bluuebunny/crossref_metadata_2025 huggingface dataset:
T-bet regulates T cell-independent plasmablast responses during intracellular bacterial infection
Authors: Newell Krista L , Levack Russell , Cabrera-Martinez Berenice , Winslow Gary M
Published: January 2023

T-bet+ B cells have emerged as a major B cell subset associated with both protective immunity and autoimmunity. T-bet is considered to be a master transcription factor in type I adaptive immune responses to intracellular pathogens, a response characterized by the production of interferon gamma. Our studies have shown that infection with the intracellular bacterium, E. muris, generates both extrafollicular T cell-independent T-bet+ CD11c+ IgM-secreting plasmablasts (PB), as well as T-bet+ CD11c+ IgM memory cells. Both of these B cell populations play key roles in antigen-specific humoral immunity, although the role of T-bet in their development had not been resolved. Although T-bet is often considered to define lineage in Type I B cells, we found that T-bet was dispensable for memory B cell development. Memory B cells from mice with B cell-specific T-bet deficiency exhibited nearly identical surface marker expression as those of wild-type mice, including CD11c, CD73, PD-L2, CD80, CD38, CD95, and CXCR3. In contrast, T-bet-deficient early CD11c+ splenic PB were significantly reduced in frequency, suggesting that T-bet regulates PB, but not memory cell development or differentiation. As has been reported, antigen-specific IgG2c was no longer the dominant serum antibody isotype in mice lacking B cell T-bet expression. Our data suggest that a critical function of T-bet in B cells is to promote the differentiation of short-lived antibody-secreting PB, and to restrict class switching to protective IgG2c antibodies. Our findings are relevant to how T-bet+ B cells function not only in pathogen-specific immunity, but also in autoimmune diseases, wherein the quality of the humoral response is of critical importance.
Characterization of the cross reactive T cell repertoire in the context of heterosubtypic influenza A virus infection
Authors: Gaevert Jessica A , Duan Susu , Thomas Paul Glyndwr
Published: January 2023

Influenza A viruses (IAVs) are a major respiratory pathogen with the capacity to rapidly mutate and escape an individual’s existing immunity to a specific strain generated by vaccination or naturally acquired infection. CD8 T cells however, target conserved proteins that could be shared across multiple subtypes. Heterosubtypic immunity to IAVs, where memory T cells generated to a previous IAV are reactivated during re-challenge with an antigenically distinct virus, may be important in generating an effective immune response to different and/or novel strains of IAV. Specifically, cross reactive T cells, where a T cell receptor (TCR) can recognize more than one epitope displayed on an MHC-I molecule, could facilitate rapid recognition of conserved or nominally divergent epitopes, may have the potential to alter the TCR repertoire over the course of heterosubtypic infections, and might be selected for by the immune system. Utilizing 8 variant influenza viruses for priming and secondary challenge, our preliminary data has shown that cross reactive TCRs seen in primary infection with one IAV can mount a robust and effective memory response to a different IAV, even if the epitope has slightly mutated. However, selection for cross reactive receptors may create a narrowed repertoire consisting of a few clonotypes over the course of many repeated heterosubtypic IAV infections, covering a narrow range of antigenic space representing the mutant epitopes encountered. Paradoxically, this selection for cross-reactivity may provide a substantial escape opportunity with future antigenic variations. Ongoing work focuses on how repeated IAV challenges shape and are shaped by these selected cross-reactive T cell pools.
Non-canonical functions of adaptive natural killer cells
Authors: Gyurova Ivayla E , Win Hannah Than , Waggoner Stephen N
Published: January 2023

Natural killer (NK) cells are critical for the control of cytomegalovirus (CMV) infections in both mice and humans. Subsets of phenotypically and functionally distinct adaptive NK cells accumulate after CMV infection and potentially represent desirable new targets for a preventive CMV vaccine owing to their putatively greater antiviral functionality. However, our lab and others have described a series of non-canonical functions of NK cells that prevent harmful disease outcomes by modulating the responses of other leukocytes. In fact, we now demonstrate that bulk human blood NK cells can suppress CMV-specific T cell responses during in vitro culture. We reason that amplification of these non-canonical functions within an expanded subset of virus-induced adaptive NK cells would be beneficial to health in the context of virus persistence. In order to compare the canonical antiviral and non-canonical immunoregulatory functions of conventional and adaptive NK cells, we first sought to determine if it was possible to expand these subsets using K562 feeder cells with membrane bound IL-21. Similar to our recent longitudinal clinical profiling of adaptive NK cells (Gyurova et al. 2019), we observed variable increase or decrease in the baseline frequencies of adaptive (i.e. FcRγneg) NK cells following ex vivo expansion of cells from disparate healthy donors. Ex vivo expanded NK subsets demonstrated a reduced capacity to kill K562 cells. These results establish a method for obtaining sufficient quantities of adaptive and conventional NK cells for functional evaluation of antiviral and regulatory activity.
SATB1 as a novel therapeutic target for methyltransferase inhibitors against Cutaneous T Cell Lymphoma
Authors: Harro Carly , Perez-Sanz Jairo , Costich Tara Lee , Payne Kyle K , Galindo Carmen Maria Anadon , Gonzalez Ricardo A Chaurio , Biswas Subir , Mandal Gunjan , Rigolizzo Kristen E , Mine Jessica A , Showe Louise C , Liu Kebin , Rodriguez Paulo C , Pinilla-Ibarz Javier Lee , Sokol Lubomir , Conejo-Garcia Jose R
Published: January 2023

Cutaneous T cell lymphoma (CTCL) is a clinically unmet need. Using conditional knockout mice, we found that ablation of the genomic organizer Special AT rich sequence binding protein 1 (Satb1) induces a progressively fatal lymphoma characterized by mature, skin homing, Notch activated CD4 and CD8 T cells. Mechanistically, Satb1 restrains Stat5 phosphorylation and the expression of skin homing chemokine receptors in mature T cells. Notably, SUV39H1 and 2 methyltransferase dependent epigenetic repression of SATB1 is universally found in human Sezary Syndrome, but not other peripheral T cell malignancies. Accordingly, H3K27 and H3K9 trimethylation occlude the SATB1 promoter in Sezary cells. Inhibition of SUV39H1 and 2 methyltransferases with novel drugs, unlike EZH2 inhibition, restores SATB1 expression, selectively abrogating the growth of primary Sezary cells more effectively than Romidepsin. Therefore, SATB1 acts as a tumor suppressor in mature T cells upon NOTCH1 deregulation, and inhibition of methyltransferases that silence SATB1 could address an unmet need for patients with mycosis fungoides and Sezary syndrome.
S. aureus persister cells have increased survival to components of innate immunity, leading to persistence within a catheter-associated biofilm infection
Authors: Daubert Trevor Alan , Kluthe Kennedy , Nuxoll Austin
Published: January 2023

Staphylococcus aureus is known for its ability to cause chronic reoccurring infections in clinical settings. The organism’s ability to thrive despite antibiotic treatment has led to the severity of disease that is often exhibited with these infections. It has been hypothesized that energy-dependent persister formation leads to antibiotic tolerance in S. aureus. Persister cells are a dormant-like phenotypic variant of S. aureus that resemble cells growing in stationary phase. Previously, it was shown that a knockout in the tricarboxylic acid (TCA) cycle gene, fumC, which reduced intracellular ATP, led to an increase in the number of persisters. While persisters have been examined in relationship to antibiotic tolerance, it is currently unknown how persisters interact with the innate immune system. Considering a vast amount of chronic reoccurring infections in a clinical environment involve a biofilm, persisters were examined in a biofilm-associated catheter infection within a mouse. Following a 9 day infection, the fumC knockout exhibited increased survival within the tissue surrounding the catheter and within the heart compared to wild type S. aureus. Fluorescent activated cell sorting (FACS) analysis revealed similar recruitment of M1 and M2 macrophages, as well as neutrophils, suggesting there was not a difference in immunogenicity between the two strains. The fumC knockout exhibited increased survival compared to wild type S. aureus within a macrophage demonstrating persisters are better equipped to tolerate the innate immune system. These results indicate persisters are not only a problem with antibiotic treatment but are also better suited to survive the host’s immune system leading to increased survival within a host.
Screening of biological biomarker for distinguishing latent and active tuberculosis by proteome microarray
Authors: Li Chuanyou , Liu Yi
Published: January 2023

This study is to identify potential serum biomarkers for distinguishing between latent tuberculosis infection(LTBI) and active tuberculosis(ATB). A proteome microarray containing 4,262 antigens was used for screening serum biomarkers of 40 serum sample from patients with LTBI and ATB. The interaction network and functional classification of differentially expressed antigens were analyzed by STRING 10.0 and the TB datebase respectively. Enzyme-linked immunosorbent assays(ELISA) were used to validate candidate antigens further using 279 clinical samples. The diagnosis performances of candidate antigens were evaluated by receive operating characteristic curve(ROC) analysis. Both antigens combination and logistic regression analysis were used to improve diagnostic ability. Microarray results showed that levels of 152 Mycobacterium tuberculosis antigen-specific IgG were significantly higher in ATB patients than LTBI patients. These differentially expressed antigens showed stronger association each other and were involved in various biological processes. Eleven candidate antigens were further validated by ELISA and showed consistent results in microarray analysis. ROC analysis showed that antigens Rv2031c, Rv1408, and Rv2421c had higher sensitivity and specificity for TB diagnosis. In addition, combination of both antigens and logistic regression analysis improved the diagnostic ability.
Dispensable role of chemokine receptors in migration of mycobacterial antigen-specific CD4+ T cells into mycobacteria-infected lung
Authors: Umemura Masayuki , Yamasaki Masatoshi , Tamura Toshiki , Matsuzaki Goro
Published: January 2023

Mycobacterial antigen-specific CD4+ Th1 cells have a pivotal role in protective immunity against mycobacterial infections, including pulmonary tuberculosis. In the course of infection, Th1 cells differentiate in the lung-draining lymph nodes and migrate into the infected lung. Chemokine receptors on T cells are involved in T cell migration into the intestine and skin. However, the role of chemokine receptors in the migration of CD4+ T cells into the lung has not yet been determined. To address this issue, the role of chemokine receptors in T cell migration into the mycobacteria-infected lung was analyzed using mycobacterial Ag85B peptide 25-specific T cell receptor-transgenic (P25) CD4+ T cells. P25 T cells in the Mycobacterium bovis BCG-infected lung and lung-draining mediastinal lymph nodes (MedLN) expressed the chemokine receptors, CCR5, CCR6, CXCR3, and CXCR5, which bind chemokines produced by the BCG-infected lung. To further analyze the role of chemokine receptors in the migration of BCG-primed P25 T cells into the lung and medLN, P25 T cells were adoptively transferred into BCG-infected wild-type mice and their migration into the lung was monitored. Unexpectedly, blocking chemokine receptor function with pertussis toxin, a G-protein inhibitor, failed to suppress migration of T cells into the infected lung. However, the treatment completely blocked migration of the MedLN P25 T cells into the recipient lymph node. These results suggest that the interaction of chemokine receptors on mycobacterial antigen-specific Th1 cells with chemokines is dispensable for their migration into the mycobacteria-infected lung.
Advancing Compensation into the Future with the Next Generation of Comp Beads
Authors: Calderon Veronica E , Montoya Leticia , Chen Yongfen , York Adam
Published: January 2023

A need for more robust compensation techniques comes with continued growth in the study of immunotherapy and immunology by flow cytometry. Compensation is a method used to address the measurement of a fluorophore in multiple detectors (referred to as spillover) by mathematically correcting signal overlap between emission spectra of different fluorochromes to determine the true level of staining/positive target signal. The signal spillover can impact data quality and becomes very important with different levels of marker expressions. This measurement can be achieved by use of cells, or more typically by use of compensation beads to save precious biological samples.

Current compensation beads have been shown to be problematic and can lead to over- or under-compensation, resulting in less or more true positive signal, respectively. Additionally, current compensation beads are incompatible with far red violet and ultraviolet dyes and may not be ideal for dim/low expressing markers.

These studies address compensation by comparing various compensation products to biological samples on multiple immunophenotyping panels of either human or murine origin. These panels compare the use of a wide range of fluorophore conjugates, including the use of violet and ultraviolet excitable dyes, low expressing targets, and intracellular targets to determine compensation accuracy. The results demonstrate that a precise compensation matrix can be obtained with a newly improved commercial bead that is comparable to a cell sample. This data validates that precious cell samples may not be needed to be used in place of commercial bead products to obtain accurate compensation and quality data regardless of a panel’s design elements.
Microfluidic generation of lipid nanoparticles to facilitate DNA entry into human mast cells
Authors: Kulka Marianna , Puga Narcy Arizmendi
Published: January 2023

Mast cells are highly granulated immune cells that can be targeted for transfection using lipid nanoparticles (LNP). Lipid nanoparticles (LNPs) were composed of cholesterol and 1-Stearoyl-2-oleoyl-sn-glycero-3-phosphocholine, the ionizable lipid 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane, and the phospholipid 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene-glycol)-2000] using microfluidic mixing and used to encapsulate a plasmid encoding enhanced green fluorescent protein (EGFP) under the control of a human cytomegalovirus promoter (pEGFP-C1). Encapsulation efficiency was determined using PicoGreen and LNP size was measured using dynamic light scattering. Human mast cells-1 (HMC-1) were transfected with loaded or empty LNPs in the presence or absence of apolipoprotein E (ApoE) for 24, 48 or 72 hours in medium. Cells were analyzed by fluorescence microscopy and flow cytometry. LNPs were approximately 100 nm in diameter (ideal) and efficiently encapsulated plasmids pEGFP-C1 (75.38%). ApoE4 appeared to slightly augment (>10%) LNP uptake after 24 hr. Our study demonstrates that LNPs generated by microfluidic mixing efficiently encapsulate a plasmid payload and can be used to successfully transfect human mast cells. Carrier molecules such as ApoE can be used to augment LNP uptake by mast cells. Further functionalization and optimization of these LNPs can provide powerful new tools for targeting other immune cells.
A Performance-Optimized Multicolor Flow Cytometry Dried-Reagent Cocktail for Memory T-Cell Analysis
Authors: Hanley Mary , Bharadwaj Mamatha , Lomas Chip , Bornheimer Scott , Saksena Suraj , Afonina Elena , Lopes Erika Barboza Prado
Published: January 2023

The reliance on flow cytometry for phenotypical and functional analyses of immune components in both research and clinical laboratories has increased the need flow cytometric assay standardization. Dried-down reagent cocktails pre-optimized with regards to fluorophore choice for each specificity as well as concentration of each conjugate can improve standardization. Use of a pre-optimized dried-down cocktail provides an efficient assay workflow which can minimize operator error in addition to offering enhanced reagent stability and reduced variability across instruments and sites.

We developed a dried-down reagent cocktail for the identification and characterization of CD4+ and CD8+ T-cell memory and naïve subsets, including stem cell memory populations. The BD Horizon™ Dri Memory T cell panel contains optimized antibody-fluorophore conjugates against CD3, CD4, CD8, CD45RA, CD197, CD27 and CD95. Additional markers can be accommodated as drop-ins for a more comprehensive analysis.

This cocktail was optimized for stain index in all channels of a red, blue, violet laser flow cytometer (BD FACSLyric™) in liquid format, then converted to dry format. The performance of the dried-down cocktail, with regards to MFI and % positivity of T-cell subsets, is comparable to that of its optimized liquid equivalents. Additionally, results are consistent across FACSLyric instruments and similarly when acquired on a BD LSRFortessa™.

The comparable performance of the dried-down cocktail tube, along with the advantages offered by using a dried reagent cocktail, and the convenience of adding liquid drop-in reagents, reveal the BD Horizon Dri Memory T cell panel tube to be a valuable option for memory T-cell flow cytometry analysis.
Inhibition of CSF-1R curtails inflammation during experimental autoimmune encephalomyelitis
Authors: Hwang Daniel , Ciric Bogoljub
Published: January 2023

Multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE) are autoimmune diseases characterized by extensive infiltration of T cells and myeloid cells into the CNS. Treatments for MS are typically either non-specific immunosuppressive drugs or target adaptive immune cells. However, mounting evidence shows that myeloid cells have essential roles in MS/EAE pathology, but few, if any, MS drugs specifically target myeloid cells. A promising strategy for bridging this gap in treatment modalities for MS may be targeting CSF-1R, a receptor tyrosine kinase with important roles in myeloid cells, including survival and differentiation. We found that blocking CSF-1R signaling attenuated EAE and our analysis of the CNS these mice revealed substantially diminished numbers of inflammatory myeloid cells. These data demonstrate that inhibiting CSF-1R signaling is a viable strategy for attenuating myeloid cell responses during autoimmune neuroinflammation. These studies also suggest that targeting CSF-1R signaling may be an effective approach for treating MS.
Taurodeoxycholate, a master key locking both priming and activation signal necessary for inflammasomal activation of microglia by Amyloid beta in Alzheimer’s diseases
Authors: ISLAM MD JAHIRUL , Cho Jong-Ah , Kim Tae-Joo , Kim Bong Jik , Moon Sung-Yoon , Lee Eun-Young , Lee Song-Jin , Seong Seung-Yong
Published: January 2023

Alzheimer’s disease (AD) is an inflammatory neurodegenerative condition characterized by deposition of amyloid beta (Abeta) in the brain and origins dementia. Neuronal apoptosis following neuroinflammation by Abeta impairs cognition and memory. In this study we focused on Taurodeoxyxcholate (TDCA) is an active bile acid derivative with potent anti-inflammatory function.

In 5× Familial Alzheimer’s disease (5xFAD) mice model, chronic administration of TDCA in 5xFAD mice improved learning and memory in different memory test compared with PBS group. TDCA treatment significantly decreased neuronal apoptosis, load of Abeta plaques, the number of activated GFAP+ astrocytes and Iba-1+ microglia in different brain region of 5xFAD mice. CD11bhiGr1intF4/80+ myeloid cells in brain and spleen increased significantly in TDCA-treated group compared to PBS-treated group when brain single cell were immune-stained. In vitro, TDCA increased phagocytosis of Abeta. TDCA suppressed NLRP3-ASC oligomerization and production of mature IL-1β/IL-18 in Microglia by Abeta. TDCA inhibited Ca++ influx incurred by ATP or BzATP (P2X7 agonists). Compared with wild type, Ca++ influx of macrophages from TGR5 KO mice was like that of P2X7 KO mice in response to BzATP, suggesting that TDCA might play role as an inverse agonist for TGR5 complexed with P2X7. TDCA activates adenylate cyclase and cAMP inhibit priming phase of inflammasomal activation and oligomerization of NLRP3-ASC complex, suggesting that TDCA suppresses both priming and activation phase of NLRP3 inflammasome. TDCA and its novel chemical derivatives could be better choice in ameliorating broad spectrum of neuroinflammation to improve cognition and memory in AD patients.
Alpha-synuclein (αS) functions as an alarmin to promote inflammatory and immune responses by activating dendritic cells and macrophages
Authors: Alam Md Masud , Yang De , Li Xiao-Qing , Liu Jia , Back Timothy Carrel , Trivett Anna , Karim Baktiar , Zasloff Michael , Oppenheim Joost J
Published: January 2023

Alpha-synuclein (αS) is causally involved in the development of neurodegenerative disorders including Parkinson’s disease (PD). Recently, αS has been shown to be a chemotactic activator of phagocytes. Here, we report that αS is a critical mediator of inflammatory and immune responses. Treatment with αS activated human and mouse dendritic cells (DCs) and macrophages at both phenotypic and functional levels. αS treatment of DCs triggered the NF-κB signaling pathway and predominantly enhanced their Th1-polarizing capacity. αS also polarized mouse macrophages in an M1 direction. Activation of APCs by αS was mediated by TLR4. Intraperitoneal administration of αS recruited DCs and macrophages into the peritoneal cavity, while inflammatory reactions in the peritoneal cavity and colons induced αS expression and production. Importantly, immunization with ovalbumin (OVA) in the presence of αS promoted OVA-specific antibody and T cell responses, whereas the induction of inflammatory and OVA-specific immune responses was markedly reduced in αS−/− mice. Thus, αS can be considered an alarmin that functions as a mediator of inflammatory and immune responses. The potent proinflammatory and immunostimulating effects of αS in the periphery may cause the accumulation of αS and inflammatory damage in the central nervous system, culminating in Parkinson disease (PD).
Dimethyl Itaconate, an Itaconate Derivative, Exhibits Immunomodulatory Effects on Neuroinflammation in Experimental Autoimmune Encephalomyelitis
Authors: Kuo Ping-Chang , Weng Wen-Tsan , Scofield Barbara A. , Paraiso Hallel C. , Brown Dennis A. , Yu I-Chen , Yen Jui-Hung
Published: January 2023

Itaconate has recently emerged as a regulator of immune cell function. Studies show that itaconate is required for the activation of anti-inflammatory transcription factor Nrf2 by LPS in macrophages. Moreover, dimethyl itaconate (DMI), an itaconate derivative, has been shown to inhibit IL-17-induced IκBζ activation in keratinocytes and modulate IL-17-IκBζ pathway-mediated skin inflammation in an animal model of psoriasis. Currently, the effect of itaconate on regulating macrophage functions and peripheral inflammatory immune responses is well established. However, its effect on microglia (MG) and CNS inflammatory immune responses remains unexplored. Thus, in this study we investigated whether itaconate possesses an immunomodulatory effect on regulating MG activation and CNS inflammation in experimental autoimmune encephalomyelitis (EAE). Chronic C57BL/6 EAE and relapsing-remitting SJL/J EAE were induced to assess the therapeutic effect of DMI. Our results show DMI ameliorated disease severity in the chronic C57BL/6 EAE model. Further analysis of the cellular and molecular mechanisms revealed that DMI mitigated blood-brain barrier disruption, inhibited MMP3 and MMP9 production, suppressed microglia activation, and repressed CNS infiltration of Th1 and Th17 cells. Strikingly, DMI also exhibited a therapeutic effect on lessening severity of relapse in the relapsing-remitting SJL/J EAE model. In conclusion, we demonstrate for the first time that DMI suppressed neuroinflammation and ameliorated disease severity in EAE through multiple cellular and molecular mechanisms, and our findings suggest that DMI can be developed as a novel therapeutic agent for the treatment of EAE through its anti-inflammatory properties.
Role of non-HLA antigens in cardiac transplant rejection
Authors: Panicker Anjali Jacob , Gates Katherine V , Biendarra-Tiegs Sherri M , Vetr Nikolai G , Higuita Manuela Lopera , Nelson Timothy J , Pereira Naveen L , Griffiths Leigh G
Published: January 2023

Although immunosuppressive drugs improve short term survival, rejection incidence and long term survival remain largely unchanged in heart transplant patients. While Human Leukocyte Antigen (HLA) is a common initiator of graft-specific immune responses, non-HLA antibody mediated rejection (AMR) also commonly occurs. Furthermore, non-HLA antibodies have been implicated in cell mediated rejection and cardiac allograft vasculopathy. Therefore, there is a need to identify the role of non-HLA antigens in heart transplant rejection.

Serum from heart transplant patients lacking donor specific anti-HLA antibodies was collected longitudinally; pre-transplantation, acute rejection, and post-rejection for patients with AMR and matched controls. Serum IgG was used to form anti-human heart affinity columns and antigenic proteins from donor heart extracts captured. Antigens were eluted from the columns and subjected to LC-MS/MS. Longitudinal graft-specific changes in immune response were calculated.

Response toward non-HLA antigens increased longitudinally, with 41 high prevalence antigens found in ≥50% of the cohort. Co-culturing cardiomyocyte and endothelial cells with patient IgG indicated cell function changes correlated with specific antigens (e.g., Prohibitin). Immunofluorescence staining of patient biopsies revealed that longitudinal increase in antigen expression correlated with non-HLA antibody response.

The non-HLA antigens identified in this study elicit acute and chronic graft-specific adaptive immune responses in heart transplant recipients. Antibodies against these non-HLA antigens induced cellular dysfunction by changing expression of their target protein in the donor heart.
Enhancing IFN-β production through synergy between the STING pathway and TLR pathways
Authors: Higgs Emily , Gajewski Thomas
Published: January 2023

Previous work from our lab has shown that tumors activate the STING pathway in antigen presenting cells. STING signaling leads to IFN-β production which is required for T cell priming and recruitment. IFN-β transcription is induced by multiple transcription factors including NF-κB and IRF3. Enhancing STING activation and downstream IFN-β production has therapeutic potential, and STING agonists are currently being evaluated in clinical trials. However, innate immune activation in response to a pathogen rarely occurs by activating only one signaling pathway, and activating multiple innate immune pathways similar to a natural infection could improve the efficacy of STING agonists. To test this, we performed experiments with the STING agonist DMXAA alone or in combination with several TLR agonists. We found that LPS + DMXAA induced significantly greater IFN-β transcription than the sum of either agonist alone. To explain this synergy, we assayed each step of STING pathway signaling. LPS did not increase STING aggregation beyond that induced by DMXAA. Additionally, LPS did not increase IRF3 phosphorylation nor IRF3 nuclear translocation in combination with DMXAA. LPS did increase the phosphorylation and nuclear translocation of the NF-κB subunit p65. These results suggest that the synergy in IFN-β production is achieved through IRF3 activation downstream of STING signaling and NF-κB activation downstream of TLR4 signaling. Intratumoral injection of LPS + DMXAA resulted in significantly improved tumor control of B16 melanoma in vivo than either agonist alone, suggesting that combining activation of these pathways improves the anti-tumor innate immune response in a way that is therapeutically beneficial.
Expression of cancer cell-associated Protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK) regulates protective anti-tumor immunity in melanoma
Authors: Mandula Jessica , Mohamed Eslam , Sierra-Mondragon Rosa , Cao Yu , Trillo-Tinoco Jimena , Rodriguez Paulo
Published: January 2023

Activation of moderated endoplasmic reticulum (ER) stress in cancer cells promotes cellular adaptation and survival in response to the adverse conditions of the tumor microenvironment (TME). However, how modulation of ER stress in cancer cells culminates in the development of an immunosuppressive TME remains poorly understood and therapeutically unexploited. Upon disruption of ER homeostasis, mediator proteins IRE1a, ATF6 and PERK induce activation of the unfolded protein response (UPR). Although maladaptive priming of UPR mediators in immune cells deactivates their capacity for anti-tumor immunity, the immunosuppressive potential of UPR mediators in cancer cells, in particular PERK, remains unknown. Thus, in this study, we hypothesized that activation of PERK in tumor cells shapes the formation of the immunosuppressive TME and impairs the development of anti-tumor immunity. Elimination of PERK in cancer cells impaired survival upon chemically-induced ER stress and promoted caspase-independent cell death. Interestingly, in vivo ablation of PERK in melanoma impaired tumor growth, which surprisingly occurred in a T cell-dependent manner. Additionally we observed that ablation of PERK shifted the TME from an immunosuppressive to immunostimulatory environment, enhanced accumulation of effector T cells and directed reprogramming of myeloid derived suppressor cells (MDSCs) into immunostimulatory cells. Additional experiments indicated that ablation of PERK induced expression of mediators associated with immunogenic cell death. Collectively, our findings demonstrate the immunoregulatory role of PERK in tumor cells and highlight the therapeutic potential of targeting UPR mediators and tumor adaptive processes.
Too Much of a Good Thing: IFN-I, Dendritic Cells, and Cancer
Authors: Vartabedian Vincent F , Kemper Esther K , Parker Christopher G , Shaabani Namir , Teijaro John R
Published: January 2023

To prime T cells to fight cancer, dendritic cells (DCs) require Type I Interferon (IFN-I) signaling. IFN-I signaling induces the transcription and translation of Interferon Stimulated Genes (ISGs), including strong induction of Usp18, which dampens further IFN-I signaling. We hypothesized that increasing IFN-I signals to DCs in vivo via conditional Usp18 deletion would enhance the ability of DCs to prime T cells, augmenting their anti-tumor activity. To test this, we used Usp18fl/fl x CD11c-cre mice and the syngeneic B16F10 melanoma model. Surprisingly, tumors grew more quickly in Usp18fl/fl x CD11c-cre mice, and intratumoral T cells were less functional post-priming by Usp18-deficient DCs. This functional decrease was coupled with an accelerated effector differentiation marked by early loss of the stem cell-like factor TCF-1. These data suggest that Usp18-deficient DCs expedite terminal effector T cell differentiation, resulting in suppressed effector functionality at later time points. Consistent with this T cell dysfunction, intratumoral STING agonism was less effective at controlling tumors in Usp18fl/fl x CD11c-Cre mice than Usp18fl/fl littermate controls. To understand the molecular drivers of these changes, we profiled DCs with tandem mass tag (TMT) mass spectrometry. We found that Usp18-deficient DCs exhibit deficiencies in mitochondrial function and cysteine biosynthesis, potentially driving their altered ability to prime T cells. Overall, this work highlights the importance of stringent IFN-I signaling regulation for mounting optimal immune responses to cancer. Our findings also identify DC-intrinsic Usp18 expression as a potential biomarker for predicting patient responsiveness to STING agonist therapy.
T cells redirected against Igβ for the immunotherapy of B cell lymphoma
Authors: Jiang Dongpeng , Chu Jianhong , Wu Depei
Published: January 2023

CD19-redirected CAR-T immunotherapy has emerged as a promising strategy for treatment of B cell lymphoma, however, many patients often relapsed due to antigen loss. Therefore, it is urgently needed to explore other suitable antigens targeted by CAR-T cells to cure B cell lymphoma. Igβ is highly expressed on the surface of lymphoma cells and mutates in part of patients resulting continuous PI3K signaling promote the survival of B lymphoma. These conclusions suggest that wild-type or mutant Igβ may become a potential therapeutic target. In this study, we engineered T cells to express anti-Igβ CAR with CD28 costimulatory signaling moiety and observed that Igβ-CAR T cells could efficiently recognize and eliminate wild-type or mutant Igβ+ lymphoma cell lines both in vitro and in two different lymphoma xenograft models. Of note, both the in vitro and in vivo effect of Igβ CAR-T cells was comparable with that of CD19 CAR-T cells. Importantly, Igβ CAR-T cells recognized and eradicated patient-derived lymphoma cells in the autologous setting. Lastly, the safety of anti-Igβ CAR-T cells could be further enhanced by introduction of the inducible caspase-9 suicide gene system. Collectively, Igβ-specific CAR-T cells may be a promising immunotherapeutic approach for B cell lymphoma.
Myeloid-derived suppressor cell subset heterogeneity drives glioblastoma progression in a sex-dependent manner
Authors: Bayik Defne , Zhou Yadi , Park Chihyun , Hong Changjin , Vail Daniel , Silver Daniel J , Lauko Adam J , Roversi Gustavo A , Watson Dionysios C , Lo Alice , Alban Tyler J , McGraw Mary , Sorensen Mia D , Grabowski Matthew M , Otvos Balint , Vogelbaum Michael A , Horbinski Craig M , Khalil Ahmad M , Hwang Tae Hyun , Ahluwalia Manmeet S , Cheng Feixiong , Lathia Justin D
Published: January 2023

Immunosuppression is a major factor facilitating glioblastoma (GBM) progression and therapeutic resistance. We previously demonstrated that myeloid-derived suppressor cells (MDSCs) expand in GBM patients, but the mechanisms by which MDSC subsets promote tumorigenesis remain unknown. Using multiple syngeneic mouse GBM models, we show that monocytic MDSCs (mMDSCs) accumulate in tumors of males and associate with poor prognosis. Consistent with preclinical observations, males, who constitute 60% of GBM patients and have a worse prognosis than females, had significantly more tumor-infiltrating mMDSCs. In contrast, female tumor-bearing mice had a two-fold increase in circulating granulocytic MDSC (gMDSC) frequency, and a high gMDSC gene signature correlated with poor prognosis of female patients. Male-to-female bone marrow transplantation indicated that immune cell-intrinsic discrepancies drive the sex differences in GBM survival. In line with the differential MDSC localization, targeting gMDSCs with anti-Ly6G neutralizing antibodies extended the lifespan of female mice without affecting males. However, mMDSCs were protected from the anti-Ly6C depletion strategy due to their systemic and local proliferation, as indicated by ex vivo Ki-67 staining and subsequently confirmed by gene expression analysis. Drug-prediction using the differential expression profiles and subsequent pre-clinical testing established that mMDSCs can be targeted by chemotherapies, while IL-1 inhibitors are effective against gMDSCs. These findings indicate that MDSC subset variation represents an opportunity for improved immunotherapy efficacy while accounting for sex as a biological variable.
Dendritic cell dysfunction precedes tumor formation during pancreatic oncogenesis
Authors: Lin Jeffrey H , Vonderheide Robert H
Published: January 2023

Dysfunction of type 1 conventional dendritic cells (cDC1s) – the dendritic cell subtype crucial for spontaneous anti-tumor T cell immunity – has long been hypothesized to develop secondarily to invasive cancer. Here, we reveal that cDC1 dysfunction instead develops in the earliest stages of cancer development upon formation of preinvasive pancreatic intraepithelial neoplasias (PanINs) in the KrasG12D Trp53 R172H Pdx1-Cre-driven (KPC) mouse model of pancreatic ductal adenocarcinoma. This results in systemically increased cDC1 apoptosis and insufficient upregulation of T cell-polarizing cytokines during cDC1 maturation. CD8+ T cell activation following vaccination is subsequently impaired in PanIN- and tumor-bearing mice. Yet, combination therapy with CD40 agonist and Flt3 ligand in tumor-bearing mice returns cDC1 abundance to normal levels and induces an IFNg response signature, repairing cDC1 maturation and restoring full CD8+ T cell activation. This study therefore reveals the unexpectedly early and systemic onset of cDC1 dysregulation while elaborating therapeutically tractable strategies towards cDC1 repair.
Distributions of non-expressed MHC class I chain related A (MICA) and NKG2D receptor polymorphism in cholangiocarcinoma
Authors: Phanabamrung Sornwit , Jumnainsong Amonrat , Leelayuwat Chanvit
Published: January 2023

MICA is an MHC class I like molecule functioning as a ligand of an activating receptor, NKG2D. This molecule is up regulated under stress conditions especially of cancerous cells. Thus, lacking of this molecule could lead to an impairment of immune surveillance and the progression of cancer. In addition to the ligand, polymorphism of NKG2D is also impact the activating function of immune cells. Thus, we have investigated the distributions of non-expressed (MICA*010) allele and deletion of the MICA gene in 365 northeastern Thais (NET) and 121 patients of cholangiocarcinoma (CCA) which is the most common form of intrahepatic cancer of NET with the highest prevalence in the world. MICA*010 and NKG2D typing were performed by PCR-SSP and the MICA deletion by multiplex PCR using three pairs of primers. The prevalence of MICA deletion was higher in CCA than that of NET (% allele frequency (%AF) of 0.25 vs 1.7: Pc = 0.036) with a relative risk of 6.21. Although the %AF of MICA*010 in CCA was not significantly different from NET (34.7 vs 34.3; p-value = 0.96), the prevalence of heterozygous MICA*010 with MICA null in CCA was significantly higher than that of NET (%AF = 0.29 VS 3; Pc = 0.037; RR = 3.39). The results suggested that individuals carrying the MICA null allele with or without the non-expressed allele were more susceptible to develop CCA implicating the involvement of immune escape via the activating receptor. Additionally, NKG2D polymorphisms were also investigated. These data will be combined with MICA results and discussed at the conference.
Distinct responses to αPD-1, αPD-L1 and αPD-L2 immunotherapy in aged versus young hosts includes T-cell stem cell effects and PD-L2 expression differences
Authors: Garcia Myrna G , Padron Alvaro S , Deng Yilun , Kancharla Aravind , Reyes Ryan M , Gupta Harshita B , Curiel Tyler J
Published: January 2023

Aging is the biggest risk factor for cancer, yet little is known about how it affects cancer immunotherapy. Melanoma response to αPD-1/αPD-L1 correlates with CD8+ TCF-1+ T cell stem cell (TCSC) generation. We tested αPD-1 (100 μg/mouse), αPD-L1 (100 μg/mouse) or αPD-L2 (200 μg/mouse) in aged (18–24 months) and young (3–8 months) mice challenged orthotopically with B16 melanoma (SQ) or ID8agg ovarian cancer (IP). Tumors and draining lymph nodes (TDLN) were analyzed by flow. We previously reported that αPD-1 treats young and aged with B16 and αPD-L1 only treats young. αPD-L2 treated B16 in aged but not young, the first single agent cancer immunotherapy exhibiting this property. Preliminary data indicate survival benefit of αPD-L2 against ID8agg in aged but not young mice. Aged mice tolerated all treatments comparable to young. B16 efficacy in young (αPD-1, αPD-L1) and aged (αPD-1, αPD-L2) correlated with increased TCSC and total TIL, but TCSC differed by age and treatment. PD-1 interacts with PD-L1 and PD-L2 while PD-L1 interacts with PD-1 and CD80. Aged mice expressed significantly more T-cell PD-1 and PD-L2 and up to 40-fold more PD-L2, PD-L1 and CD80 vs. young on myeloid cells and NK cells. We generated bone marrow-derived DC with GM-CSF. Aged DC expressed much high PD-L2 versus young, suggesting a cell-intrinsic PD-L2 age bias. Treatment differences in aged versus young could depend on these immune checkpoint or TCSC differences. We are now identifying mechanisms for increased PD-L2 and contributions to αPD-L2 efficacy in aged, and testing TCSC effects on treatments. Our work can improve cancer immunotherapy in aged hosts and provide models for treatment resistance in young hosts.
A phosphotyrosine switch controls antitumor activity of estrogen receptor b
Authors: Yuan Bin , Clark Curtis A , Yang Jing , Wu Bogang , Hu Yanfen , Curiel Tyler J , Li Rong
Published: January 2023

Recently, we identified a phosphotyrosine residue in human ERb (pY36), which is highly conserved in all mammalian ERb orthologs, but not present in ERa. In addition, this phosphotyrosine switch in ERb controls its tumor-intrinsic (tumor cells) activity. However, the weakness of the current literature concerns limited information about the ERb tumor-extrinsic antitumor activity. To investigate ERb tumor-extrinsic antitumor activity, we generated a whole-body knock-in (KI) mouse model in which the tyrosine residue Y55 of endogenous mouse ERb, which corresponds to Y36 of human ERb, is mutated to phenylalanine (Y55F). We found that B16 melanoma grew more robustly in KI recipient mice than in their WT counterparts, as well as MC38 and M-Wnt tumor models. These data demonstrate that the phosphotyrosine switch is important for ERb tumor-extrinsic antitumor activity in multiple tumor types and in both sexes of recipient mice. In the following mouse bone marrow chimera experiment, we found B16 melanoma growth was significantly faster in KI>WT chimeras versus male WT>WT control. This suggests that KI-derived immune cells poorly deterred tumor growth. Analysis with enriched subpopulations of immune cells from spleen, CD8 T cells have ERb protein expression. Adoptive transfer of CD8 T cells from WT donor but not KI donor significantly reduced tumor growth in Rag1 KO mice, a function attributed to reduced infiltration of KI CD8 T cells into tumors. Accordingly, increased infiltration of CD8 T cells was positively associated with ESR2 expression in various cancer types from TCGA datasets, including breast cancer and melanoma.
Adrenergic stress regulates the exhausted phenotype of T cells in the tumor microenvironment
Authors: Qiao Guanxi , Chen Minhui , Mohammadpour Hemn , Bucsek Mark , MacDonald Cameron , Hylander Bonnie , Repasky Elizabeth
Published: January 2023

We have shown previously that host adrenergic stress (model of standard ambient temperature-induced chronic stress) slows tumor progression by enhancing CD8+ T-cell activation and limiting the suppressive function of myeloid-derived suppressor cells in the tumor microenvironment (TME). We also found that reducing/blocking β-adrenergic receptor (β-AR) signaling significantly improves anti-tumor immune responses and efficacy of immune checkpoint immunotherapy. It has been reported that expression of immune checkpoint receptors (e.g. PD-1, TIM3, LAG3) is characteristic of exhausted T-cells and has been linked to failure of immune checkpoint inhibitors. Now we report that host adrenergic stress plays a previously unrecognized role in regulating T-cell exhaustion in the TME. In murine melanoma and colon cancer models, we found that propranolol (a pan β-AR blocker) significantly reduces the number of exhausted T-cells expressing immune checkpoint receptors in the TME and increases numbers of T-cells expressing effector cytokines. The overexpression of immune checkpoint receptors has also been associated with mitochondrial dysfunction. Our previous work shows that β-AR signaling during CD8+ T-cell activation in vitro impairs metabolic reprogramming. In new in vivo data, we observed that propranolol treatment increases both glycolysis and oxidative phosphorylation in tumor infiltrating CD8+ T-cells. Together these data suggest that β-AR signaling is a significant factor regulating the functional status of CD8+ T-cells in the TME. Blockade and/or reduction of adrenergic stress has the potential to improve anti-tumor immunity as well as the efficacy of immune checkpoint inhibitors.
Adjuvant combinations activate dendritic cells and contribute to antigen-specific CD4 T cell expansion
Authors: Bauer David Laurence , Kurtz Jonathan R , Grant Samuel B , Immethun Vicki E , McLachlan James B
Published: January 2023

Adjuvants are important mediators of immune responses and enhance the activation of innate immune cells, leading to protective T and B cell responses. Only a few vaccine adjuvants are currently approved for use in the US and, along with the discovery of novel adjuvants, combination adjuvants are likely to have a major impact on future vaccine development. Here, we investigated the ability of both the double mutant Escherichia coli heat labile toxin R192/L211A (dmLT) and the TLR4 agonist monophosphoryl lipid A (MPL-A) to drive innate and adaptative immune responses independently and in combination. We found that the combination adjuvant induces more robust activation of dendritic cells (DCs) than either adjuvant alone, leading to the formation of the NLRP3 inflammasome complex and increased secretion of IL-1β, as well as other hallmark pro-inflammatory cytokines. Furthermore, RNA sequencing and ingenuity pathway analysis support that the combination adjuvant may allow these DCs to polarize a multi-faceted Th1/2/17 CD4 T cell response. To determine how this adjuvant combination might regulate CD4 T cell responses, we intradermally immunized mice with a model T cell antigen in conjunction with dmLT or MPL-A alone or combined and, using MHC class II tetramers, found that vaccination with the combination demonstrated the greatest expansion and activation of endogenous, vaccine-specific CD4+ T cells in all lymphoid tissues assessed which included a Th1/Th17 response. These results reveal how combination adjuvants can be used to not only potentiate better vaccine responses, but to potentially bias the immune response to one that is more capable of combating infection.