bluuebunny/crossref_metadata_2025 huggingface dataset:
Discrete modeling of macrophage differentiation
Authors: Butchy Adam A , Miskov-Zivanov Natasa
Published: January 2023

The process through which macrophages differentiate into either an M1 or M2 state is thought to be well elucidated. Literature suggests that only a handful of receptors and their ligands play a role in deciding macrophage fate. These receptors share many signaling pathways and work together to create either an M1 or M2 phenotype. While these phenotype generalizations are broad, the M1 and M2 states have been used as placeholders to describe drastically different metabolic phenotypes.

To determine if this complex process could be controlled by a handful of ligands and receptors, we used discrete modeling—a modeling method that uses Boolean logic and a simple notation that qualitatively describe a system—to create a model of the differentiation pathway as it is currently understood. From there, we began testing the model, seeing if we could elicit either M1 or M2 states by altering the levels of cytokines and chemokines in the model to simulate a macrophage being exposed to different stimuli. According to literature, M1 activation is generally elicited by: LPS, RAGE, and Interferon gamma; and M2 activation by: CSF-1, IL-13, and IL-4. In order to determine if a phenotype was successfully observed, we defined M1 activation as high expression of TNF-alpha, IL-12 and CD-48; and M2 activation as high expression of IL-10, TGF-beta and CD-36.

With this model, we were able to simulate the process of M1 and M2 differentiation using changes in ligand concentrations to elicit the metabolic phenotypes of each activation state. This means that the current understanding of the differentiation pathway is sufficient to build functional differentiation models. This opens up opportunities to explore the broad metabolic variation within activation states.
MicroRNA-30 modulates metabolic inflammation by regulating Notch signaling in adipose tissue macrophages.
Authors: Miranda Kathryn , Nagarkatti Prakash , Nagarkatti Mitzi
Published: January 2023

Macrophages are innate immune cells that play integral roles in maintenance of adipose tissue homeostasis. Visceral obesity stimulates pro-inflammatory macrophages to infiltrate into adipose tissue causing chronic low-grade inflammation that can lead to insulin resistance and cardiometabolic disorders. Notch signaling is elevated in obesity and serves as a form of communication between macrophages and adipocytes. Blockade of this pathway promotes adipose tissue browning while reducing inflammation and therefore, has therapeutic potential. MicroRNAs (miRNA, miR) are non-coding RNAs that bind the 3′UTR of target mRNAs to repress their translation and miRNA based therapies are presently being developed for clinical purposes. In the current study, we identified differentially expressed miRNAs in adipose tissue macrophages (ATMs) between lean and obese mice by microarray. Array analysis and PCR validation revealed miRNAs -30a-5p, -30c-5p, and -30e-5p were downregulated in obese ATMs suggesting the miR-30 family plays an important role in macrophage phenotype. Pathway analysis demonstrated that miR-30 targeted Notch signaling genes including Delta-like ligand-4 (DLL4), a previously identified therapeutic target for cardiometabolic disorders. It was noted that DLL4 expression was increased on obese ATMs and in vitro miRNA transfection studies further demonstrated that miR-30 modulates Notch signaling-induced inflammation. These data demonstrate for the first time that the miR-30 family may play a critical role in the regulation of DLL4-mediated Notch signaling in ATMs, thereby modulating metabolic inflammation. (Supported in part by NIH grants P01AT003961, R01AT006888, R01ES019313, R01MH094755 and P20GM103641).
The HMGCR inhibitor Fluvastatin induces apoptosis and autophagy in primary and neoplastic mast cells
Authors: Barnstein Brian O , Paez Patrick A , Kolawole Elizabeth Motunrayo , Ryan John J
Published: January 2023

Mast cells are best known for their role in inflammatory and allergic disease. Mast cell leukemia is a rare subtype of acute myelogenous leukemia, typically expressing the c-Kit mutation, D816V, that yields factor-independent growth. Understanding and treating mast cell hyperplasia and leukemia could be beneficial to both inflammatory and malignant diseases. Recently the family of statin drugs, widely employed as HMG CoA Reductase (HMGCR) inhibitors to lower serum cholesterol, have also been found to be immunosuppressive. However, statin effects on mast cell survival are not known. We report that Fluvastatin induces apoptosis in mouse bone marrow-derived mast cells (BMMC) and P815 mastocytoma cells in a dose- and time-dependent manner. Apoptosis was evidenced by DNA fragmentation and caspase activation. Importantly, the mastocytoma line P815, which expresses mutant c-Kit, was readily killed by Fluvastatin, with an IC50 of 5–10μM. P815 cell death was marked by c-Kit upregulation preceding mitochondrial membrane instability and Caspase-9 activation. Chemical blockade of autophagy reduced Fluvastatin-mediated apoptosis in primary mast cells, but augmented apoptosis in transformed mast cells. These data suggest that the HMGCR pathway may be targeted to suppress mast cell survival, and that its combination with autophagy inhibitors may be useful for treating mast cell neoplasias.
A20 regulates NF-κB activation through K48 linked polyubiquitination of NEMO
Authors: Lee Younglang , Lee Eries , Zilberman-Rudenko Jevgenia , Chen Yong , Gucek Marjan , Siegel Richard M. , Hanson Eric Paul
Published: January 2023

Activation of the NF-κB family of transcription factors is related to diverse biological processes, such as development, immune responses, and inflammation. As a result, mutations affecting the NF-κB signaling pathway cause syndromes characterized by immunodeficiency and autoinflammaotory disease. Recently, we identified the C-terminus of NEMO to be a regulator of NF-κB signaling, as a class of NEMO mutations lacking the C-terminus (ΔCT-NEMO) confer gain-of-function properties to the IKK complex due to impaired recruitment of A20 to the TNFR. Here, we investigated details by which A20 recruitment to the TNFR suppresses NF-κB signaling. The C-terminus of NEMO has been previously identified to preferentially recognize K63-linked polyubiquitin that in its unanchored form can mediate NEMO/A20 interaction. We found that the IKK complex containing ΔCT-NEMO forms but not other mutant forms of NEMO exhibit impaired K63 polyubiquitin binding ability. In addition to unanchored K63-lilnked polyubiquitin, we hypothesized that direct polyubiquitination of A20 could enable a functional interaction between A20 and NEMO. Using mass spectroscopy, we identified 4 lysine residues on A20 to be ubiquitination sites. Mutation of these lysines to arginine impaired A20’s ability to suppress TNF induced NF-κB activation. We noted stabilization of NEMO in A20 deficient cells following TNF stimulation. Biochemical studies in Jurkat and 293 cells revealed A20 dependent K48 linked polyubiquitination of NEMO at the TNFR following receptor stimulation. Our data suggest a mechanism of NF-κB regulation in which A20 is directly K63 polyubiquitinated to functionally interacts with NEMO which it targets by K48 linkages proteasomal degradation.
Identification of Nuclear Matrix Protein 4 (Nmp4) as a novel regulator of innate immunity
Authors: Bidwell Joseph , Morris David L , Adaway Michele , Huang Shengping , Shao Yu , Wek Ronald C , Sun Jie
Published: January 2023

Mesenchymal stem/progenitor cells (MSPCs) have immunomodulatory activity. We previously observed that osteoprogenitors and CD8+T cells are increased in the bone marrow (BM) of mice lacking the transcription factor Nmp4. Transcriptome analysis of MSPCs from Nmp4−/− and wild type (WT) mice revealed significant changes in genes related to innate immune responses, including reduced expression of Tlr3 and Mda5 in the null MSPCs. Loss of Nmp4 increased mRNA translation, ribosomal biogenesis, and Gadd34 expression in MSPCs; collectively, these processes likely promote the unfolded protein response (UPR), expanding the processing capacity of the endoplasmic reticulum. Therefore, we hypothesized that disabling Nmp4 would improve innate immune cell function. To test this, Nmp4−/− mice (C57BL6) were infected intranasally with influenza A/PR8 (H1N1; 150pfu). Despite having similar virus load in the lung, H1N1-infected Nmp4−/− mice lost less body weight than WT mice post infection and had improved survival. Infected Nmp4−/− mice had fewer infiltrating neutrophils (PMNs; CD11b+/Ly6G+) and monocytes (CD11b+/SiglecFNeg/Ly6GNeg/Ly6C+) in bronchoalveolar lavage (BAL) but more BAL macrophages (CD11c+/SiglecF+); CD4+ and CD8+ T cells were not different between genotypes. Ex vivo, Nmp4-deficiency did not alter the differentiation of BM marrow-derived macrophages; however, Chil3/Ym1 and Alox15 mRNA expression was reduced in Nmp4−/− macrophages polarized with IL-4/IL-13, suggesting cell-autonomous regulation of M2 macrophage activation by Nmp4. Our data point to Nmp4 as a novel regulator of innate immunity and influenza response in mice.
Discovery of two meningeal macrophage populations with differential roles during homeostasis and inflammation
Authors: Rua Rejane , Johnson Kory , McGavern Dorian B
Published: January 2023

The meningeal and perivascular spaces of the central nervous system (CNS) are inhabited by specialized macrophages. Under steady state conditions, we discovered two populations: immature macrophages (MHC-II−) and mature macrophages (MHC-II+). Microarray analyses revealed that IL-10 and TGFb were upstream regulators of the immature macrophage transcriptome, which included stem cell-specific genes. These data suggest that immature macrophages represent local progenitors maintained by anti-inflammatory cytokines within the meninges. Interestingly, in naïve mice MHC-II+ macrophages were enriched upon aging and upregulated inflammatory genes, suggesting age-based maturation. To better understand the dynamics of these macrophages, we triggered CNS inflammation by inoculating mice with lymphocytic choriomeningitis virus (LCMV). Both myeloid populations were infected by the virus, and intravital imaging studies revealed that they were targeted by infiltrating virus-specific CD8+ T cells, which promoted their depletion. Following viral clearance, myeloid repopulation of the meninges was derived largely from infiltrating monocytes that engrafted this CNS niche and adopted a transcriptomic signature of mature resident meningeal macrophages. In stark contrast, sterile depletion of meningeal macrophages without infection induced massive local proliferation of immature macrophages that transformed into mature macrophages and repopulated the meninges. This occurred in the absence of peripheral monocyte engraftment. Collectively, these data indicate that the CNS meninges are inhabited by two macrophage populations with a differential ability to repopulate the niche based on the inflammatory milieu.
A viral immunoevasin controls innate immunity by targeting the prototypical natural killer cell receptor family (NK1.1/NKR-P1)
Authors: Carlyle James R , Berry Richard , Rahim Mir Munir A , Reichel Johanna J , Popovic Branka , Tanaka Miho , Fu Zhihui , Balaji Gautham R , Lau Timothy NH , Tu Megan M , Kirkham Christina L. , Mahmoud Ahmad B , Mesci Aruz , Krmpotic Astrid , Allan David S.J. , Makrigiannis Andrew P , Jonjic Stipan , Rossjohn Jamie , Aguilar Oscar A
Published: January 2023

Natural killer (NK) cells play a key role in innate immunity by detecting alterations in self and non-self ligands via paired NK cell receptors (NKR). Despite identification of numerous NKR-ligand interactions, physiological ligands for the prototypical NK1.1 orphan receptor remain elusive. Here, we identify a viral ligand for the inhibitory and activating NKR-P1 (NK1.1) receptors. This murine cytomegalovirus (MCMV)-encoded protein, m12, restrains NK effector function by directly engaging the inhibitory NKR-P1B receptor. However, m12 also interacts with the activating NKR-P1A/C receptors to counterbalance m12 decoy function. Structural analyses reveal that m12 sequesters a large NKR-P1 surface area via a “polar claw” mechanism. Polymorphisms in, and ablation of, the viral m12 protein and host NKR-P1B/C alleles impact NK cell responses in vivo. Thus, we identify the long-sought foreign ligand for this key immunoregulatory NK cell receptor family and reveal how it controls the evolutionary balance of immune recognition during host-pathogen interplay.
Regulation of immune gene expression by the zinc finger DNA binding protein, CNBP/ZNF9
Authors: Chen Yongzhi , Jiang Zhaozhao , Caffrey Daniel R. , Shruti Sharma , Fitzgerald Katherine A.
Published: January 2023

An inducible program of inflammatory gene expression is a hallmark of antimicrobial defenses. This response is controlled by a collaboration involving signal-dependent activation of transcription factors, transcriptional co-regulators, and chromatin-modifying factors. Here we have identified a highly conserved Zinc finger DNA binding protein CNBP (also called ZNF9) upregulated in myeloid cells exposed to lipopolysaccharide. CNBP resides primarily in the cytosol and upon TLR4 engagement, CNBP translocate to the nucleus. To investigate the functional consequences of these events, we generated mice lacking CNBP and characterized the role of CNBP in controlling the inducible transcriptional program using a combination of RNA-sequencing and multiplex gene expression analysis (Nanostring). In response to an array of signals such as LPS, CNBP-deficient macrophages were impaired in their ability to induce important immune genes including IL12p40 and IL6 amongst others. CNBP-deficient cells showed normal activation of TBK1 and p65. However, the nuclear translocation of c-Rel that is critical for the inducible expression of IL-12p40 was significantly compromised in cells lacking CNBP. Furthermore, Chromatin Immunoprecipitation experiments revealed that CNBP itself bound the IL12 promoter and c-Rel binding to this promoter was compromised in CNBP-deficient macrophages. Lastly, ectopic expression of wild type CNBP but not a mutant form that could not be phosphorylated together with c-Rel led to synergistic activation of an IL12 promoter driven reporter gene. Collectively, these findings identify CNBP as a novel DNA binding protein that acts as a transcriptional regulator controlling the inflammatory program in macrophages.
Sterile type 2 inflammation triggered by microparticles is abrogated by inhibition of IL33 through blockade of Bruton’s tyrosine kinase
Authors: Mishra Pankaj Kumar , Palma Mark J , Millman Ariel , Hallab Nadim J , Beebe Kathleen , Benevenia Joseph , Gause William C
Published: January 2023

Wear debris microparticle release associated with total joint arthroplasty may contribute to harmful inflammation, osteolysis and implant failure. We examined the role of different molecular signaling pathways in microparticle-mediated inflammation. Intraperitoneal inoculation of cobalt chrome (CoCr) in mice induced a robust innate type 2 response with increases in IL4, IL5, IL13, IL33, M2 macrophages, eosinophils and neutrophils compared to controls given vehicle only. Blockade of the SYK signaling pathway with the inhibitor BAY 61-3606 or the down stream BTK signaling pathway with Ibrutinib abrogated the CoCr-mediated innate type 2 inflammation. Further studies in CBA/Nxid mice, a genetic BTK loss of function mutant also showed decreases in type 2 inflammation, confirming a critical role for BTK in CoCr mediated sterile type 2 inflammation. Inoculation of JH−/−, B cell deficient mice, or depletion of T cells by anti-CD4 (GK1.5) antibody, had no impact on CoCr mediated type 2 inflammation. We further demonstrated that the major source of IL33 was macrophages and intra-peritoneal administration of recombinant IL33 rescued the CoCr mediated type 2 response after BTK blockade. These studies indicate that the type 2 immune response caused by solid particles is abrogated by blockade of BTK signaling pathways and that exogenous IL33 is sufficient to rescue this response. These studies elucidate the nature of wear debris-mediated type 2 sterile inflammation and provide potential targets for future therapy.
Downregulation of the O-GlcNAc signaling promotes activation of the innate immune response in microbial sepsis
Authors: Li Xinghui , Gong Wei , Li Lupeng , Wen Haitao
Published: January 2023

Sepsis is the most common cause of death in intensive care units. Acute systemic inflammatory responses mediate morbidity in microbial sepsis. Studies of immune system metabolism have recently identified the tight link between metabolic reprogramming and immune system activation. A robust increase in glucose metabolism is observed in the initial phase of sepsis, indicating the involvement of glucose metabolism in the pathogenesis of sepsis. However, the role of individual glucose metabolism pathways in the regulation of septic inflammation is poorly understood. The hexosamine synthesis pathway (HBP) is a unique glucose metabolism pathway leading to protein modification by the O-linked b-N-acetylglucosamine (O-GlcNAc), a process known as O-GlcNAcylation. Here we report a critical role of HBP-associated O-GlcNAc signaling in modulating septic inflammation. Activation of TLR4 signaling results in decreased HBP flux activity and protein O-GlcNAcylation in macrophages. Decreased O-GlcNAc signaling is also observed in peritoneal macrophages following cecal ligation and puncture (CLP)-induced experimental sepsis, indicating that decreased O-GlcNAc signaling may exacerbate septic inflammation. Myeloid-specific deletion (OgtΔmye) of O-GlcNAc transferase (OGT), the key enzyme mediating protein O-GlcNAcylation, leads to increased activation of the NF-κB signaling and cytokine production upon the stimulation with various TLR agonists. Furthermore, OgtΔmye mice show increased susceptibility to LPS-induced endotoxin shock and CLP-induced sepsis. These findings identify a novel link between glucose metabolism and innate immune activation and suggest OGT as a therapeutic target for treating microbial sepsis.
Tissue resident memory B cells established in lungs in allergic asthma
Authors: Turner Damian L , Verter Jacob , Turner Ronak , Cao Minwei
Published: January 2023

Allergic asthma is a disease driven by the adaptive immune system in the lungs. B cells play a critical role during the allergen sensitization process by producing IgE and possibly through antigen presentation. While most current research has focused on the role of CD4+ tissue resident memory (TRM) cells, it is not known whether TRM B cells exist within the lungs and what role they might play. To investigate this question, we sensitized mice to house dust mite (HDM) intranasally for three weeks to induce asthmatic symptoms. We in vivo labeled mice with circulating fluorescent anti CD45 mAb and assessed the lung B cells’ susceptibility to labeling. We found that B cells, that are resistant to in vivo labeling, are a major population of lung lymphocytes following HDM sensitization. We were further able to detect these non-circulating B cells in the lungs as late as 10 weeks after treatment in sensitized mice. Furthermore, these cells were found in the same anatomic locations as CD4+ TRM cells and underwent expansion during HDM challenge. These data suggest that B cells do form a tissue resident population in the lungs and that these cells are involved in allergic asthmatic responses. This resident memory population of B cells are also locally activated upon allergen rechallenge. Whether these B cells are reactivated by HDM-derived antigens or by T cell-derived cytokines is yet to be determined.
Induction of CXCR5+ follicular CD8 T cells by CD40L adjuvanted DNA/MVA vaccination is associated with enhanced control of pathogenic SHIV infection.
Authors: Velu Vijayakumar , Styles Tiffany , Reddy Pradeep Babu Jagadeesh , Hicks Sakeenah , Gangadhara Sailaja , Amara Rama Rao
Published: January 2023

A significant challenge to HIV eradication is the elimination of viral reservoirs in germinal center (GC) resident T follicular helper cells. Therapeutic interventions that target viral reservoirs should induce anti-viral CD8 T cells that home to GC. We and others recently described a unique population of CD8 T cells that express the chemokine receptor CXCR5 (required for homing to GC) during chronic HIV/SIV infections. A critical question that remains to be addressed is if follicular homing CD8 T cells can be induced by vaccination and if so how will they impact control of HIV. Here we show that vaccination of rhesus macaques (RM) with DNA/MVA (modified vaccinia Ankara) SHIV vaccine induces low levels of vaccine-specific CXCR5+ CD8 T cells in blood. However, adjuvanting the DNA vaccine with CD40L dramatically enhanced the induction of CXCR5+ SHIV-specific CD8 T cells up to 10-fold with a frequency of about 5% of total CD8. These CXCR5+ CD8 T cells expressed higher levels of cytolytic molecules perforin and granzyme-B with higher proliferative capacity (ki-67) compared to CXCR5-subset. Following a pathogenic SHIV infection, the CXCR5+ CD8 subset expanded rapidly. Importantly, the frequency of SHIV-specific CXCR5+ CD8 T cells post vaccination and post infection showed a significant inverse correlation with viral load (p=0.003, R=−0.66), these associations were not observed with CXCR5-CD8 T cells. These data demonstrate that CXCR5+ CD8 T cells can be induced by vaccination, their induction can be augmented by CD40L, and the vaccine-induced CXCR5+ CD8 T cells contribute to control of pathogenic immunodeficiency virus infection. These findings have important implications for developing HIV cure strategies.
CAR NK and CAR T cells versus CTL: factors limiting the sensitivity and efficiency of engineered cytolytic effectors
Authors: Mazzanti Nicholas William , Anikeeva Nadia , Sato Takami , Sykulev Yuri
Published: January 2023

Factors limiting highly sensitive and efficient responses of virus- and cancer-specific CTL have been extensively studied. However, the sensitivity and effectiveness of T cells transduced with CAR specific for target molecules on cancer cells have been less characterized. We endowed NK92 cells and T cells purified from human cord blood with second generation CAR specific for High Molecular Weight Melanoma Associated Antigen (HMW-MMA) and compared the ability of CAR-NK92 and CAR-T cells to kill skin melanoma and uveal melanoma cell lines in in vitro cytolytic assay. We also evaluated the ability of established human virus-specific CTL clones to kill the same target cells presenting cognate pMHC ligands. Skin melanoma cells were more sensitive to specific lysis than uveal melanoma cells. The extent of the specific lysis strongly depended on the level of HMW-MMA expression. CTL clones appeared to be more superior effectors than CAR-NK92 or CAR-T cells as they lysed the melanoma cells that displayed much lower levels of cognate pMHC ligands. CAR-NK92 cells proliferated more vigorously than NK92 cells and maintained the level of CAR expression in culture. In contrast, CAR-CD8+T cells exhibited lower proliferative capacity than the control T cells and were losing cell surface CAR expression. We are currently evaluating the extent of TCR vs CAR clustering and co-clustering of CAR with TCR at the cell surface. We are also comparing Ca2+ flux and granule release by CAR-NK92, CAR-CD8 T cells and CTL in response to CAR or TCR stimulation. Disparities in these parameters could account for differential sensitivity and efficiency of engineered and natural cytolytic effectors.
Combination of adoptive T cell therapy with an EGFR x EpCAM-specific antibody for the treatment of a murine melanoma model
Authors: Klaus Richard Carl Philipp , Lampert Christopher , Schmollinger Jan C. , Niederfellner Gerhard , Sustmann Claudio , Klein Christian , Endres Stefan , Kobold Sebastian
Published: January 2023

Background

Adoptive T cell therapy is a new powerful treatment of cancer - however limited by T cell infiltration to the tumor. Transduced antigen-specific T cells with a marker antigen (EGFR) not expressed by normal T cells combined with an EGFR x EpCAM bispecific Antibody (biAB) mediate potent anti-tumor effects. To test the applicability of this approach to different T cell antigens and tumor models, we hypothesized that this synergy could be seen in the B16-OVA-melanoma model using ovalbumin-specific T cells (OT-1).

Methods and Results

We generated a B16-OVA-mEpCAM cell line through retroviral transduction. We expanded CD8+ T cells from OT-1 transgenic mice in vitro. C57Bl/6 mice were subcutaneously injected with B16-OVA-EpCAM melanoma cells and were treated with OT-1 T cells or the combination of the biAB with T cells (n=6 per group). Tumor growth was delayed in the combination group (mean tumor size on day 15, 77 mm2 vs 137 mm2). However we found that the treatment success relied entirely on the biAB alone, while the T cells did not contribute to efficacy (mean tumor size on day 18, 8 mm2 in antibody-treated vs 106 mm2 in untreated mice). We hypothesized that antibody dependent cellular cytotoxicity (ADCC) may be the mode of action of the biAB. We thus sublethally irradiated (5 Gy) mice 24 h after subcutaneous injection of B16-OVA-EpCAM tumor cells and found that irradiation completely abrogated the effect of the biAB treatment (mean tumor size on day 29, 146 mm2 in irradiated vs 117 mm2).

Conclusions

Combination of antigen-specific OT-1 T cells and a biAB does not synergize in the B16-OVA-EpCAM model. The biAB was, however, effective in this model, most likely due to ADCC. Other strategies need to be investigated to enhance ACT in this model.
Low antigen dose in adjuvant-based vaccination selectively induces CD4 T cells with enhanced functional avidity and protective efficacy
Authors: Billeskov Rolf , Wang Yichuan , Solaymani-Mohammadi Shahram , Frey Blake , Kulkarni Shweta , Agger Else Marie , Sui Yongjun , Berzofsky Jay A
Published: January 2023

T cells of high functional avidity sense and respond to low levels of antigen (Ag), and their superior protective efficacy was first described using CD8 T cells cultured in vitro with low levels of Ag resulting in higher avidity and anti-tumor/viral efficacy compared to low avidity T cells cultured with high Ag concentrations. Selectively inducing high avidity by low Ag concentrations has required in vitro expansion so far, as in vivo attempts with low-dose vaccination gave no response.

We show for the first time that functionally high avidity CD4, but not CD8, T cells are selectively primed in vivo by low concentrations of Ag in the strong crosspriming liposomal cationic adjuvant formulations (CAF) DDA/TDB or DDA/MMG/pIC from SSI. Increased functional avidity observed after low-dose vaccinations correlated with lower surface expression of PD-1 and higher per cell IFN-gamma production compared to low avidity CD4 T cells. We further show that IL-15, known to increase functional avidity of CTLs, is essential for high avidity of vaccine specific CD4 T cells.

Importantly, high avidity CD4 T cells offered superior protection in a murine model of latent infection with Mycobacterium tuberculosis compared to low avidity T cells. Furthermore, adoptive transfers of HIV-IIIB gp140-specific CD4 T cells of high functional avidity resulting from low-dose immunizations in combination with TCR transgenic HIV-IIIB gp120 specific CTLs conferred superior protection against viral (vaccinia-HIV-IIB-gp140) challenge compared to TCR-Tg CTLs alone or in combination with low avidity CD4 T cells induced by high-dose immunization. Thus, inducing high avidity CD4 T cells by low-dose immunization is important in clearing viral and bacterial infections.
Study of specific plasmablasts and plasma cells to lipidic particles in the mouse models of lupus generated by liposomes bearing lipidic particles stabilized with different inducers
Authors: Mora Albany Reséndiz , Pardo Elena Sanchez , Galicia Viridiana Galicia , Barbosa Sandra Sanchez , Wong Carlos , Baeza Isabel , Baeza Carlos Wong
Published: January 2023

Systemic lupus erythematosus (SLE) is an inflammatory, multisystem, chronic an autoimmune disease of unknown etiology. In our research group, we developed a mouse model of lupus by the administration of liposomes bearing lipid particles stabilized by chlorpromazine, promazine or manganese in BALB/c mice. In this model, we have detected IgG antibodies against lipid particles, so we are studying the immune mechanisms of how IgG antibodies are generated against a lipid antigen. It is noteworthy that the production mechanisms of anti-lipid IgG antibodies are practically unknown and have not been studied in autoimmune diseases such as SLE. In previous works, we showed that B cells mainly respond through germinal center to generate IgG antibodies against lipid particles. Moreover, against protein antigens after germinal center reaction, B cells differentiate into secondary lymphoid organs to plasmablasts that are maintained in cell cycle, can be divided and are in circulation. Eventually these plasmablasts can differentiate into plasma cells that are no longer in the cell cycle and are set in a niche (mainly in bone marrow or less in secondary lymphoid organs) where they begin to secrete antibodies. In this work, we propose to study the mechanisms of plasmablasts and plasma cells, specific to a lipid antigen, that leads to the production of IgG antibodies. This is very important because the IgG autoantibodies are the main causes of autoimmunity and particularly the anti-lipid particles antibodies, which have been found in patients with SLE, leprosy and preeclampsia, are those that trigger the lupus like disease in the mouse.
Comparative analyses of PAD expression and activity in myeloid cell lines
Authors: Rumble Julie M , Fackelman Elli M , Mobley James L
Published: January 2023

Protein arginine deiminase (PAD) enzymes and the arginine to citrulline reaction that they catalyze have increased in prominence in recent years, as dysregulation is associated with multiple inflammatory and autoimmune diseases. Under normal circumstances, PAD4 is required for the generation of neutrophil extracellular traps (NETs) by neutrophils. PAD4 citrullinates histone H3, initiating chromatin decondensation and subsequent extrusion of nuclear DNA to form a net-like structure. These NETs are important in innate immune responses to infection, as loss of components of this process can lead to the inability to eradicate usually harmless pathogens. We have developed tools to better understand the complete picture of PAD2 and PAD4-catalyzed citrullination and its effects in various systems. HL-60 and THP-1 cells differentially expressed PAD2 and PAD4, depending on differentiation and activation stimuli, as shown by ELISA. The activity of these PAD enzymes was also highly dependent on the differentiation and activation status of these cells. The PAD4 target histone H3 was found to be citrullinated only upon activation of differentiated cells, although PAD4 was expressed in differentiated cells which had not been activated. These data help to elucidate appropriate model systems for researchers looking to investigate PAD activation and downstream targets in immune cells.
The role of Ndfip1 in macrophage-mediated bacterial clearance
Authors: Field Natania S , Moser Emily K , Chandramouleeswaran Prasanna , Subudhi Ipsita , Vasquez Nikoff , Oliver Paula M
Published: January 2023

Macrophages are essential components of the innate immune response to bacteria. While these cells must mount an inflammatory response to harmful pathogens, they require tight regulation to prevent host damage. One way in which macrophages attenuate inflammatory signaling cascades is through ubiquitination. This post-translational modification can target a protein substrate for downstream signaling or for degradation. E3 ubiquitin ligases are required to facilitate the formation of these chains. Nedd4-family interacting protein 1 (Ndfip1) activates multiple E3 ubiquitin ligases, including Itch, which is crucial for attenuating cytokine production by T cells, and Nedd 4-2, which is required for iron regulation by neurons. While Itch has been shown to limit macrophage activity, it is unclear how Ndfip1 functions in macrophages, and how it affects macrophage antibacterial responses.

We generated mice lacking Ndfip1 in myeloid cells (Ndfip1fl/fl LysM Cre). Unlike mice lacking Ndfip1 in T cells, these mice have no evidence of inflammation at baseline. Upon i.n. infection with Klebsiella pneumoniae, Ndfip1 fl/fl LysM Cre mice show reduced bacterial loads three days post infection, despite normal innate cell recruitment. This result could be recapitulated when mice were infected intraperitoneally. Peritoneal macrophages lacking Ndfip1 have unchanged cytokine responses when stimulated with K. pneumonia, suggesting Ndfip1 attenuates macrophage activity through a mechanism other than limiting cytokine production. This project may reveal new mechanisms used by myeloid cells in eliminating bacteria, providing insight into the regulatory mechanisms that prevent excess inflammation.
The dual roles of membrane and soluble forms of gC1qR and C1q in the breast cancer microenvironment.
Authors: Kandov Evelyn , Pednekar Lina , Hosszu Kinga K , Nanda Japbani , Galanakis Dennis K , Preissner Klaus T , Kishore Uday , Peerschke Ellinor I.B , Ghebrehiwet Berhane
Published: January 2023

There is robust experimental data, which show that C1q and gC1qR play a pro- as well as anti-proliferative role in cancer. This opposing role in turn depends on whether each molecule is expressed on the cell surface or pericellular milieu. The aim of the present studies was therefore to more closely examine the functions of soluble and membrane forms of gC1qR and C1q using the SkBr3 cell line as a model for breast cancer. Our results show that: (1) In addition to gC1qR and cC1qR, SkBr3 cells express membrane C1q, and these molecules appear to be co-localized with each other as well as with fibrinogen (FGN) and vitronectin (VN). (2) Co-culture of SkBr3 cells with either purified C1q or with pAb directed against the gC1qR site on the A-chain of gC1q (ghA) resulted in a time-dependent inhibition of cell growth. (3) Co-culture of SkBr3 cells with individual C1q head modules-–ghA, ghB and ghC–showed significant inhibition, with the inhibitory hierarchy being ghA>ghC >ghB. (4) While SkBr3 cells do not secrete C1q, they are able to secrete gC1qR, and the latter in turn is able to negate the anti-proliferative effect of exogenously added C1q supporting the postulate that gC1qR secreted into the tumor cell microenvironment serves as an important molecular checkpoint. Collectively, the data show that both membrane-associated gC1qR and C1q on their own are pro-proliferative. Whereas the proliferative function of gC1qR can be abrogated by blockade of its C1q binding site, the proliferative function of C1q can be abrogated by blockade of its gh domains with specific antibodies indicating therefore that the pro-proliferative function of C1q resides in the its gh domains, with the ghA domain playing a central role.
Endosomal RNA-sensing toll-like receptor stimulated T cells play a unique role in autoimmune manifestations in murine model of DNA accrual.
Authors: Pawaria Sudesh , Marshak-Rothstein Ann
Published: January 2023

DNaseII is a lysosomal DNase that plays a critical role in immune homeostasis. DNaseII deficiency in mice causes embryonic lethality, due to severe anemia associated with excessive production of type I interferon (IFN), and can be rescued by IFN receptor knockout (IFNaR). The DNaseII/IFNaR double knockout (DKO) mice develop a late-onset inflammatory arthritis, dependent on the cytosolic DNA sensor STING (Stimulator of Interferon Genes). They also develop early-onset clinical manifestations of systemic autoimmunity including disrupted hematopoiesis and B cell lymphopoiesis, splenomegaly (due to increased accumulation of Ter119+ cells), and the production of anti-nuclear antibodies (ANA), through a mechanism that is STING-independent, Unc93B1-dependent and likely to result from endosomal RNA-sensing TLRs. Bone marrow (BM) chimera studies have shown that the DNaseII deficiency is essential in both radioresistant host cells and hematopoietic donor cells for all disease manifestations. Development of arthritis requires the presence of STING in both host and donor. The TLR-dependent defects require TLR expression in only the hematopoietic compartment but still need DNaseII−/− recipients. Remarkably transplantation of DKO spleen fragments or injection of DKO spleen cells alone into DNaseII-sufficient Rag−/− mice leads to splenomegaly and disrupted hematopoiesis and lymphopoiesis. Moreover DKO T cells are sufficient to transfer these TLR-associated defects to the Rag−/− mice. As activated T cells are known to be radioresistant, T cells activated in a DKO environment may account for the radioresistant host component required to precipitate the TLR-dependent disease manifestations.
High dimensional analysis of immune signatures in melanoma patients
Authors: Krieg Carsten , Nowicka Malgorzata , Guglietta Silvia , Schindler Sabrina , Hartmann Felix J. , Weber Lukas M. , Dummer Reinhard , Robinson Mark D. , Levesque Mitchell P. , Becher Burkhard
Published: January 2023

Checkpoint inhibitor therapy (CIT) has revolutionized cancer therapy. Notably, inhibition of programmed cell death protein 1 (PD-1) showed benefit in the treatment of metastatic melanoma and has even been promoted to first line therapy in advanced melanoma. Despite an increase in overall survival a minority of patients profits from CIT with anti-PD-1. Therefore, immune components or markers predicting responsiveness are urgently needed. Using phenotypic and functional markers we used high-dimensional single cell cytometry by time of flight (CyTOF), to measure blood samples from anti-PD-1 treated patients before and during therapy. CyTOF uses metal-tagged instead of fluorochrome-tagged antibodies, thus eliminating spectral overlap and, in combination with barcoding, enables the measurement of more than 30 parameters on a single cell in multiple samples in parallel. Due to the complexity of the data set and in order to reduce analytical bias we adapted a custom bioinformatic workflow. Using this approach we find immune signatures which allow us to separate responders from non-responders and might provide insights into the biology of immune therapy.
Cancer derived peptide of vacuolar ATPase ‘a2’ isoform plays immunosuppressive role by recruiting myeloid derived suppressor like cells
Authors: Sahoo Manoranjan , Kulshrestha Arpita , Katara Gajendra K , Beaman Kenneth D
Published: January 2023

Cancer cells express vacuolar ATPase (V-ATPase), a multi-subunit proton pump on the plasma membrane and intracellularly on vesicular membranes. The V-ATPases are responsible for acidification of intracellular vesicles and in certain activated cells are expressed on the surface where they appear to acidify the extracellular environment. The N-terminal domain of the a2 isoform of V-ATPase (a2NTD) is cleaved and secreted in exosomes. a2NTD has immunomodulatory effects on monocytes, macrophages and neutrophils. The changes elicited by a2NTD play an important role during pregnancy and cancer/tumor progression. To further investigate whether a2NTD has an effect on recruitment or stimulation of MDSCs in vivo, we injected a2NTD into the peritoneum of mice and analyzed CD11b+ myeloid cell populations. Here we observed that, intra-peritoneal (i.p.) administration of recombinant a2NTD protein results in recruitment of significantly higher number of CD11b+Ly6G+Ly6C+ MDSC like cells to the peritoneal cavity. We show that these cells are different from neutrophils by their expression of Ly6C on their surface. However, the total percent of CD11b+ cells or CD11b+F4/80+ macrophages remained unchanged. In vitro results demonstrate that addition of recombinant a2NTD protein to primary bone marrow derived macrophages (BMMs) from C57BL/6 mice leads to secretion of anti-inflammatory cytokine IL-10. Collectively, these results demonstrate that a2NTD administration leads to an immunosuppressive environment that might facilitate tumor progression. Further study is needed to understand the mechanism by which a2NTD modulates the MDSC like cells and creates an immunosuppressive pro-tumorigenic environment.
HDAC8 inhibition modulates liver tumor microenvironment: Rationale for combined epigenetic and immunotherapy
Authors: Cheng Alfred Sze , Feng Yu , Zhou Jingying , Yang Weiqin , Sun Hangyong , Chen Zhiwei , Goodwin Tyler , Liu Rihe , Huang Leaf
Published: January 2023

The tumor microenvironment plays an instrumental role in cancer development and treatment response/resistance. Accumulating evidence is underscoring the fundamental importance of epigenetic regulation in tumor immune evasion. Epigenetic modification agents represent a ‘double-edged sword’, as they can exert divergent effects on cancer cells and the milieu of immune and stromal cells. Our integrative epigenomics analysis has elucidated previously unexplored functions of histone deacetylase 8 (HDAC8) in promoting β-catenin-dependent hepatocarcinogenesis through interacting with another critical chromatin regulator enhancer of zeste homology 2 (EZH2). Given the strong oncogenicity of HDAC8, we further investigated the therapeutic potential of a potent and highly-selective HDAC8-specific inhibitor PCI-34051. Using a hepatocellular carcinoma (HCC) orthotopic model in immunocompetent mice, we demonstrated that HDAC8 inhibition exerted a strong anti-tumorigenic effect (Ctrl vs. PCI: 6.32 vs. 0.33*107 ROI intensity; p<0.01), which was comparable with programmed death-ligand 1 (PD-L1) blockade. Intriguingly, PCI-34051 significantly increased tumor-infiltrating multi-functional CD8+ T cells (~3-fold) and specifically reduced regulatory T cells (~2-fold). However, the level of PD-1+CD8+ T cells also increased, implicating potential T cell exhaustion. Our data suggest that selective chromatin modifications by HDAC8 and EZH2 alter the tumor immune surveillance program. By understanding the impact of epigenetic control on the liver tumor microenvironment, rational combinatorial epigenetic and immune checkpoint targeting has the potential to fully unleash T cell responses against HCC.
Immunotherapy against experimental melanoma is improved by beta-adrenergic blockade
Authors: Kokolus Kathleen M , Drabick Joseph J , Repasky Elizabeth A , Schell Todd D
Published: January 2023

Despite recent advances in immunotherapy which have expanded cancer treatment regimens to target a patient’s own immune cells, a significant proportion of patients fail to achieve long-lasting clinical benefit. The stress and immune responses are closely integrated yet few cancer treatment strategies account for this relationship. Activation of stress signaling releases norepinephrine which binds β-adrenergic receptors (βARs) on immune cells leading to regulation of immune functions. βAR signaling is reduced with antagonists (β-blockers) and these drugs, often prescribed for hypertension, are associated with improved cancer outcomes. We determined how βAR blockade impacts the efficacy of two immunotherapies used to treat melanoma: IL-2 and anti-(α)PD-1 which promote T cell proliferation and function, respectively. Mice with established melanomas were treated with β-blockers and immunotherapy (αPD-1, IL-2 or αPD-1/IL-2). Tumor growth was monitored and immune cell accumulation within tumors and lymphoid tissues was evaluated. Blockade of βAR signaling alone had no impact on tumor growth. Attenuation of tumor growth by each immune-based therapy was enhanced with administration of β-blockers, but this effect was most significant with dual αPD-1/IL-2. Likewise, survival of tumor-bearing mice was significantly extended by addition of β-blockers to αPD-1 or αPD-1/IL-2. Importantly, our results from a parallel retrospective clinical analysis of metastatic melanoma patients support a potential role for β-blockers in enhancing immunotherapy. This novel strategy could be translated to potentially increase the number of melanoma patients experiencing long term benefit from immune-based therapies.
Harnessing the immune response for the treatment of cancer by inducing immunogenic cell death through inhibition of a group II chaperonin
Authors: Khaled Annette R , Showalter Anne , Limaye Arati , Bassiouni Rania , Khaled Amr S , Oyer Jeremiah , Igarashi Robert , Copik Alicja
Published: January 2023

Immunotherapy is a promising treatment for cancer but lacks broad application. One way to enhance immunotherapy is by inducing immunogenic cell death (ICD). ICD is characterized by a unique molecular signature, involving the release of molecules that sensitize cancer cells to killing by the immune system. However, most ICD-inducing agents are not effective at clinically relevant doses. We developed a novel cytotoxic peptide, CT20p, that is delivered to cells using nanoparticles (NPs), which causes regression of tumors in mice at nanomolar concentrations. CT20p-treated cancer cells upregulated markers characteristic of ICD, such as calreticulin (Crt) and HSP70. The intracellular target of CT20p is a group II chaperonin called chaperonin containing-TCP-1 (CCT) that is essential for the folding of actin, tubulin and other proteins like STAT3. The CCT complex is composed of eight subunits (CCT1-8). That CT20p cytotoxicity is driven by CCT was proven by inhibiting or overexpressing CCT2 in susceptible or resistant cells, respectively. Disruption of CCT activity by CT20p caused endoplasmic reticulum (ER) stress that initiated intracellular pathways, such as PERK and IRE-1, generating danger signals associated with ICD. As a result, increased phagocytosis of dying cancer cells and enhanced NK cell killing was observed. Importantly, normal epithelial cells, macrophages or NK cells were unaffected. Using the CRISPR-CAS9 system, the genes for CCT were edited. Loss of CCT2 altered the levels of other CCT subunits as well as client proteins such as STAT3, while increasing membrane-exposed Crt. These studies revealed that inhibiting CCT induces ICD to stimulate immune functions essential for generating a robust anti-cancer immune response.
Targeting CARP-1 in breast cancer to decrease tumor growth and enhance immunogenicity
Authors: Shissler Susannah Claire , Drechsler Jennifer M , Derakhshandeh Roshanak , Rishi Arun K , Webb Tonya J
Published: January 2023

Natural killer T (NKT) cells are important in antitumor immunity, but these cells are significantly reduced in breast cancer (BC) patients. The tumor-associated factors that lead to the reduction in NKT cells are unclear; however, it is known that during malignancy BCs upregulate pro-survival proteins in order to proliferate and evade immune detection. Cell Cycle and Apoptosis Regulatory Protein (CARP)-1, a perinuclear phosphoprotein that regulates cell growth and apoptosis, is overexpressed in chemotherapy-treated cancers and several BC cell lines, such as MCF-7. CARP functional mimetics (CFMs) are small molecule inhibitors that interfere with the interaction of CARP-1 and the anaphase promoting complex, causing cell cycle arrest and apoptosis. Thus, we hypothesize that targeting CARP-1 through the use of CFMs will induce apoptosis and restore anti-tumor immune responses. In this study, BC cell lines were treated with CFM-4&5 and proliferation, cytotoxicity, and immunogenicity were assessed. It was found that pretreatment of human MCF-7 and murine E0771 BC lines with 20μM CFM-4&5 resulted in 80–100% cell death within 48–72h. In order to assess immunogenicity in the absence of cytotoxicity, BC cell lines were pretreated with 10μm for 24h, washed and cultured in fresh medium. Conditioned medium was collected and used to treat CD1d-expressing cells for 4h prior to coculture with NKT cell hybridomas. To test the effects of CFMs on immune cell activation, peripheral blood mononuclear cells (PBMC) from healthy and BC patients were treated with CFM-4&5 in the presence of stimuli and immuno-activity was assessed by ELISA. Collectively, these data implicate a novel role for targeting CARP-1 for the treatment of BC.