bluuebunny/crossref_metadata_2025 huggingface dataset:
Safety, tolerability, pharmacokinetics and pharmacodynamics of VTP-43742, a RORγt inhibitor, in normal healthy volunteers
Authors: McGeehan Gerard M , Palmer Sally A , Bryson Catherine C , Zhao Yi , Shi Meng , Lipinski Kerri K , Bukhtiyarov Yuri , Guo Joan , Claremon David A , Lala Deepak S , Gregg Richard E
Published: January 2023

VTP-43742 is an orally active RORγt inhibitor being developed for the treatment of autoimmune disorders, including psoriasis, through inhibition of IL-17A production and down regulation of the IL-23 receptor. Safety, tolerability, pharmacokinetic (PK) and pharmacodynamic (PD) profiles of VTP-43742 were assessed in two, randomized, double-blind, placebo-controlled studies in which VTP-43742 was administered to normal healthy volunteers. The first study was a single ascending dose (SAD) study (N = 53) in which participants were randomized to a one-time administration of VTP-43742 (7 dose levels, 30–2000 mg) or placebo. The second study (NCT02555709) was a multiple ascending dose (MAD) study in which participants (N = 40) received VTP-43742 (5 dose levels, 100–1400 mg/d) or placebo once daily for 10 days. VTP-43742 was shown to be safe and generally well tolerated at all dose levels in both studies. No serious adverse events were reported, and all study subjects completed dosing. No clinically significant clinical chemistry, hematologic or ECG abnormalities were observed, and dose proportionality was demonstrated across all dose levels. In the SAD study, VTP-43742 had a terminal plasma half-life of ~30 hours. In an ex vivo whole blood assay (WBA) of IL-17A secretion, VTP-43742 suppressed RORγt dependent secretion of IL-17A in a dose-responsive manner; >90% inhibition was observed at the higher doses which was mostly maintained over 24 hours. In the MAD study, those receiving VTP-43742 showed >90% inhibition of RORγt dependent IL-17A secretion in the WBA for a full 24hrs in all but the lowest dose cohort. These data support the further development of VTP-43742 for the treatment of autoimmune disorders.
Disruption of tumor subline cooperation may be a new way to understand EMT, dormancy and metastasis
Authors: Cohen Stefan A. , Everson Paul , Kim Untae
Published: January 2023

Tumors are heterogeneous since they contain sublines. Immunodeficient mice can function as an in vivo cell sorter (USP#7989001) to separate SW480 parental tumor cells (PTC) into 2 distinct sublines. Subline 1 TC (S1TC) are lymphotropic, hybrid non-EMT TC and are sensitive to cytostatic NK cells (cNK). S2TC are hematotropic, EMT TC and are resistant to cNK. Mice have a mechanism to distinguish non-EMT from EMT TC. PTC injected SC into SCID mice form local tumors and colonize lymph nodes (LN) and lungs (LU). In contrast, S1TC colonize the LN but do not form local tumors or colonize the LU. cNK suppress local S1TC growth and induce dormancy. Ablation of cNK by anti-NK leads to local S1TC growth while suppressing LN invasion. Stoppage of cNK ablation leads to an intense local anti-tumor response of S1TC. S2TC form local tumors that grow faster than PTC but do not colonize the LU or LN. S1TC cooperate with S2TC to colonize the LU. Some biomarkers (ZAP70, CXCR4) are expressed only on PTC and S1TC while others (SRC, CD21) are only on PTC and S2TC. CD26 has been linked to decreased invasiveness of some TC. Only S2TC expressed CD26 and not PTC or S1TC. S1TC may have suppressive effects on S2TC surface proteins. CD26 on S2TC may result in reduced invasion by peptidase action on SDF1 (CXCR4 ligand). S1TC may inhibit CD26 expression on S2TC resulting in SDF1/CXCR4 signaling and LU invasion. Disruption of tumor subline cooperation may be a new way to control metastasis.
Processed Aloe vera gel inhibits colitis-related colon carcinogenesis by inhibiting both chronic inflammation and cell cycle progression in the colon
Authors: Im Sun-A , Kim Heesuk , Kim Jiwhan , Shin Eunju , Do Seon-Gil , Kim Kyungjae , Lee Chong-Kil
Published: January 2023

The processed Aloe vera gel (PAG) contains immunomodulatory polysaccharides, which consists of a mixture of polymer chains of b-(1,4)-linked acetylated galactomannan with different lengths. PAG was shown to inhibit azoxymethane(AOM)-initiated and dextran sodium sulfate(DSS)-promoted colon carcinogenesis model in mice. In the present study, we examined the molecular mechanisms for the inhibition of colitis-associated colon carcinogenesis by PAG. PAG administration significantly inhibited the production of pro-inflammatory cytokines and mediators in the colon. Western blot analysis using the colon extracts showed that PAG administration reduced the activation of NF-kB resulting in the inhibition of iNOS and COX-2 expression. PAG appeared to inhibit NF-kB activation through the PPARγ activation. PAG administration also inhibited the expression and phosphorylation of STAT3, which was known to connect inflammation and cancer. PAG administration also inhibited cell cycle progression-related transcription factors such as the proliferating cell nuclear antigen (PCNA), ERK, cyclin-dependent kinase (CDK) 4 and cyclin D1. In contrast, PAG administration increased the expression of CDX2, which was known to be a tumor suppressor in colorectal cancers. These findings show that PAG suppresses colitis-related colon carcinogenesis by inhibiting both chronic inflammation and cell cycle progression in the colon.
Modulation of the myeloid tumor microenvironment in chronic lymphocytic leukemia by targeting CSF-1R
Authors: Yazdanparast Haniyeh , Hanna Bola , Rößner Philipp , Haderk Franziska , Haegel Helene , Lichter Peter , Seiffert Martina
Published: January 2023

Chronic lymphocytic leukemia (CLL) is highly dependent on the tumor microenvironment, which comprises monocyte-derived nurse-like cells (NLCs) as key players that possess tumor-supportive properties and resemble tumor-associated macrophages. Our main goal is to understand and target the tumor supportive properties of myeloid cells in CLL.

CSF-1R is known to be important for proliferation, differentiation, and survival of myeloid cells. Targeting CSF-1R has revealed clinical relevance in solid tumors. However, its role in hematological malignancies has not been assessed so far. Therefore, we tested a unique novel monoclonal antibody, TG3003 (Transgene S.A.), against this receptor in cocultures of primary CLL cells and NLCs and observed a significant decrease in CLL cell survival as well as a change in the concentration of inflammatory factors such as CCL2 and IL-6 in the cell culture supernatant. Moreover, we found that these changes were connected to a decrease in NLC numbers. Therefore, we aimed at exploring the in vivo activity of TG3003 in a well-established mouse model of CLL, that is the adoptive transfer of malignant splenocytes from leukemic Eμ-TCL1 mice into syngeneic recipient animals. Targeting CSF-1R in these mice resulted in a significant decrease of splenic monocytes but not macrophages and dendritic cells, indicating that monocytes are the main target of the antibody. More detailed investigations of monocyte subsets revealed that the previously described CLL-associated enrichment of patrolling monocytes was significantly but incompletely reversed. As TG3003 treatment had no effect on tumor development in this model, our future work will focus on combination approaches including other immunomodulatory drugs.
Microencapulated rapamycin prevents carcinogen and inflammation driven colon cancer through immune mechanisms
Authors: chen wanjiao , Dao Vinh A , Cardenas Kim , Lao Shunhua , Padrón Álvaro , Hasty Edward P. , Sharp Zelton Dave , Hurez Vincent , Curiel Tyler J.
Published: January 2023

We tested microencapsulated rapamycin (eRapa, ~2.2 mg/kg rapamycin/mouse/day) in carcinogen (azoxymethane, AOM) + inflammatory agent (dextran sodium sulfate, DSS) colon cancer. WT BL6 mice fed eRapa before, and during AOM/DSS had significantly fewer colon tumors and tumor burden than control fed mice (empty microcapsules). eRapa prevented colon cancer in δ TCR KO mice lacking γδ T cells but not in IFN-γ KO mice. In carcinogen (DMBA) + inflammatory agent (TPA) skin cancer, IFN-γ and γδ T cells were both needed for eRapa cancer prevention, showing tumor-specific and common immune requirements for eRapa-mediated cancer prevention. In βδ TCR KO mice lacking all T cells, AOM/DSS induced no cancer or only few tumors, suggesting αβ T cells are required for colon neoplasia and cancer in the AOM/DSS model. Protection from acute colitis in this model usually predicts colon cancer protection. Strikingly, however, eRapa did not prevent acute clinical or histologic colitis induced by DSS, despite cancer protection, suggesting effects on chronic colitis, or induction of cancer-protective but not acute colitis-protective mechanisms. In acute colitis, eRapa significantly decreased spleen and colon weights and CD3+CD4+ T cells, and increased mesenteric lymph node γδ T cells (with decreased Vγ1.1+ and increased Vγ2+ subsets) consistent with altered inflammation and reduced CD4-CXCR3+ and CD4-α4β7 T cells (likely γδ T cells) consistent with altered trafficking but did not affect CCR9+ T cells. In chronic colitis, eRapa significantly increased γδ T cells (with no changes in Vγ1.1+ or Vγ2+ subsets) that could mediate cancer protection. These novel immune effects of rapamycin help define its cancer prevention mechanisms and define novel clinical uses.
Conditioned media from the renal cell carcinoma cell line 786.O drives human blood monocytes to a monocytic myeloid-derived suppressor cell phenotype
Authors: Okada Shannon , Simmons Randi , Franke-Welch Secil , Gilbertson Deb
Published: January 2023

Myeloid-derived suppressor cells (MDSCs) are immature myeloid cells that can be found in high numbers in the tumor microenvironment and are critical in mediating immune suppression in cancer patients. To develop an in vitro assay system that functionally mimics the tumor microenvironment, we cultured human blood monocytes with conditioned media from several cancer cell lines. Conditioned media from 4 out of 8 tumor cell lines tested induced survival and differentiation of monocytes into cells with characteristics similar to macrophages and MDSCs. Media from one cell line in particular, 786.O (a renal cell carcinoma line), induced the monocytes to acquire a monocytic MDSC phenotype characterized by a decrease in cell surface expression of HLA-DR, an increase in nitric oxide (NO) production, and changes in morphology similar to an immature myeloid cell. Additionally, these in vitro generated MDSCs suppressed autologous CD3+ T cell proliferation. The tumor conditioned MDSCs were compared, functionally and phenotypically, to MDSCs derived using GM-CSF and IL-6 and to M1 and M2 differentiated and polarized macrophages. We further demonstrated that the in vitro MDSCs are phenotypically and functionally similar to patient-derived MDSCs. MDSCs differentiated in vitro from 786.O tumor conditioned media represent a platform to identify potential therapeutics that inhibit MDSC activities.
6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) is necessary for human melanoma MDSC differentiation and function
Authors: Grewal Jaspreet , Ricthie Jamaal , Al Rayyan Numan , Telang Sucheta , Yaddanapudi Kavitha , Chesney Jason
Published: January 2023

Myeloid derived suppressor cells (MDSCs) inhibit the expansion of tumor antigen-specific effector CD8+ T cells via arginase, transforming growth factor – β (TGF – β) and indoleamine 2,3-dioxygenase (IDO). Recently, MDSCs were found to over-express hypoxia inducible factor 1 alpha (HIF-1α) which is required for their differentiation. An essential transcriptional target of HIF-1α is 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) which synthesizes fructose 2,6-bisphosphate, an allosteric stimulator of glycolysis and of proliferation via stimulation of cyclin dependent kinase-1 (CDK1). We hypothesized that MDSCs might over-express PFKFB3 which in turn might be required for their function as T cell suppressors. We demonstrate that monocytic MDSCs (M-MDSCs) induced by co-culture with A375 melanoma cells express increased PFKFB3 and that exposure to the PFKFB3 inhibitor, PFK-158, blocks the suppressive function of these M-MDSCs on T cell activation. Furthermore, we analyzed three advanced cancer patients for circulating MDSCs before and after PFK-158 administration as part of a multi-center phase 1 clinical trial. And, we found that the MDSCs were markedly reduced in each patient. Taken together, these data indicate that selective inhibition of PFKFB3 may be a novel approach to target MDSCs and combinations of PFKFB3 inhibitors with immunotherapies may be a rational strategy to promote durable immune-mediated remissions in cancer patients.
CITN11-02 phase I trial of subcutaneous recombinant human IL-15 (rhIL-15) is associated with expansion of circulating CD56+ NK cells and CD8+ T cells
Authors: Morishima Chihiro , McNeel Douglas G , Patel Manish R , Kohrt Holbrook E.K. , Waldmann Thomas A , Thompson John A , Conlon Kevin C , Sondel Paul M , Wakelee Heather A , Apodaca Minjun C , Fling Steven P , Disis Mary L , Creekmore Steven P , Miller Jeffrey S
Published: January 2023

Background

IL-15 is a high priority for clinical development because of its homeostatic effects on both NK cells and CD8+ T cells, and minimal effect on regulatory T cells. The Cancer Immunotherapy Trials Network is conducting a phase I dose-escalation study of subcutaneous (SQ) rhIL-15 in advanced melanoma, renal cell, non-small cell lung and squamous cell head and neck carcinoma patients.

Methods

A cycle consists of 5 daily SQ injections of rhIL-15 (E.coli-derived, NCI) given Monday-Friday for two weeks, then 2 weeks observation. The absolute lymphocyte count is tested every injection day, and whole blood flow cytometric analyses are conducted on Days 1, 11 and 15 of each cycle.

Results

Three patients each were treated at the 0.25, 0.5, 1.0 and 3.0 mcg/kg dose levels and six patients at 2.0 mcg/kg/dose (N=18). Seventeen patients completed ≥1 cycle, with one dose limiting toxicity (DLT) at 3.0 mcg/kg and one serious adverse event at 2.0 mcg/kg. Flow cytometric data indicate a consistent increase in the frequency of CD56+CD3− NK cell frequencies peaking at Day 15 of Cycle 1 (Day 12=last dose), with lesser increases in subsequent cycles. The median fold-increase in circulating NK cells during Cycle 1 demonstrated dose responsiveness and peaked at 11.5-fold with 3.0 mcg/kg. By contrast, the maximum fold-increase in circulating CD8+ T cells was 2.6-fold.

Conclusion

SQ rhIL-15 has been well tolerated and the 3.0 mcg/kg dose cohort is being expanded. Higher doses of rhIL-15 were associated with profound increases in circulating NK cells with significant but smaller increases in CD8+ T cells. Outpatient use of SQ rhIL-15 is safe and will be combined with other cancer immunotherapies.

Study support: NIH 1U01 CA154967-01 (Clinical Trials.gov NCT01727076).
Immunogenicity and biodistribution of nanoparticles in vivo
Authors: Tsirikis Peter , Wilson Kirsty , Xiang Sue , Wei Wei , Ma Guanghui , Selomulya Cordelia , Plebanski Magdalena
Published: January 2023

Nanoparticles have been widely used in vaccine design as both adjuvants and antigen delivery vehicles. In a seminal study, 40–50 nm nanoparticles with conjugated antigen were shown to induce high antibody titers and IFN-γ production in mice but with no added inflammatory stimuli. Subsequent research has shown that similar levels of immunogenicity can be achieved via the co-injection of naked 40–50 nm nanoparticles adjuvants and larger 500 nm nanoparticles with conjugated antigen. Furthermore, recent works indicate that particle shape can also influence the immune response. As such, we investigate the influence of surface morphology using 40–50 nm smooth and rough surfaced nanoparticle adjuvants and report their differential immunogenicity via ELISA, ELISpot and flow cytometry. Further, we determine the biodistribution of fluorescent 40–50 nm nanoparticle adjuvants with smooth and rough surfaces and larger 500 nm nanoparticles. Nanoparticle size is shown to be a discriminating factor in lymph node drainage, using a Carestream FX PRO in vivo imaging system and fluorescence microscopy of lymph nodes sectioned ex vivo. To elucidate the safety profile of this vaccine construct, we also investigate the biodistribution of nanoparticles within the major organs. The outcomes from this study provide key design criteria in the development of novel nanoparticle immunotherapeutics for the treatment of disease.
Small molecule agonists of IRF3 activation function as influenza vaccine adjuvants by modulating the humoral and cellular anti-viral immune response
Authors: Probst Peter , Grigg John B , Hemann Emily A , Loo Yueh-Ming , Knoll Megan L , Ireton Renee , Gale Michael J , Iadonato Shawn P , Bedard Kristin M
Published: January 2023

We have identified a panel of small molecule immunomodulators that activate IRF3 and induce innate immune signaling to drive an antigen-specific protective immune response against viral infections. Our lead adjuvant candidate, KIN1148, binds to retinoic acid inducible gene-I (RIG-I) and induces RIG-I signaling to drive IRF3 activation. Studies using the H1N1 influenza virus challenge model demonstrate that immunization with monovalent influenza split vaccine (SV) and KIN1148 is dose sparing and protects mice against a lethal H1N1 A/California/04/2009 challenge. The SV-H1N1/KIN1148 adjuvant system induces functional antibodies neutralizing viral infectivity and inhibiting influenza hemagglutinin-mediated blood agglutination. SV-H1N1/KIN1148 prime/boost immunizations mediate a strong influenza-specific Th2 response and enhance the production of the immunoregulatory cytokine IL-10 by lung and draining lymph-node-derived T cells from challenged mice. In addition, prime immunization with SV-H1N1/KIN1148 alone provides protection against a 10xLD50 H1N1 challenge by reducing the viral load in the lungs of infected mice. Passive transfer experiments suggest that the protection after prime immunization is at least partially mediated by a cellular immune component. In summary, the KIN1148 split vaccine adjuvant system controls influenza virus infection by boosting the production of functional antibodies, reducing viral load, and inducing a Th2-type immune response. The induction of Th2 and immunoregulatory cytokines by KIN1148-adjuvanted SV might be beneficial to ameliorate the immunopathogenesis of an immune response to highly pathogenic avian influenza virus in infected individuals.
Intrapulmonary delivery of TLR agonists to treat metastatic cancer
Authors: Goguet Emilie , Tross Debra , Klinman Dennis M
Published: January 2023

The TLR7/8 agonist 3M-052 and the TLR9 agonist CpG ODN both trigger innate immune responses that support the induction of tumor-specific immunity. Previous studies showed that intra-tumoral delivery of this agonist combination was synergistic and led to the eradication of large primary tumors and the establishment of long-term protective immunity in murine tumor models. We explored the effect of delivering 3M-052 plus CpG ODN to the lungs of mice bearing breast-derived lung metastases. 66Cl4-Luc cells (a luciferase-expressing variant of the 4T1 murine mammary carcinoma) were orthotopically implanted in the fourth mammary fat pad of Balb/c mice. Progression of the primary tumor and lung metastases was monitored by bioluminescence. TLR agonist treatment by intratracheal instillation was initiated after mets were detected in the lungs. Results show that intra-pulmonary treatment with the combination of TLR7/8 and TLR9 agonists slowed the growth of the lung metastases compared to either agonist alone. This treatment decreased pulmonary tumor burden and significantly prolonged survival when compared to control (untreated) animals. In contrast, pulmonary therapy had no significant effect on the primary tumor. Results from ongoing studies examining the effect of higher/more frequent lung delivery combined with direct treatment of the primary tumor will be presented.
Enterobactin, a metallophore, mitigates the immune responses of macrophages
Authors: Yeoh Beng San , Saha Piu , Xiao Xia , Singh Vishal , Vijay-Kumar Matam
Published: January 2023

Iron is required to facilitate the redox activity of heme proteins expressed in both immune and non-immune cells. In this study, we investigated whether the chelation of iron by enterobactin (Ent; a prototypical bacterial siderophore) can affect the immune and nitrosative responses of RAW 264.7 and bone marrow-derived macrophages. We observed that Ent dose-dependently inhibited the LPS-induced secretion of cytokines (i.e., serum amyloid A, IL-6, lipocalin 2) and nitrite in the macrophages. The LPS-induced expression of inducible nitric oxide synthase (iNOS; a heme protein) was also inhibited by Ent at both the mRNA and protein levels. To demonstrate the physiological relevance of these findings, we next investigated whether Ent could protect the intracellular pathogen Salmonella enterica serovars typhimurium in the gentamycin protection assay. Ent reduced the nitrite levels in Salmonella-infected macrophages, but upregulated the expression of iNOS (mRNA, protein) and Arginase-1 (mRNA). More importantly, Ent-treated macrophages were impaired in mediating the killing of Salmonella. Ent-sufficient Salmonella also survived better compared with their isogenic Ent-deficient strains, whereas the addition of exogenous Ent substantially improved the survival of both strains. The inhibitory effects of Ent, however, were abrogated when saturated with iron (1:1 ratio), thus indicating that Ent is required to be in its iron-free form to mediate efficient inhibition on the macrophages. Taken together, our findings advance the concept that Ent not only facilitate bacterial iron uptake, but also serve to safeguard bacteria against macrophage immune responses.
The role of LPC and lipid transporter MFSD2A in CD8 T cells
Authors: Piccirillo Ann , Hawse William F , Menk Ashley V , Wallace Callen T , Buechel Heather M , Watkins Simon C , Wendell Stacy G , Delgoffe Greg M , Silver David L , D’Cruz Louise M
Published: January 2023

Little is known about how effector CD8 T cells obtain exogenous long chain fatty acids (LCFAs) during T cell activation. LCFAs assist with membrane biogenesis and are required for rapid proliferation as well as effector molecule generation. One potential mechanism of fatty acid import is in the form of lysophophatidylcholine (LPC) by lipid carrier major facilitator superfamily domain containing 2a (MFSD2A). Here, we show that MFSD2A is highly expressed in activated CD8 T cells. Conditional loss of MFSD2A caused an altered effector response and resulted in defective memory cell formation. Taken together, these data show MFSD2A and LPC play a role in CD8 T cell metabolomics. Our future studies will use mass spectrometry lipidomics and gene expression analysis to identify the mechanisms by which CD8 T cells use LCFAs to mount a robust immune response to infection.
Role of Bcl-3 in T cells upon challenge with lymphocytic choriomeningitis virus
Authors: Jaiswal Hemant , Siebenlist Ulrich
Published: January 2023

Bcl-3 is an atypical member of the IkB family. Classical IkBs inhibit trans-activating NF-kB dimers such as p50/RelA largely via cytoplasmic retention; they are degraded in response to many signals, which allows nuclear entry of NF-kB. By contrast, Bcl-3 is not degraded and acts in the nucleus to modulate transcription of NF-kB target genes by associating with p50/NF-kB1 and p52/NF-kB2 homodimers. The latter complexes do not trans-activate by themselves, and association with Bcl-3 may promote or inhibit transcription in a highly gene- and context-dependent manner. Bcl-3 has profound biologic roles. It is associated with recurrent translocations in some B-cell tumors and Bcl-3−/− mice exhibit notably impaired adaptive and innate immune responses. Previously, we have shown that Bcl-3 regulates plasticity and pathogenicity of autoimmune CD4 T cells in the T cell transfer-induced colitis model as well as in EAE, a model for multiple sclerosis. However, the detailed molecular controls of Bcl-3 in T cell differentiation and its antigenic responses are not well understood. To evaluate the possible role of Bcl-3 in antigen-specific T cell responses, mice lacking Bcl-3 specifically in T cells were challenged with acute lymphocytic choriomeningitis virus (LCMV) infection. Preliminary data revealed that Bcl-3−/− CD8 T cells had comparable primary antiviral responses at day 8 post infection. However, loss of Bcl-3 in T cells significantly reduced the proportion of LCMV-specific CD8 T cells in spleens 6 weeks post infection. This suggests a crucial role of Bcl-3 in long-term maintenance of antigen-specific CD8 T cells. We will present initial investigations into mechanisms by which Bcl-3 may help to maintain antiviral T cell responses over time.
Inflammatory monocytes hinder antiviral B cell responses
Authors: Kuka Mirela , Sammicheli Stefano , Di Lucia Pietro , De Oya Nereida Jimenez , De Giovanni Marco , Fioravanti Jessica , Cristofani Claudia , Maganuco Carmela G. , Fallet Benedict , Ganzer Lucia , Sironi Laura , Mainetti Marta , Ostuni Renato , Larimore Kevin , Greenberg Philip D. , de la Torre Juan Carlos , Guidotti Luca G , Iannacone Matteo
Published: January 2023

Antibodies are critical for protection against viral infections. However, several viruses, such as lymphocytic choriomeningitis virus (LCMV), avoid the induction of early protective antibody responses by poorly understood mechanisms. We analyzed the spatiotemporal dynamics of B cell activation to show that, upon subcutaneous infection, LCMV-specific B cells readily relocate to the interfollicular and T cell areas of draining lymph nodes, where they extensively interact with CD11b+Ly6Chi inflammatory monocytes. These myeloid cells were recruited to lymph nodes draining LCMV infection sites in a type I interferon– and CCR2-dependent fashion, and they suppressed antiviral B cell responses by virtue of their ability to produce nitric oxide. Depletion of inflammatory monocytes, inhibition of their lymph node recruitment, or impairment of their nitric oxide–producing ability enhanced LCMV-specific B cell survival and led to robust neutralizing antibody production. Our results identify inflammatory monocytes as critical gatekeepers that restrain antiviral B cell responses and suggest that certain viruses take advantage of these cells to prolong their persistence within the host.
Type I IFN mediates cell-intrinsic host protective effects during acute gram-negative bacterial infection of macrophages
Authors: Dorrington Michael G , John Sinu , Fraser Iain D.C.
Published: January 2023

Many bacteria require entrance into host immune cells, such as macrophages, in order to replicate. These intracellular bacteria have evolved complex strategies for avoiding the antimicrobial programming of these immune cells to create replicative niches. While we know a great deal about various virulence factors deployed by intracellular bacteria as well as how cells try to kill these pathogens, how bacteria and host cells modulate each others’ responses during infection is less well understood.

Using the Gram-negative bacteria Burkholderia cenocepacia, we have uncovered a negative relationship between type I IFN signaling and the replicative potential of invading bacteria. Firstly, cells infected with these bacteria produce significant levels of IFN as well as the products of IFN-stimulated genes (ISGs). Cells pre-treated with IFNβ are less permissive to bacterial replication, while cells from mice lacking the type I IFN receptor (IFNAR) have increased levels of bacterial replication compared to wild-type cells. Interestingly, this phenotype is type I IFN-specific, as pre-treatment with IFNγ has no effect on bacterial replication. Additionally, siRNA knockdown of select ISGs, such as IFIT1, leads to increased bacterial replication within macrophages. A rigorous, concurrent transcriptomic profiling of both infected cells and intracellular bacteria is providing insight into how the bacteria and their host cells interact with one another during acute infection, providing a basis for targeted CRISPR-based screening experiments to unravel critical regulators of the host-pathogen interaction.
IL-27 limits neutrophil mediated pathology during pulmonary infection with Cryptococcus neoformans
Authors: Sun Donglei , Zhang Mingshun , Shi Meiqing
Published: January 2023

Cryptococcus neoformans is a pathogenic encapsulated fungus and a leading cause of fatal meningitis in immunocompromised individuals. IL-27 is a recently identified cytokine affecting both innate and adaptive immunity. Both proinflammatory and inhibitory functions on T cells by IL-27 have been reported in different disease models. Nevertheless, the effect of IL-27 during Cryptococcus infection has never been studied. In current study, IL-27 receptor alpha knockout mice (IL27R KO) was infected with a high virulent cryptococcal strain. We found that mice deficient in IL-27 signaling have significantly increased fungal burden in the lung and shortened survival time. Surprisingly, infected IL-27R KO mice showed unchanged Th1 and Th17 cytokines but significantly reduced Th2 cytokines including IL-4, IL-5 and IL-13. The reduced Th2 response is unlikely to be a cause of increase fungal burden since Th2 was generally known as detrimental during Cryptococcus infection. We next studied the immune cell composition by flow cytometry, no difference in cell number was found for all cell types except neutrophil which is increased during infection indicating more inflammation. The increase of neutrophils is associated with increased chemokines KC and MIP-2. Cryptococcus infection causes sustained production of TNF alpha, we next found that IL-27 suppressed the chemokine release by immune cells as a response to TNF-alpha. Lastly, depletion of neutrophils improved the outcome in infected IL-27R KO mice proving that immune pathology mediated by neutrophils can be detrimental during Cryptococcus infection.
Interaction of both SH2 domains of SHP-2 with a PD-1 homodimer is required for PD-1- mediated inhibition of T cell responses
Authors: Patsoukis Nikolaos , Council Asia , Berg Anders , Bardhan Kankana , Weaver Jessica , Mahoney Kathleen M , Freeman Gordon J. , Boussiotis Vassiliki A
Published: January 2023

PD-1 is a checkpoint receptor, which promotes peripheral tolerance and restrains anti-viral and anti-tumor immunity. PD-1 blockade promotes anti-tumor responses by eliminating PD-1 mediated inhibitory signaling. PD-1-mediated inhibition relies on the interaction of its ITSM motif with SHP-2 but the mechanism is unknown. We examined how PD-1:SHP-2 interaction inhibits T-cell responses. Pull-down assays revealed that PD-1 interacted with GST-SHP-2 fusion protein after PD-1 ligation with simultaneous TCR/CD3-mediated activation and this interaction was mediated via the SH2 domains of SHP-2. SHP-2 contains two SH2 domains (N-SH2 and C-SH2). To determine which SH2 domain is important for PD-1 interaction, we mutagenized the functional sites of N-SH2 and C-SH2. We transfected COS cells with cDNA of SHP-2 wild type or each SH2 mutant together with PD-1 and TCR proximal kinase Fyn, which is required for phosphorylation of PD-1 ITSM. Immunoprecipitation experiments showed that mutagenesis of either SH2 domain abrogated PD-1:SHP-2 interaction, indicating that both SH2 domains are required for interaction with PD-1. We confirmed this finding by Biacore studies. These results indicate that SHP-2 brings together two ITSM-phosphorylated PD-1 molecules by interaction with N-SH2 and C-SH2 domains. Culture of human T cells in the presence of a soluble dimeric PD-L1 inhibited T cell proliferation and IFN-γ production whereas monomeric PD-L1 had the opposite effect. Our results reveal a new mechanism of PD-1:SHP-2 interaction with implications for the development of PD-1-binding compounds to selectively suppress T cell responses by dimerizing PD-1 or to enhance T cell activation by disrupting PD-1 homodimerization.
Intratumoral IL-12 gene electrotransfer-mediated regression of established B16F10 melanoma protects against rechallenge
Authors: Shi Guilan , Edelblute Chelsea , Lundberg Cathryn , Heller Richard
Published: January 2023

Melanoma is an aggressive tumor with limited treatment options and poor prognosis. Interleukin-12 (IL-12) is a potent cytokine mediating antitumor activity. However, it has only achieved modest antitumor effects in clinical trials, often accompanied by unacceptable adverse events. We evaluated whether intratumoral delivery of a plasmid encoding IL-12 using gene electrotransfer (GET) to control the dose could induce rejection of an established poorly immunogenic tumor and avoid dose dependent toxicities.

We treated B16F10 murine melanoma 3 times in 1 week by intratumoral injection of pUMVC3mIL-12 plasmid (pmIL-12, 50 μg) followed by GET. The GET protocols were; EP1 (six 300 V/cm, 100 μs pulses with an array of 6 penetrating electrodes), EP2 (ten 600 V/cm, 5ms pulses with non-penetrating caliper electrodes).

Localized delivery of pmIL-12 with GET in the tumor microenvironment, significantly delayed disease development and prolonged disease free survival and induced long term immune memory protecting against rechallenge. The response was associated with a significant reduction in Treg and MDSC infiltration in B16F10 melanoma and increase of Granzyme B. Mice treated with pmIL-12 EP1 that achieved protection against rechallenge were observed to have CD62LlowCD44high memory T cells. Interestingly, pmIL-12 demonstrated stronger anti-tumor effects than pmIL-12 delivered by EP2.

By manipulation of GET parameters the onset, level and duration of IL-12 protein expression can be controlled, resulting in meaningful therapeutic effects without associated IL-12 toxicities. Furthermore, the approach induced systemic antitumor immune activity and memory and therefore might have an impact against other types of metastatic disease.
Autoreactive T cells display altered CD25 expression that changes responses to low dose IL-2 and antigen delivery.
Authors: Iwamura Chie , Benck Charles , Coley William , Liu Yi , Zhao Yongge , Esposito Juan Antonio Quiel , Tarbell Kristin V
Published: January 2023

Dendritic cells (DCs) are important for T cell tolerance induction and treatment of autoimmune diseases such as type 1 diabetes. Chimeric antibodies that deliver self-antigens to DCIR2+ DCs can delay but not completely prevent diabetes development in non-obese diabetic (NOD) mice. These DCIR2 antibodies induce T cell tolerance via antigen-specific deletion and anergy, but do not increase antigen-specific Foxp3+ regulatory T cells (Tregs). Because low dose (LD) IL-2 administration can preferentially expand Tregs, we asked if delivering antigens to tolerogenic DCIR2+ DCs along with IL-2 would boost antigen-specific Tregs and block autoimmunity. But, surprisingly, adding IL-2 did not increase efficacy of DC-targeted antigens to inhibit diabetes. To date, the effect of LD-IL-2 on autoreactive Treg and effector T cells has not been fully elucidated. By using tetramer staining to identify islet-specific CD4+ T cells in NOD mice, we now show the effects of LD-IL-2, with or without specific antigen delivery to DCIR2+ DCs, on both polyclonal and autoreactive effector and regulatory T cells. As expected, LD-IL-2 increased total Tregs, but autoreactive Tregs required addition of antigen and LD-IL-2 to elicit significant expansion. We found that islet-specific Tregs had lower CD25 expression compared to polyclonal Tregs, but islet-specific Foxp3− cells had higher CD25 expression. IL-2 increased activation and expansion of Foxp3− cells, and this effect was more pronounced for autoreactive cells after treatment with IL-2 + islet antigens. Therefore, IL-2, especially when combined with self-antigens, may not be an effective treatment for chronic autoimmunity, and increased Foxp3+ cells may not be a good biomarker for treatment efficacy.
Targeting the VEGF/GD3 immunosuppressive axis in ovarian cancer
Authors: Bollino Dominique , Muthuswamy Ravikumar , Odunsi Kunle , Webb Tonya J
Published: January 2023

Ovarian cancer is the fifth leading cause of cancer deaths among women, thereby accounting for more deaths than any other cancer of the female reproductive system. It is known that ovarian cancer tissue and ascites contain lymphocytic infiltrates, suggesting that immune cells traffic to tumors, but are then inhibited by immunosuppressive molecules within the tumor microenvironment. Moreover, vascular endothelial growth factor (VEGF) expression is inversely correlated with survival in ovarian cancer patients. Since avoiding detection by the host’s immune system is crucial for the growth and metastasis of cancer, we investigated the effects of ovarian cancer-associated VEGF on CD1d-mediated antigen presentation to natural killer T (NKT) cells. Pretreatment of antigen presenting cells with ascites or conditioned medium from OV-CAR-3 and SK-OV-3 ovarian cancer cell lines suppressed CD1d-mediated NKT cell activation. Ovarian cancer-associated ascites and conditioned medium from ovarian cancer cell lines contain high levels of VEGF and the ganglioside GD3. Importantly, inhibiting VEGF production by ovarian cancer cell lines led to a reduction in GD3 expression and restored NKT cell activation. To elucidate the mechanisms by which VEGF induces GD3 shedding by ovarian cancer cells, we are examining signaling through the VEGF-receptor, assessing its impact in vivo using the ID8-VEGF ovarian cancer mouse model, as well as measuring levels in human blood and ascites. Collectively, these studies identify novel mechanisms through which VEGF impairs anti-tumor immune responses and thus have the potential to enhance therapeutic strategies for the treatment of ovarian cancer.
Colon tumorigenesis mediated by Tollip in neutrophils.
Authors: Lee Christina , Zhang Yao , Yuan Ruoxi , Chen Keqiang , Li Liwu
Published: January 2023

Innate neutrophils have ability to confer either pro- or anti-tumor immune responses. However, molecular mechanisms responsible for the cell polarization are not well understood. Here, we aim to identify and characterize the neutrophil phenotypes in chronic colitis (CC) mouse model with use of AOM and DSS. We identify that Toll-interacting-protein (Tollip) may play a key role in modulating the long-term polarization and function of neutrophils as well as pro-tumorigenesis. We observed that Tollip deficient mice had improved clinical score and reduced generation of colon polyps when subjected to prolonged challenges with AOM and DSS. At the cellular level, Tollip deficient mice challenged with AOM-DSS had distinct neutrophil activation profiles as compared to wild type mice, including the differential expression of cell surface co-stimulatory molecules, migratory behaviors, as well as cytokine expression profiles. Adoptive transfer of neutrophils from Tollip deficient mice to wild type mice recapitulated the outcome of Tollip deficient mice. Based on the in vivo and in vitro studies, we conclude that Tollip may play a key role during the polarization of innate neutrophils involved in the modulation of chronic colitic tumor environment. Further studies with regard to the molecular mechanisms may enable a better understanding of neutrophil modulation in colon tumorigenesis.
Ligation of iNKT TCRs as a novel approach to activate iNKT cells for cancer immunotherapy
Authors: Yuan Weiming , Wen Xiangshu , Schaub Robert
Published: January 2023

Invariant natural killer T (iNKT) cells are innate-like T cells with an invariant TCRa chain and are restricted by CD1d. The activation of iNKT cells has demonstrated potent anti-tumor activity. Current anti-tumor clinical trials using the iNKT cell ligand, a-galactosylceramide (a-GalCer) have had limited success. Major limitations of a-GalCer-based therapies include the bioavailability of lipid drugs and potential over-stimulation of iNKT cells and ensued iNKT cell anergy. Therefore alternative approaches for iNKT cell stimulation are needed to optimize iNKT cell-based anti-tumor therapies. We have engineered a murine iNKT TCR-targeting monoclonal antibody (NKT14m) that directly activates iNKT cells. Based on preliminary results that the potency of NKT14m is less than that of a-GalCer, we reasoned that the addition of IL-12 stimulation would be beneficial for iNKT cell activation. Nevertheless, the toxicity of IL-12 has previously restricted its use in clinical trials. We evaluated the anti-tumor function of a non-toxic low dose of IL-12 in combination with NKT14m. Mice were inoculated with B16 melanoma cells and then treated with IL-12, NKT14m or combination of NKT14m and IL-12. We detected a synergistic anti-tumor effect in the combination of NKT14m and IL-12. These findings represent a new paradigm of iNKT cell activation for their anti-tumor function and future translation into human iNKT cells may provide a novel approach to anti-tumor immunotherapy.
NKT cell subsets involved in host defense from pulmonary Streptococcus pneumoniae infection
Authors: Crosby Catherine Marie , Mikulski Zbigniew , Kronenberg Mitchell
Published: January 2023

Invariant natural killer T cells (iNKT) are a population of innate-like T lymphocytes that have crucial roles in protective responses to infectious agents, particularly Streptococcus pneumoniae. Despite their abundance in the lungs, there is limited understanding of how iNKT cells mediate protection there. Prior work from our group has shown that after pulmonary S. pneumoniae infection, iNKT cells are rapidly activated in a CD1d-dependent manner. Further, others have defined iNKT1, 2, and 17 cell subsets, analogous to CD4+ Th1, Th2, and Th17 subsets. In this study we investigated the roles of lung iNKT cell subsets in protection from S. pneumoniae. In naïve C57BL/6 mouse lungs, we found NKT1 and NKT17 cells were the predominant iNKT cell subsets, with most NKT17 cells in the tissue and NKT1 cells in both the tissue and vasculature. We then utilized fluorescent bacteria, flow cytometry, and intravital microscopy to analyze the lungs during pulmonary infection. At 24 hours, the bacteria localized within the tissue, and were taken up primarily by alveolar macrophages and neutrophils. At 14 and 24 hours, a majority of the lung NKT17 cells were producing IL-17, while only a minor percentage of NKT1 cells were producing IFNγ. This correlated with data from Nur77GFP reporter mice that indicate TCR-mediated activation. At 24 hours, approximately 75% of NKT17 but only 30% of NKT1 cells had been TCR-activated; suggesting NKT17 cells may play a dominant role early in infection. Yet, by 24 hours, NKT1 cells expanded to become the major subset in the lungs, proposing they are also relevant for protection. Experiments are underway to determine when and where subsets are first activated, roles of other cytokines, and consequences of removing individual subsets.
Effector CD4 T cells with stem cell-like properties mediate chronic intestinal inflammation
Authors: Harrington Laurie E , Shin Boyoung , Darley-Usmar Victor , Bellis Susan
Published: January 2023

Increased IFNγ production is a hallmark of mucosal CD4 T cells in inflammatory bowel disease (IBD) patients; however, the role of this cell population in intestinal inflammation remains controversial. To elucidate this, viable IFNγ producing and non-producing CD4 T cells were isolated from mice with colitis and examined for the ability to confer disease. Paradoxical to the proinflammatory nature of IFNγ, CD4 T cells producing IFNγ cause attenuated pathology upon transfer, while IFNγ non-producing cells result in severe intestinal inflammation. The pathogenic IFNγ non-producing cells exhibit a “stem cell-like” gene expression pattern, maintaining genes associated with multipotency including Sell, Tcf7, Lef1, Klf2, and Wnt10a. Consistent with the “stem cell-like” gene signature, IFNγ non-producing CD4 T cells show higher cell survival, superior mitochondrial function, as well as functional plasticity. Conversely, IFNγ producing CD4 T cells express terminal differentiation markers, such as Klrg1, Tim3, Zeb2 and Prdm1, and are more susceptible to cell death. These divergent phenotypes correlate with the glycosyltransferase ST6Gal1. IFNγ nonproducing cells maintain higher levels of St6gal1 mRNA during colitis, while IFNγ producing cells downregulate St6gal1 gene expression. Furthermore, when IFNγ nonproducing cells lose ST6Gal1 activity, they become terminally differentiated, more susceptible to apoptosis and less pathogenic compared to IFNγ nonproducing cells with high ST6Gal1 activity. Together, these data indicate that effector CD4 T cells exist in a spectrum of differentiation states during IBD, from stem cell-like to terminally differentiated, which are associated with IFNγ production and ST6Gal1 activity.
Cross-talk between human glial cells and B cells help propagation of CNS-compartmentalized in progressive MS
Authors: Touil Hanane , Li Rui , Moore Craig , Antel Jack P , Bar-Or Amit
Published: January 2023

B cell depleting therapies efficiently decrease new multiple sclerosis (MS) relapses. Previously, we demonstrated that MS patients harbor abnormally higher proportions of pro-inflammatory effector B cells (Beff), producing high IL-6. TNF and GM-CSF levels compared to controls. B cells are fostered within the MS central nervous system (CNS). Persistence of B cells was reported within lesions and meningeal aggregates, which are adjacent to subpial cortical injury, involving neuronal loss and microglia/macrophage activation, and known to be associated with progressive MS. However, how distinct B cells persist and potentially propagate CNS-local inflammation potentially contributing to disease progression (unmet need) remains unsolved. First, we demonstrated that supernatant from astrocytes supported HC and MS B cell survival (including CD27+ B cells), while M2c-polarized human microglia appeared cytotoxic to B cells. Activated astrocytes increased CD86 expression on B cell surface. Similar results are obtained when B cells were exposed to supernatants of M1-polarized microglia. Furthermore, B cells previously exposed to activated astrocytes increased T cell proliferation. In turn, Beff but not Breg supernatants down-regulate IL-10 production by microglia/macrophage, and substantially enhance pro-inflammatory cytokines (IL-12, TNF & IL-6), independent of phenotypic changes of microglia/macrophage. These results indicate a potential bi-directional interaction between disease-relevant human B cell subsets and astrocytes CNS resident microglia and infiltrating myeloid cells, which may influence the propagation of MS-CNS compartmentalized inflammation associated with disease progression.
CD19 cytoplasmic tyrosine residues Y482 and Y513 are sufficient to promote normal B cell development and differentiation but induce a skewed humoral immune response.
Authors: VanValkenburg Arthur James , King R. Glenn , Justement Louis B
Published: January 2023

CD19 amplifies BCR signal transduction by recruiting SH2 domain-containing proteins to its nine cytoplasmic phosphorylated tyrosine residues. Previous studies have shown that Y482 and Y513, which recruit PI3K are required for normal B cell development/differentiation and a productive T-dependent humoral response. However, it has not been determined if CD19 Y482 and Y513 are sufficient to promote normal B cell differentiation or antibody response to immunization. Furthermore, the effects of upstream CD19 tyrosine-mediated Vav/PLCγ2 and Grb2/Sos signaling are not well characterized. Using mice expressing CD19 mutated to only express tyrosines Y482 and Y513, we demonstrate that CD19 mediated Lyn/PI3K signaling is sufficient for B1 B cell development, marginal zone B cell maturation, and germinal center B cell formation. However, these mice exhibit an increased antibody response against T-independent antigens and are impaired in their ability to generate class-switched germinal center B cells during a T-dependent immune response, as well as long-lived, class-switched antibody secreting cells. These findings suggest that cross-talk between signal effectors recruited to Y482 and Y513 and those recruited to other CD19 tyrosine residues is important for determining the qualitative nature of the humoral response.