabeth
Published: January 2023

Discovery of antibiotics has revolutionized the 20th century medicine, and has been one of the most potent methods to treat bacterial infections. Before antibiotics, infectious disease was the number one killer worldwide, in fact, bacterial infections killed more people than World War I. Hence, it took the world by a storm when certain bacteria started to develop their own defense in their war against antibiotics. Through a phenomenon called antibiotic resistance many bacteria commonly known as “superbugs” are able to overcome the treatment, and still can pose a threat to humanity.

Dr. Greguske’s research team at Saint Anselm College are turning to a novel approach of phage therapy to combat these lethal infections. The aim of the lab is to study the host-pathogen interface pathogenesis in order to utilize that mechanism the virus uses to specifically target and kill the bacteria, to ultimately treat the bacterial infection.

We isolated, purified, and conducted a biostability test of the newly found bacteriophage from the environmental sample. We extracted the phage’s DNA and optimized a protocol so that Pulse Field Gel Electrophoresis (PFGE) can be used to determine the bacteriophage DNA’s molecular weight. Upon doing so, we also have registered the phage into the virus database as SH1, and our next step is to get the bacteriophages’ genome sequenced at Dartmouth College, cross examine the database for the phage, and annotate its genome.
Intravenous immunoglobulin G (IVIg) concurrently promotes Th2 and T regulatory cell development while abrogating Th17 cells
Authors: Dembele Marieme , Tao Shao , Xue Di , Kaufman Gabriel Nathan , Mendonca Laura , Divangahi Maziar , Mazer Bruce David
Published: January 2023

We aimed to further elucidate the mechanisms by which IVIg modulates the balance between Th17, Th2, and Treg.

Using an OVA-driven murine model of allergic airway disease exhibiting a mixed Th17/Th2 response, we examined lung inflammation by H & E staining and histology, and performed cellular phenotyping of lung homogenates using flow cytometry. In addition, we assessed cytokine production by intracellular staining and ELISA, and performed co-culture studies.

IVIg treatment reduced lung inflammation and diminished the frequency and absolute number of lung Th17 cells, IL-17A production, and neutrophilia. In contrast, the frequency and absolute number of eosinophils and basophils was increased, while IgE production was abrogated in the IVIg group. This was accompanied by a significant increase in IL-13+ and IL-5+ T cells and in IL-5 concentration in the BAL. The frequency of Foxp3+IL10+ + cells within CD4+ T cells (1.43 % ± .061 vs 2.57 % ± .30, p < .01) and IL-10 secreting dendritic cells (0.28 % ± 0.060 vs 1.20 % ± 0.27, p < .05) was augmented. We also observed an increase in the frequency of CD206 expressing macrophages, or M2 macrophages, in the IVIg group (10.81% ± 2.21 vs 19.80 % ± 1.81, p < .01).

Using an in vitro DC: T cell co-culture, IVIg-treated DC inhibited IL-17A production by T cells and increased IL-13 production and Treg differentiation.

In conclusion, IVIg modulates Th2/Th17/Treg balance by favoring Treg and Th2 cells while inhibiting Th17 cells. Furthermore, IVIg renders both dendritic cells and macrophages tolerogenic.
Vitamin D supplementation modulates Th2 immune response by inducing T regulatory cells in allergic conjunctivitis
Authors: Lee Hyun Soo , Kwon Ji Young , Joo Choun-Ki
Published: January 2023

Allergic conjunctivitis (AC) is one of the most common diseases encountered in eye clinics. Recently, Vitamin D deficiency has been shown to be associated with allergic AC. This present study was designed to evaluate the efficacy of vitamin D to suppress ovalbumin (OVA)-sensitized AC in a murine model. BALB/c mice were sensitized with OVA and aluminum hydroxide. Two weeks later, mice were challenged by OVA eyedrops for 12 days with intraperitoneal injection of vehicle or 1,25(OH)2D3. We evaluated clinical signs, the infiltration of inflammatory cells, regulatory T cells (Treg), serum IgE, and Th2 cytokines through flow cytometry and ELISA. In addition, to evaluate inhibitory function of Treg cells, anti–CD25 blocking antibody or isotype control antibody was injected intravenously during topical OVA challenge. AC development and conjunctival infiltration of eosinophils and mast cells were significantly impaired with 1,25(OH)2D3 treatment. In addition, 1,25(OH)2D3 suppressed production of OVA-specific IgE in serum and Th2 cytokines in vitro T cell assays, such as IL-4 and IL-13, compared to vehicle group. Interestingly, 1,25(OH)2D3 led to increase Treg cells population in draining LNs and suppressive levels of clinical signs and inflammatory cells infiltration into conjunctivae were reversed by depleting Treg cells. Our results suggest that vitamin D supplement alleviate allergic conjunctivitis, indicated by suppressing Th2 response in draining LNs and inflammatory cells infiltration into conjunctiva through increasing population of Treg cells in draining LNs. Therefore our results demonstrated the therapeutic potential of vitamin D for allergic diseases including asthma or atopic dermatitis.
Diagnosis of red meat allergy with antigen-specific IgE tests in serum
Authors: Brestoff Jonathan R , Zaydman Mark A. , Scott Mitchell G. , Gronowski Ann M.
Published: January 2023

Red meat allergy is a tick-associated hypersensitivity reaction to galactose-α-1,3-galactose (α-gal) and is characterized by anaphylaxis, angioedema, urticaria and/or gastrointestinal symptoms occurring 3–6 hours after ingesting red meat such as beef, pork, or lamb. Diagnosis of red meat allergy is challenging due to the unusually long delay in symptom onset and poor sensitivity of skin prick tests with commercial meat extracts. The primary diagnostic tools available for this disease are quantification of α-gal-, beef-, pork-, and/or lamb-specific IgE in serum, however the diagnostic performance of these tests has not been reported. Using patient data for n=135 patients with red meat allergy and n=37 controls, we found that measurement of α-gal-specific IgE using the bovine thyroglobulin (bTG) ImmunoCAP method had the best overall sensitivity (100%) and specificity (92.3%) for diagnosis of red meat allergy. Measuring α-gal-specific IgE using the streptavidin (SA)-CAP technique or beef- or pork-specific IgE using ImmunoCAP were also effective tests with high sensitivities (89–92%) and variable specificities (65–82%). Lamb-specific IgE and total IgE had essentially no diagnostic value for red meat allergy. Positive and negative predictive values mirrored these trends. Taken together, these findings indicate that the α-gal-specific IgE test by bTG ImmunoCAP is the most useful for establishing a diagnosis of red meat allergy, although α-gal-specific IgE by SA-CAP and beef- and pork-specific IgE by ImmunoCAP are also effective tests.
RNA binding protein HuR is required for robust antigen-mediated CD4+ T cell allergic airway inflammation
Authors: Atasoy Ulus , Ellis Jason S , Techisintana Patsharaporn , Glascock Jacqueline , Ridenhour Suzanne
Published: January 2023

RNA binding protein HuR (ELAVL1) controls helper T cell differentiation by modulating the stability of multiple target mRNAs through interactions with 3′ UTR AU-rich elements. We hypothesized that HuR regulates antigen induced T cell activation and initiation of Th2 driven allergic airway inflammation. HuR was conditionally ablated in developing T cells prior to thymic egress (distal Lck-Cre ROSA HuRfl/fl mouse). Upon in vitro activation, YFP+ (HuR KO) T cells produced significantly less IL-4, IL-5, and IL-13 than control cells and considerably more IL-2. We also observed reductions in Gata-3, IL-4 and IL-2Rα (CD25) nascent transcription. The OVA challenge model of airway inflammation was used to ascertain the effects of HuR KO in vivo. While only 50% of CD4+ T cells in HuR KO mice lack HuR, there was total suppression of Th2 responses and these mice had complete amelioration of lung inflammation. Lung infiltrating CD4+ T cells had reductions in IL-2 and CD25 expression, and OVA challenged HuR KO mice had significantly reduced BAL IL-13. In parallel, mice were immunized with either OVA or KLH, boosted 10 days later, and antigen specific proliferative responses measured in vitro to investigate antigen-specific T cell responses in the absence of HuR. HuR KO T cells had profound proliferation defects compared to controls, and CD3/CD28 stimulation only partially restored proliferation defects. HuR KO T cells had reductions in STAT5/STAT6 signaling, pathways which are key for Th2 differentiation and function. We conclude HuR plays an indispensable role in regulating antigen specific allergic airway inflammation and Th2 differentiation.
EMPD-specific chimeric antigen receptor mediates T cell cytotoxicity to B cells expressing membrane IgE
Authors: Ma Zhengyu (Mark) , Fay Brittany F
Published: January 2023

IgE is the key mediator of allergic immune responses. Targeting the source of IgE, the IgE-expressing B cells, using adoptive T cell therapy (ACT) represents an attractive therapeutic approach for severe allergic diseases such as severe allergic asthma. To develop ACT for allergy, we designed chimeric antigen receptors (CARs) that recognize the extracellular membrane-proximal domain (EMPD) of membrane IgE (mIgE). EMPD serves as an ideal marker for IgE-expressing B ells since it exists only on mIgE but not on secreted IgE, and all IgE-expressing B cells express mIgE, although at varying levels. To construct EMPD-specific CARs, variable regions of four EMPD-specific monoclonal antibodies were cloned to assemble single chain variable fragments (scFvs) for EMPD binding. EMPC-specific CARs were efficiently expressed on the surface of primary human T cells. One of the CARs, 2E3E10, mediated potent cytotoxicity and INFγ production in response to U266 cells and Daudi cells expressing mIgE. The responses to U266 cells are especially encouraging since the cells express very low levels of mIgE. Our study demonstrates the proof of concept for using T cells expressing EMPD-specific CARs to eliminate IgE-expressing B cells. The rapid advances in the efficacy and safety of ACT should facilitate the development of CAR T cell immunotherapy for severe allergic diseases. Taking advantage of the ability of CAR T cells to persist and continuously eliminate IgE-expressing B cells, the approach may relieve symptoms for an extended period of time with a single treatment.
Role of Rab7 in activating NF-kB in B cells to mediate class-switching and plasma cell survival in antibody and autoantibody responses and to promote B cell lymphomagenesis
Authors: Yan Hui , Wang Rui , Lam Tonika , Kulp Dennis , Rivera Carlos Eduardo , Taylor Julia R. , Wang Zhonghua , Zan Hong , Zhong Guangming , Ivanov Dmitri , Casali Paolo , Xu Zhenming
Published: January 2023

NF-kappaB activation mediates B cell class-switching to generate IgG, IgA and IgE as well as survival of plasma cells, as differentiated from B cells to secrete antibodies at a high rate. Here we have identified a B cell-intrinsic role of Rab7, a small GTPase located in endosomes, in activating NF-kappaB for class-switching and plasma cell survival. Mice that are deficient in Rab7 specifically in B cells or treated with CID 1067700, a Rab7-specific small molecule inhibitor, cannot mount T-independent or T-dependent switched antibody responses, despite normal B cell proliferation/survival and T cell/myeloid cells functions. Rab7 co-localizes, on endosomes, with the CD40 receptor that is internalized upon engagement and directly interacts with TRAF6, an adaptor of CD40 and TLR signaling for NF-kB activation, thereby forming complexes that transduce signals. Disruption of such complexes by CID 1067700 hampers NF-kappaB activation in normal B cells and abrogates NF-kappaB dysregulation in lupus B cells and B lymphoma cell lines, in which Rab7 is upregulated. Treatment with CID 1067700 reduces pathogenic IgG autoantibody responses in murine lupus models, leading to life-span extension, and prevents tumorigenesis in nude mice transplanted with B lymphoma cells. These, together with the rescue of class-switching/plasma cell survival in normal B cells and proliferation/survival of B lymphoma cells by enforced NF-kappaB activation, indicate that Rab7 plays an important role in NF-kappaB activation to mediate B cell differentiation and dysregulation and is a potential therapeutic target in lupus and B cell lymphomagenesis.

Supported by NIH grants AI 079705, AI 105813/105813S1 (to P. C.) and AI 124172 (to Z. X.).
ADAM10 is a novel sheddase of Inducible Costimulatory Ligand (ICOSL).
Authors: Luker Andrea , Lownik Joseph Cornelius , Conrad Daniel
Published: January 2023

ICOSL is an important co-stimulatory molecule involved in the development of TH2 responses through germinal center reactions. Although not well defined, the regulation of ICOSL is known to occur through its interaction with ICOS. Evidence suggests surface expression levels are regulated by a catabolic event, and not through internalization. Here we confirm that regulation of ICOSL is mediated by the metalloproteinase ADAM10. Cleavage of recombinant ICOSL results in an amino-terminal fragment of approximately 3kDa, which is blocked by an ADAM10-specific inhibitor. Recombinant and cell-based cleavage assays suggest that ADAM17 can also cleave ICOSL. In vivo, we observed a ten-fold increase in ICOSL expression in ADAM10B−/− mice compared to wildtype, a phenomenon that was absent in ADAM17B−/− mice. This suggests that ADAM10 is the primary physiological sheddase of ICOSL, while ADAM17 may serve as a secondary sheddase. Additionally, ADAM10B−/− mice develop a significantly diminished T follicular helper (TFH) cell population in response to NP-KLH/alum immunization. Together, these data provide insight into the regulatory mechanism between ICOS/ICOSL and suggest ADAM10 may be a potential therapeutic target for attenuating antibody driven diseases.
Alpha(v) Integrins Engage Autophagy Components to Regulate B cell Immune Response
Authors: Acharya Mridu , Sokolovska Anna , Tam Jenny M , Raso Fiona , Stuart Lynda , Jackson Shaun , Rawlings David J , Lacy-Hulbert Adam
Published: January 2023

Signaling through Toll-like receptors (TLR) has been implicated in activation of immune cell populations such as B lymphocytes. TLR ligands associated with self-antigens can drive increased self-reactive B cell responses however TLR ligands present in pathogenic products or vaccines can also enhance protective antibody responses. Our recent work has identified a novel mechanism by which αv integrins, a family of adhesion molecules and the autophagy proteins limit excessive B cell TLR signaling.

Specifically, we have found that αvβ3 integrin regulates TLR signaling (NFKB and IRF7 activation) by directing maturation of TLR containing endosomes, through activation of components of the autophagy pathway (LC3 and Atg5). B cells lacking either αv integrins or autophagy components show enhanced TLR signaling and increased proliferative responses to TLR ligands. Mice with disruption in this pathway develop increased autoantibodies with age and develop accelerated autoimmunity in murine lupus models. Therefore, we propose that αv-mediated regulation of TLR signaling exists to limit excessive B cell responses to self-antigens.

In addition, we find that mice in which αv is specifically deleted from B cells mount stronger antibody responses when immunized with exogenous antigens containing TLR-ligand adjuvants. Immune responses to these antigens are characterized by increased expansion of germinal center cells, increased somatic hyper-mutation and higher levels of high-affinity class-switched antibodies. Therefore this αv mediated regulation of TLR signaling not only regulates B cell responses to self-antigens but it is also important for regulating germinal center B cell responses and production of high-affinity antibodies.
TRPV4 attenuates M1 polarization and subsequent NASH progression by stalling CYP2E1-mediated redox toxicity via eNOS.
Authors: Seth Ratanesh K , Chandrashekaran Varun , Dattaroy Diptadip , Alhasson Firas , Nagarkatti Mitzi , Nagarkatti Prakash , Diehl Anna Mae , Liedtke Wolfgang , Chatterjee Saurabh
Published: January 2023

M1 macrophage polarization in nonalcoholic steatohepatitis (NASH) liver has been impacted by several exogenous or endogenous factors including inflammatory stimuli, oxidative stress, and cytokines. In absence of an active endogenous defense mechanism, the M1 polarization bias potentiates NASH progression by intensified inflammation and intermittent fibrosis in the rodent model of liver injury. We introduce an endogenous defense mechanism in the liver that is mediated by TRPV4, a transient receptor potential calcium-permeable ion channel that responds to the cytotoxic liver environment and negatively regulates CYP2E1. We hypothesized that CYP2E1-mediated oxidative stress causes M1 polarization in experimental NASH and active TRPV4 inhibits CYP2E1 mediated inflammation with concomitant attenuation of M1 polarization. Since CYP2E1 takes center stage in redox toxicity we use a toxin model of NASH which uses pyrazole, a ligand and a substrate of CYP2E1 for inducing liver injury with NASH-like phenotype. Using both cyp2e1−/− and trpv4−/− mice, we show that in the absence of active TRPV4, CYP2E1 induced oxidative stress causes M1 polarization bias, that includes a significant increase in IL-1β, IL-12, IL-6 and IL-23 while CYP2E1 null or diallyl sulfide treated mice prevent it. Recently, we discovered that the TRPV4 modulates eNOS activation and nitric oxide release from Kupffer cell during an early stage of liver injury. Based on the adaptive NO increase in trpv4+/+ mice, NO donor administration in trpv4−/− mice abrogated CYP2E1-induced oxidative stress, M1 polarization, and NASH progression. Thus, a novel endogenous defense molecule TRPV4 can be a promising immunotherapeutic approach against chronic liver injury.
Reduced circulating MAIT cells in obesity
Authors: Liu Jianyun , Brutkiewicz Randy R
Published: January 2023

Obesity is a major risk factor for certain types of cancer and the mechanisms by which obesity contributes to tumor metastasis are not well understood. Whereas the immune system is essential for fighting against tumors, in the context of obesity, systemic chronic inflammation with altered immune responses are found. Recent studies have revealed that the gut microbiota also contributes to obesity and the pathology associated with that disease. Mucosal-associated innate T (MAIT) cells are a unique subpopulation of T cells that are characterized by the expression of a semi-invariant TCR a chain (Vα19 in mice; Vα7.2 in humans). Although the development and maturation of MAIT cells require the gut microbiota and antigen presenting molecule MR1, the actual function of MAIT cells has not been well studied. We have found reduced circulating MAIT cells in obese patients as compared to age- and gender-matched healthy donors. Using real-time PCR to determine the expression levels of the invariant TCR of MAIT cells and classical T cell transcription factors, we found that, although no difference was observed in any gene expression analyzed between samples from obese patients or healthy donors, mRNA expression of the Vα7.2 TCR positively correlated with the transcription factor TBX21 (found in T helper 1 cells) and BCL6 (found in follicular helper T cells). Our work suggests that obesity alters the number of MAIT cells. The correlation of MAIT cell TCR gene expression with transcription factors of classical effector T cells, suggests there may be transcriptional pathways linking MAIT cells to the function of conventional T cells. Future work will focus on determining how obesity impacts the function of MAIT cells.
TWEAK/Fn14 activation is instrumental in Ro52-mediated photosensitization in cutaneous lupus erythematosus
Authors: Liu Yale , Xu Meifeng , Min Xiaoyun , Wu Kunyi , Zhang Ting , Li Ke , Xiao Shengxiang , Xia Yumin
Published: January 2023

Background

TWEAK, a member of the TNF superfamily, binds to the Fn14 receptor, participating in many pathological alterations. Ro52 autoantigen mediates the photosensitization of lupus erythematosus. Recently, TWEAK/Fn14 activation was found in the lesion of cutaneous lupus erythematosus (CLE).

Objectives

To further show the potential role of TWEAK/Fn14 pathway in Ro52-mediated photosensitization.

Methods

The expression of TWEAK and Fn14 was detected in skin tissues from patients with LE and healthy donors. The expression of Ro52 was determined in murine keratinocytes that were treated with TWEAK or plus UVB irradiation. Some cells were pretreated with siRNA transfection or specific inhibitors of signaling pathways. The chemotaxis of J774.2 macrophages was evaluated on TWEAK stimulation of cocultured keratinocytes. The expression of TNF receptor associated factor 2 (TRAF2) and TNF receptors (TNFR) was determined in keratinocytes upon UVB or plus TWEAK treatment.

Results

TWEAK, Fn14, and downstream cytokines were highly expressed in CLE lesions that overexpressed Ro52. Moreover, TWEAK promoted the enhancement effect of UVB on Ro52 expression in keratinocytes. Meanwhile, TWEAK stimulation of keratinocytes favored the migration of macrophages. Furthermore, nuclear factor-kappa B inhibitor or Fn14 siRNA transfection abrogated the TWEAK effect on Ro52 regulation. Similarly, TRAF2 siRNA attenuated the TWEAK effect on Ro52 expression. Interestingly, UVB irradiation increased the expression of TNFR1 but decreased TNFR2 expression in keratinocytes.

Conclusion

TWEAK/Fn14 activation is instrumental in Ro52-mediated photosensitization and involves the function of the Fn14-TRAF2-TNFR axis.
Noncanonical NF-κB signaling is elevated in inflammatory bowel disease patients and may be associated with therapeutic response
Authors: Allen Irving C , Eden Kristin , Nguyen Vu , Knight Kristin , Sorrentino Dario
Published: January 2023

The canonical NF-κB signaling pathway is well characterized in the context of inflammatory bowel disease (IBD). However, noncanonical NF-κB signaling has not been evaluated. This pathway is key for the production of specific chemokines with diverse immunological effects in the gut. To evaluate this pathway, biopsy specimens from control patients and patients with IBD (CD and UC) were collected and pathways associated with inflammation were assessed via custom RT-PCR arrays. These expression data were compared to lesion status, IBD subtype (CD or UC), and treatment. Our analysis revealed significant up-regulation of several key genes associated with noncanonical NF-κB signaling in lesion tissue from IBD patients compared to controls and non-lesion areas. Interestingly, CD patients displayed increased noncanonical signaling compared to both control and UC patients in over 20 genes directly involved in the noncanonical pathway. Treatment with the anti-TNF biologic infliximab resulted in down-regulation of these and other noncanonical NF-κB signaling molecules in CD patients. Subsequent bioinformatics analysis of CD patients revealed that elevation of chemokines associated with noncanonical NF-κB signaling are indicators of infliximab responsiveness, with patients refractory to infliximab showing significantly elevated levels of gene expression. Together, these data indicate that noncanonical NF-κB signaling is elevated in IBD patients and may have links to therapeutic response.
Correlation of sentinel lymph node immune cells with disease-free survival and metastasis in breast cancer patients using 4-color chromogen-based immunohistochemistry and quantitative imaging microscopy
Authors: Blenman Kim RM
Published: January 2023

In this study, we assessed whether the cell densities of in situ sentinel lymph node (SLN) CD3+ T cells, CD20+ B cells, and CD1a+ dendritic cells correlated with cancer cell invasion, disease-free survival, or distant metastasis in breast cancer patients. Multiplexed chromogen-based IHC and quantitative microscopy imaging were performed on SLNs from 115 breast cancer patients.

Results

In our cohort, breast SLN T cells were reduced in microenvironments invaded by cancer cells (positive) compared to microenvironments that were cancer cell free (negative) (p = 0.03). The T cell reduction was dependent on cancer cell invasion (pearson correlation = −0.21, p = 0.03). There were no differences in dendritic cells or B cells between positive and negative microenvironments. Low numbers of T cells and B cells were associated with shorter disease-free survival in univariate analysis (p < 0.0001, HR = 0.43; p = 0005, HR = 0.46, respectively). Logistic regression revealed that B cells were the only immune cells that were associated with an increased odds of metastasis in both univariate and multivariate analysis (p = 0.02, OR = 2.41; p = 0.01, OR = 5.42, respectively). SLN T cells were the only immune cells that were significantly reduced in patients with local metastases (p = 0.04). SLN B cells were the only immune cells that were significantly reduced in patients with distant metastases (p < 0.0001).

Conclusion

In our breast cancer cohort, reduced numbers of SLN T cells predicted cancer cell invasion, shorter disease-free survival, and local metastases. Reduced numbers of SLN B cells predicted shorter disease-free survival and distant metastases.
hetIL-15 treatment of rhesus macaques is safe and systemically increases effector cytotoxic lymphocytes that infiltrate the lymph nodes.
Authors: Watson Dionysios C , Valentin Antonio , Moysi Eirini , Petrovas Costas , Bergamaschi Cristina , Felber Barbara K. , Pavlakis George N
Published: January 2023

Background

Heterodimeric interleukin-15 (hetIL-15) is presently evaluated in humans for the treatment of cancer. hetIL-15 activates and expands cytotoxic T and NK cells, which suggests that the cytokine could be useful for treating diseases such as malignancies and HIV infection.

Methods

Rhesus macaques received six s.c. injections of hetIL-15 over 2 weeks using increasing doses of cytokine (step-dosing). Animals were sacrificed after treatment and hetIL-15 effects on lymphocytes isolated from necropsy tissues were monitored by multiparametric flow cytometry and quantitative multiplexed confocal microscopy imaging (Histo-cytometry).

Results

This regimen was found to be safe in macaques and resulted in systemic proliferation and expansion of CD8+ and NK cells with higher granzyme B content. These expanded cells were found in both effector sites, such as liver, vagina and rectum, and secondary lymphoid tissues. A significant increase in effector memory CD8+ T cells was found in peripheral lymph nodes (LN) from all hetIL-15-treated macaques. Imaging analysis by Histo-cytometry revealed that effector CD8+ T cells infiltrated the B cell follicles where chronically infected follicular helper CD4+ T cells are located.

Conclusions

Step-dose administration of hetIL-15 is a well-tolerated regimen that results in systemic activation and expansion of cytotoxic leukocytes that infiltrate areas where chronic HIV-infected cells reside. These results suggest that hetIL-15 could be useful in disrupting or eliminating long-term viral reservoirs in HIV-1 infected individuals, contributing to a functional cure of the infection. Work assessing the impact of hetIL-15 on the size of the viral reservoir in sampled LN is currently ongoing.
Gemcitabine-generated ROS promotes neutrophil chemotactic activity through CXCL8 production via PKCδ/ERK pathway in a murine 4T1 breast cancer model
Authors: Jeong Jinseon , Kim Yong-Jae , Yang Kwang Hoon , Yoon Sun Young , Sohn Ki-Young , Kim Heung-Jae , Kim Jae Wha
Published: January 2023

Chemotherapy-induced neutropenia (CIN) is a common side effect that necessitate dose reductions during treatment of cancer patients. Preventing CIN is critical in chemotherapy because a rapid decline of neutrophil counts increase susceptibility to infection of cancer patients. In spite of its importance, the mechanism of CIN still remains unclear. This study is aimed to investigate the mechanism of CIN in an aspect of neutrophil activation by chemotaxis in gemcitabine-treated breast tumor model. We hypothesized that gemcitabine-generated ROS promotes CXCL8 or MIP-2 production by upregulating ERK pathway in PKCδ-activated intratumoral macrophages and thus neutrophil recruitment to the chemokine gradient. In 4T1 breast tumor mouse model, we found that the basal level of PKCδ activity was higher than in normal mice. The number of blood and peritoneal leukocytes was analyzed at 24h after intraperitoneal administration of gemcitabine. The neutrophil counts decreased in the blood but increased in the peritoneal cavity. In addition, intratumoral neutrophils were elevated in the gemcitabine-treated group. The mRNA levels of neutrophil attracting chemokines/receptors were increased in the peritoneal and tumoral cells. ROS inhibitors N-Acetyl-L-cysteine and Apocynin blocked gemcitabine-induced neutrophil migration, mRNA expression of the chemokines and receptors and PKCδ/ERK phosphorylation. CXCR1/2 inhibitor reparixin decreased neutrophil recruitment to the peritoneal cavity and the tumor tissue. Together, our results suggest that gemcitabine treatment promotes neutrophil chemotactic activity by increasing ROS/PKCδ/ERK/CXCL8 pathway. These findings will provide new insights for developing a therapeutic for CIN.
Cooperative anti-tumor effect of soluble NKG2D ligand MIC neutralization and PD-1 pathway blockade
Authors: Basher Fahmin , Wu Jennifer
Published: January 2023

Shedding of the human NKG2D ligand MIC from tumor cell surfaces correlates with progression of many epithelial cancers. Soluble MIC enables tumor immune escape through multiple immune suppressive mechanisms, such as disturbing natural killer (NK) cell homeostatic maintenance, impairing NKG2D expression on NK cells and effector T cells, and facilitating the expansion of arginase I+ MDSCs. Our recent studies have demonstrated that sMIC is an effective cancer therapeutic target. We thus sought to investigate whether targeting sMIC using a nonblocking monoclonal antibody would enhance immunotherapies currently in use, such as PD-1 blockade therapy, using genetically engineered transplantable syngeneic sMIC+ tumor models. We demonstrate that combination therapy of a sMIC-neutralizing antibody and anti-PDL1 improved the survival of animals bearing sMIC+ tumors, in comparison to monotherapy. We further demonstrate that combination therapy augmented the activation and functional potential of NK cells, CD8 T cells, and CD4 T cells. Combined therapy significantly enhanced antigen-specific CD8 T and CD4 cell responses. Combined therapy also re-modulated the tumor microenvironment to be more immune activating and reduced neovascularization. Our findings provide a rationale for co-targeting sMIC to enhance the therapeutic efficacy of PD-1 blockade.
Immune modulatory effects of chemotherapy increase the effectiveness of anti-PD1 immunotherapy in a poorly immunogenic murine model of osteosarcoma
Authors: Ladle Brian H , Phillips Maggie J , Yu Colburn , Gamper Christopher J
Published: January 2023

While great excitement surrounds the success of anti-PD1 cancer immunotherapies, few responses are observed treating sarcomas with these agents. The low mutation burdens and minimal T cell infiltrates present in sarcomas, especially pediatric sarcomas, may explain the lack of response to anti-PD1 therapy. We investigated combining chemotherapy treatment with a sarcoma vaccine (GVAX) and anti-PD1 treatment in a mouse model of osteosarcoma. We targeted osteosarcoma as patients treated with standard of care chemotherapy agents (doxorubicin, cisplatin, and methotrexate (MTX)) maintain robust lymphocyte counts, making combination with immunotherapy plausible. Consistent with the poor immunogenicity of sarcomas, mice challenged with the syngeneic osteosarcoma cell line K7M2 showed poor responses to GVAX alone (irradiated K7M2 tumor cells mixed with irradiated GM-CSF-secreting bystander cell line), anti-PD1 alone, or combining GVAX with anti-PD1 treatment. Treating K7M2 tumors with doxorubicin or cisplatin alone had no impact on tumor growth. However, treatment with MTX alone did show shrinkage of tumors in 30–40% of mice with kinetics consistent with an immune-mediated response. Further supporting an immune-mediated effect, MTX alone had no impact on K7M2 tumor growth in NSG immune deficient mice. Combining MTX with GVAX and anti-PD1 resulted in complete rejection of established K7M2 tumors in 70% of mice. These experiments support further investigation of combining chemotherapy agents (such as MTX) with immunotherapies. Our findings also indicate that poorly responsive tumors to anti-PD1 monotherapy can be rendered responsive, broadening the potential use of checkpoint blockade to include poorly immunogenic tumors.
Stereotactic radiotherapy increases functionally suppressive regulatory T cells in the tumor microenvironment
Authors: Muroyama Yuki , Nirschl Thomas R. , Kochel Christina M. , Lopez-Bujanda Zolia , Theodros Debebe , Mao Wendy , Carrera-Haro Maria A. , Ghasemzadeh Ali , Marciscano Ariel E. , Velarde Esteban , Tam Ada J. , Thoburn Christopher J. , Uddin Muniza , Meeker Alan K. , Anders Robert A. , Pardoll Drew M. , Drake Charles G.
Published: January 2023

Radiotherapy (RT) enhances innate and adaptive anti-tumor immunity; however, the effects of RT on immune-suppressive regulatory T cells (Tregs), in the tumor microenvironment (TME) have not yet been fully elucidated. Although previous reports suggest a post-RT increase in Tregs, whether these Tregs are functionally suppressive has not been determined. To test the hypothesis that RT enhances the suppressive function of Tregs in the TME, we selectively irradiated implanted tumors using the Small Animal Radiation Research Platform (SARRP), which models stereotactic RT in human patients– followed by flow-cytometric and functional analyses of tumor-infiltrating lymphocytes (TIL). Our data showed that RT significantly increased tumor-infiltrating Tregs (TIL-Tegs), and that these cells have higher expression of CTLA-4, 4-1BB, and Helios, consistent with activated/suppressive phenotype. This observation held true across several tumor models (B16/F10, RENCA, MC38). Notably, we found that post-RT, TIL-Tregs had equal or improved suppressive capacity compared with non-irradiated tumors. Our data also indicated that Tregs proliferate post-RT, more robustly than the other T cell subsets in the TME. In addition, the post-RT Treg expansion occurred when T cell migration was inhibited by Fingolimod - suggesting that the post-RT Treg increase is likely due to preferential proliferation of intratumoral Tregs. Our data also suggest that post-RT Treg expansion is independent of TGF-β and IL-33. Collectively, these data demonstrate that RT increases the phenotypically and functionally suppressive Tregs in the TME, and provide a rationale for treatment regimens that combine RT with Treg-targeting agents to maximize anti-tumor efficacy.
Guided delivery of the “alarming” cytokine IL-33 to tumor by chimeric antigen receptor T cells
Authors: Chen Yuang , Lu Binfeng
Published: January 2023

Current immune checkpoint-based cancer therapy is limited by low levels of spontaneous antitumor T cells and immune suppressive microenvironment (TME). Interleukin-33 (IL-33) is a member of the IL-1 family of cytokines and is released by damaged tissue cells or activated innate immune cells such as macrophages upon tissue damage or infection. Thus, IL-33 is thought to serve as an endogenous “alarming signal”, which triggers inflammation and cell-mediated immune responses in affected tissues. We have found that IL-33 is expressed in many normal human lining tissue cells but drastically down-regulated in advanced cancer cells indicating the role of IL-33 in inhibiting tumor progression. Thus, IL-33 is a candidate cytokine whose down-regulation is an important mechanism for tumor “immune ignorance”, which is crucial for immune evasion by cancer cells. Consistent with this idea, we have furthered shown that expression of IL-33 in tumor cells inhibit tumor growth. Thus, tumoral delivery of IL-33 can serve as a novel therapy for immune therapy of cancer. In the current study, we “armed” CAR T cells with IL-33 expression cassette, which allows guided IL-33 expression in the tumor site specifically. The antitumor efficacy and toxicity of such strategy is evaluated in a humanized mouse model. The data will show whether and how tumor-site delivery of IL-33 increases efficacy of CAR T cells and immunogenicity of the TME.