bluuebunny/crossref_metadata_2025_split huggingface dataset:
Macronutrient composition regulation of nutrient sensing factors and neurotransmitters affects anti-tumor immunity and subsequently HCC development
Authors: Shalapour Shabnam , Ryujin Nicolas T , Huang Jian , Damania Ashish V , Ajami Nadim , Rosario Spencer , Long Mark 
Published: August 2023

Abstract
Cells alter their metabolic state in response to changes in nutrient availability through the induction of transcriptional programs that affect the levels of nutrient transporters and metabolic enzymes. Cell-intrinsic mechanisms of nutrient sensing are intimately linked to adaptive metabolic responses and play critical roles in shaping the complex and dynamic nutrient environment of a growing tumor. We aim to understand how food composition, in combination with alcohol consumption, impact host metabolism, and the development of steatohepatitis induced hepatocellular carcinoma (HCC). We further elucidated how macronutrient composition, dysbiosis, and integrated metabolic responses influence HCC development specifically through the axis of adaptive immune cell metabolic status. Here we present how diet induced chronic inflammation promotes a bidirectional relationship between the immune system and systemic metabolism, to further influence the effector function of immune cells, through a gut-liver-brain axis by induction of neurotransmitter like serotonin and GABA. This relationship between changes in whole body metabolism and immunometabolism drives immense changes in humoral and cellular immune response, and further HCC development. Technologies such as Met-Flow, Mass spectrometry, FC and scRNA/ATAC-seq were used for comprehensive analysis, in conjunction with long term diet intervention. In summary, we show how the regulation of nutrient sensing pathways by dietary intervention modulate immunoediting and support tumor development, providing knowledge to advance effective strategies for treatment and prevention
U01AA027681
Bromodomain and extraterminal (BET) domain proteins regulate immune checkpoints through AMPK-dependent and -independent pathways in peripheral T cell subsets
Authors: Denis Gerald V , Ennis Christina 
Published: August 2023

Abstract
Immune checkpoint blockade unleashes anti-tumor immunity of tumor-infiltrating CD8+ T cells. Yet, many cancer patients are refractory: unknown mechanisms resolve responders from non-responders. Analysis of transcriptional networks regulating immune checkpoint genes may provide insight. We used multi-parameter FACS to explore expression of several checkpoint inhibition molecules, focusing on functional roles of 5′-Adenosine Monophosphate-activated Protein Kinase (AMPK) and BET transcriptional co-regulators (BRD2, BRD3 and BRD4), both known to be key signaling pathways disturbed in obesity and cancer, resulting in exhaustion of effector CD8+ T cells and tumor progression. Here, human primary PBMCs from normal, healthy, adult donors were activated ex vivo with anti-CD3 and anti-CD28. We then measured expression of inhibitory receptors PD1, CTLA4, TIM3 and TIGIT on T cell subsets. Small molecule inhibitors of BET protein expression, including pan-BET inhibitor JQ1 and BRD4-selective PROTAC degrader MZ1, identified BET protein-regulated targets. We found that BRD4 regulates PD1, CTLA4 and TIM3 (but not TIGIT) in both CD4+ and CD8+ T cells. Treatment with Compound C, a modulator of AMPK, defined a unique PD1 and CTLA4 network. We confirmed that T cells with elevated expression of inhibitory receptors were impaired in cytokine secretion and proliferation. We conclude that separate signaling pathways likely converge on BRD2 and/or BRD3 and/or BRD4, acting independently as effectors at each promoter. Patterns of AMPK-regulated metabolism and BET gene expression are personalized and must be considered when evaluating immune checkpoint blockade in cancer therapy.
Supported by grants from the Cancer Systems Biology Consortium of the National Cancer Institute (U01CA182898,U01CA243004; PI Denis), NCI Cancer Moonshot (R01CA222170; PI Denis) and Boston University’s Immunology Training Program (T32AI007309-32; PI Kepler).
Endogenously high Omega-3 levels lead to a less suppressive tumor microenvironment
Authors: Tiwary Shweta , Xia Zheng , Olkhanud Purevdorj B , McKinnon Katherine , Berzofsky Jay A 
Published: August 2023

Abstract
Cellular transformation leading to cancer development involves multiple changes in the cellular pool of nutrients and macromolecules, including lipids. Tumors switch to the de novo lipid pathway and preferentially accumulate some lipids while excluding others, such as omega-3 (n-3) fatty acids and bioactive lipids derived from them, from the tumor microenvironment. These lipids, known as specialized pro-resolving lipids (SPM), may affect cancer development. Although their roles in decreased cancer risk and overall inflammation are well studied, their effects on anti-tumor immunity are still poorly understood. Here, we report the consequences of high n-3 levels on tumor growth and anti-tumor immunity. We have utilized a FAT-1 transgenic mouse model with an endogenously high n-3: n-6 ratio due to a transgenic enzyme making n-3 fatty acids and EO771 mammary tumor model. Our data show a decreased tumor growth rate in FAT-1 mice associated with increased tumor-infiltrating cells (TILs). In preliminary studies, we further discovered that CD8 and CD4 T-cells in TILs isolated from tumors growing in FAT-1 mice display a more active and proliferative phenotype. Also, the T-cells had decreased exhaustion markers such as TIM-3, LAG-3, and TIGIT in the TILs isolated from tumors growing in FAT-1 animals, and there were fewer FOXP-3 positive T-cells. Together our data suggest a less suppressive anti-tumor immune environment in FAT-1 animals. Currently, we are performing mechanistic studies to understand the role of high omega-3 in the expression and function of these exhaustion markers. We are also exploring the therapeutic benefit of checkpoint inhibitors and cancer vaccines in combination with high omega-3 fatty acids.
Single cell RNA sequencing analysis of tumor responses to triple drug oral immunotherapy in dogs with osteosarcoma
Authors: Ammons Dylan , Kurihara Jade , Weishaar Kristen , Gardner Heather , London Cheryl , Dow Steven 
Published: August 2023

Abstract
Osteosarcoma constitutes a rare childhood cancer that is associated with dismal outcomes. As such, there is a need to develop non-invasive immunotherapeutic approaches to treat this aggressive malignancy. To address this need, we described the cellular heterogeneity in spontaneously occurring canine osteosarcoma tumors then evaluated how treatment with adjuvant immunotherapy altered the tumor microenvironment. We investigated the effects of a triple drug immunotherapeutic (orally administered losartan, ladarixin, and toceranib) that was designed to deplete immune suppressive macrophages and promote antitumor immunity. The treated cohort of dogs were given the adjuvant immunotherapy for 14 days prior to amputation, while naïve samples were collected from unrelated dogs undergoing amputation to treat a primary osteosarcoma tumor. In total, we completed single cell RNA sequencing on 6 naïve dogs and 6 treated dogs to profile just under 100,000 cells. Unsupervised clustering revealed 30 unique cell populations with T cells, macrophages, and neutrophils making up the majority of immune infiltrates. Evaluation of changes to the cellular proportions following treatment revealed a marked reduction in macrophages, which suggests the combination immunotherapy was able to alter the tumor microenvironment. Overall, this study provides unique insights into the heterogeneity within canine osteosarcoma tumors and highlights the impacts of adjuvant immunotherapy on tumor infiltrating immune cell transcriptional programs.
T32 OD012201 U01 CA5341458 Shipley family foundation
Examining Transcriptomic Differences Underlying Mycosis Fungoides in Skin of Color and White Patients
Authors: Nagarkar Akanksha Dhananjay , James Nia E R , Nwade Nina , Desse Sachi , Yildiz-Altay Ummugulsum , Shokrani Babak , Deng April , Dresser Karen , Byrd Angel S , Okoye Ginette A , Richmond Jillian M 
Published: August 2023

Abstract
Mycosis Fungoides (MF), the most common type of cutaneous T-cell lymphoma, typically presents with an erythematous or dyspigmented rash that can progress to tumors and ulcerations on the skin. MF is more common in skin of color (SOC) patients, where it presents earlier and at a higher T-stage. This has led to poorer prognosis in SOC patients, with the poorest prognosis in African Americans. The pathogenesis regarding this racial disparity remains poorly understood. We compared MF skin samples from SOC and White patients to determine differentially expressed genes and elucidate transcriptomic differences. An immune panel of 343 genes were hybridized to 8 MF SOC and 8 MF White skin samples. The resulting gene signatures were analyzed to determine significant differentially expressed genes. Analysis revealed that SOC has significant downregulation of critical immune response genes involved in the recruitment of immune cells to sites of inflammation, including CCR1, CCR2, and CCR3. Genes involved in ‘chemokine signaling,’ ‘differentiation and maintenance of myeloid cells,’ and ‘cell migration and adhesion,’ were downregulated in SOC MF biopsies as compared to White skin MF biopsies. Furthermore, SOC MF biopsies have a greater abundance of mast cells, whereas White skin MF biopsies have a greater abundance of neutrophils. These transcriptomic differences suggest that MF may have a unique pathogenesis in SOC that involves significant downregulation of the host’s immune response. This potentially provides insight into the presentation of MF at a more advanced stage and aggressive course in SOC as compared to White skin. This insight may facilitate the development of immunotherapies targeted to SOC to address this racial disparity.
SD - recipient of the Dermatology Foundation DRSA; JMR - recipient of the Skin of Color Society Reseach Grant; ASB - recipient of the American Skin Association Milstein Research Scholar Award for Melanoma/Non-Melanoma Skin Cancer
Allograft TLR9 is required for endogenous donor-reactive memory CD8 T cell activation to mediate CTLA-4Ig resistant rejection of high-risk cardiac allografts
Authors: Koritzinsky Erik H , Ueda Daisuke , Tsuda Hidetoshi , Miyairi Satoshi , Fairchild Robert L 
Published: August 2023

Abstract
Organ cold ischemic storage (CIS) is inherent to deceased-donor solid organ transplantation and CIS time correlates with poorer transplant outcomes. Prolonged CIS enhances ischemia-reperfusion injury (IRI) following graft revascularization, generating an inflammatory environment that promotes endogenous donor-reactive memory T cell activation leading to CTLA-4Ig costimulatory blockade-resistant rejection. Using a murine heterotopic heart transplant model, we tested the impact of prolonged (8hr) versus minimal (0.5hr) CIS on cell-free mitochondrial DNA (cf-mtDNA) release and activation of the cf-mtDNA sensor TLR9 to understand their contributions to early post-transplant donor-reactive endogenous memory T cell activation and CTLA-4Ig resistant rejection. Plasma cf-mtDNA was significantly elevated in 8hr versus 0.5hr CIS allograft recipients as early as 12hr post-transplant. Flow cytometry analysis of graft infiltrating leukocytes showed reduced memory CD4 and CD8 T cell proliferation on D2 following 8hr CIS in TLR9KO compared to WT allografts. In contrast, graft TLR9KO did not impact the de novo donor-reactive T cell response. 8hr CIS TLR9KO and WT allografts had similar survival in untreated recipients (MST 7.5 vs 7 days) and there was no difference in IFN-γ producing donor-reactive T cells in recipient spleens on D7. When recipients of allografts subjected to 8hr CIS were conditioned with CTLA-4Ig to inhibit de novo T cell priming, graft TLR9KO significantly prolonged graft survival compared to WT (MST 59 vs 28 days). These findings indicate that IRI-induced cf-mtDNA release and TLR9 activation are novel targets for abrogating CTLA-4Ig resistant donor-reactive memory T cell activation within high-risk allografts.
This work was supported by NIH grants R01AI40459, T32AI089474, and T32GM007250.
Purine metabolic pathway regulates regulatory T cell stability and function
Authors: Lee Young S , Saxena Vikas , Piao Wenji , Li Lushen , Wu Long , WillsonShirkey Marina , Kensiski Allison , Gavzy Sam , Ma Bing , Bromberg Jonathan S 
Published: August 2023

Abstract
Regulatory T cell (Treg) lymphatic migration and maintenance of transcription factor Foxp3 expression are required for suppressor function and allograft protection, and migrated Tregs express ectonucleotidase CD39hi. Purine metabolism is implicated in Treg phenotype but the precise functions of purine metabolites on Treg function and stability have remained unclear.
Sorted Foxp3+ Tregs were used to test Treg stability, migration, and cellular viability in a transendothelial migration (TEM) in vitro model. Intracellular metabolome of Tregs was analyzed by mass spectrometry, and differentially expressed metabolites between Tregs and exTregs were identified and tested.
Foxp3hi Tregs displayed higher suppressive function and migration across LECs compared to Foxp3lo exTregs, but cellular viability was similar. Tregs showed differential metabolic profiles compared to exTregs, and the top 16 most differentially expressed metabolites involved the tricarboxylic acid cycle, polyamines, and purine metabolism. Ten metabolites including nicotinamide and inosine monophosphate (IMP) were downregulated, while six metabolites including putrescine, cadaverine, and N-acetylglycine were upregulated in Tregs compared to exTregs. A purine ectonucleotidase CD73 inhibitor decreased Treg conversion to exTregs and adenosine increased Treg migration and suppressive function concomitant with decreased exTreg conversion. Polyamine metabolites showed only subtle effects on exTreg conversion and migration.
These results suggest that purine metabolism is a potent regulator that maintains Treg Foxp3 expression and suppressive function during Treg TEM and thus, could be a promising target for therapeutic interventions.
NIH grant RO1 A1062765
PKN1 is a novel therapeutic target to prevent graft-vs-host disease after allogeneic hematopoietic cell transplantation
Authors: Choi Jaebok , Kim Sena , Lim Sora , Razmkhah Farnaz 
Published: August 2023

Abstract
We were the first to demonstrate that targeting JAK1/2 using baricitinib (BARI) and ruxolitinib (RUX) prevents and treats GVHD while enhancing multi-lineage hematopoietic reconstitution and graft-vs-leukemia (GvL) effect in mouse models of allogeneic hematopoietic cell transplantation (allo-HCT). However, unlike BARI, RUX fails to completely prevent or treat GvHD. To elucidate the mechanism underlying the superiority of BARI to RUX, we performed both mouse and human kinome analyses. We found that BARI, but not RUX, is a potent inhibitor of both murine and human PKN1, suggesting that the superiority of BARI to RUX in preventing and treating GvHD may result from the off-target effects of BARI on PKN1 in addition to its known inhibitory effects on JAK1/2. While the function of PKN1 in GvHD remains unexplored, PKN1 has been suggested as a protein kinase in signaling pathways involving S1PR1/ PPP1R14A, LFA-1/STK4 and LFA-1/RPH3A for immune cell adhesion/trafficking and the balance of regulatory T cells and effector T cells.
To define the role of PKN1 in GvHD, we examined if OTS167, a PKN1 inhibitor, alters the downstream signaling of these candidate pathways. We found that OTS167 significantly reduces the phosphorylation of PPP1R14A and STK4 in murine T cells in a dose-dependent manner. Of note, RPH3A or pRPH3A is not detectable in T cells. In addition, we found that in vivo administration of OTS167 reduces GvHD in a mouse model of allo-HCT (p&lt;0.05). Furthermore, we found that allo-HCT recipient mice transplanted with S1PR1 KO T cells demonstrate significantly improved overall survival (p&lt;0.05). These data suggest that PKN1 and S1PR1/PPP1R14A (and also LFA-1/STK4 which is currently under investigation) are critical components in inducing GvHD.
This research was supported by the Alvin J. Siteman Cancer Center through The Foundation for Barnes-Jewish Hospital.
Mesenchymal stem cell therapy to promote functional recovery in a limb transplant model
Authors: Vu Michael M , Bingham Jason R , Gorantla Vijay S , Salgar Shashikumar K 
Published: August 2023

Abstract

Introduction
Battlefield trauma leads to limb amputations. We investigated whether Mesenchymal Stem Cell (MSC) therapy can improve nerve regeneration and functional outcome in limb transplantation (Tx).

Methods
We performed orthotopic syngeneic hind-limb Tx in Lewis rats. Following Tx rats received syngeneic MSC (2×106; passage ≤6) or vehicle i.v. and locally via hydrogel. Limb sensory function (SF) and motor function (MF) were assessed weekly and at study endpoint (16 weeks) limb tissues were harvested for cell/molecular analysis.

Results
SF in the tibial, peroneal/sural and saphenous nerve boundaries recovered as early as 5, 6 and 8 weeks, respectively. Total limb SF did not vary (P&gt;0.05) between MSC and control groups; recovery was gradual at week 8 (Grade 1.4), week 12 (Grade 2.3) and week 16 (2.6) on a scale of Grade 0–3 (0, no function; 3, optimal function). A novel grading system was used to assess MF based on walking track footprints: Grade 0–4 (0, no footprint; 4, heel and 4–5 toe prints). MF was significantly (P&lt;0.05) improved with MSC compared to controls at week 8 (Grade 1.33 Vs 0.5) and at week 16 (Grade 2.66 Vs 0.6). Ex vivo expanded rat MSCs were CD29+, CD31−, CD34−, CD44+, CD45low, CD90+, MHC Class I+, Class II− and pluripotent. Neurofilament M (axonal marker), S100 and Krox20 (myelinating Schwann cells), Oct6 (Pre-myelinating Schwann cells) and GAP43 (regeneration marker) expressions were increased (P&lt;0.05) and GFAP (non-myelinating Schwann cells) were reduced (P&lt;0.05) in the distal portion of the sciatic nerve in MSC treated rats compared to controls.

Conclusion
MSC administration following limb Tx appears to promote limb function recovery and is attributed to enhanced nerve regeneration/myelination/innervation.

DoD –CDMRPL-19-0-DM190329 grant (PI, Salgar)
Pro-tolerogenic gut microbiota induce rapid and distinct gut metabolic changes after cardiac allografting
Authors: Gavzy Samuel J , Kensiski Allison , Lakhan Ram , Iyyathurai Jegan , Lee Zachariah L , Wu Long , Li Lushen , Saxena Vikas , Mongodin Emmanuel , Ma Bing , Bromberg Jonathan S 
Published: August 2023

Abstract
Desulfovibrio desulfuricans (Desulfo) induces pro-inflammatory effects, while Bifidobacterium pseudolongum (Bifido) induces pro-tolerant effects. Bifido results in decreased cardiac allograft inflammation and fibrosis compared to Desulfo. Lymph node (LN) laminins α4 and α5 are differentially regulated in transplant tolerance (α4&gt;α5) and immunity (α4&lt;α5). We investigated whether specific microbiota alter LN architecture, immune cell content, and gut metabolites.
B6 mice received antibiotics followed by bacteria gavage (Bifido, Desulfo, or PBS control), BALB/c heart transplants, and daily tacrolimus. LNs were assessed by immunohistochemistry. Stool metabolome profiling used capillary electrophoresis-mass spectrometry with partial least square-discriminant analyses.
Desulfo decreased LN laminin α4:α5 ratios and Foxp3+ regulatory T cells (Tregs) after 7 days. After 14 days, Desulfo treated animals had greater allograft fibrosis while Bifido increased LN laminin α4:α5 ratios and Tregs. Gut metabolomes demonstrated highly distinct clustering between groups after 7 days. Bifido treatment enriched compounds related to polyamine metabolism (spermine, spermidine, and putrescine), as well as by pathway analysis (arginine and proline plus spermidine and spermine metabolism) compared to Desulfo and control.
Overall, Desulfo induced rapid pro-inflammatory changes (decreased LN laminin ratios and Tregs, increased graft fibrosis), while Bifido induced rapid pro-tolerant changes (increased LN laminin ratios and Tregs). Bifido modulated distinct polyamine pathway metabolites, compounds critical for T cell reprogramming. These results suggest polyamines as mediators of Bifido pro-tolerogenic effects.
This work is supported by grants from NIH (R01HL148672, U01AI170050, and T32AI95190-10).
Immunosuppressants regulate immunity by altering lymph node structure
Authors: Wu Long , Gavzy Samuel J , Kensiski Allison , Lakhan Ram , Li Lushen , Iyyathurai Jegan , Lee Zachariah L , Bromberg Jonathan S 
Published: August 2023

Abstract
Lymph node (LN) stromal cell-derived laminins α4 and α5 are differentially regulated in tolerance (α4&gt;α5) and immunity (α4&lt;α5). We previously showed that depleting fibroblastic reticular cell (FRC)-derived laminin α4 reduced Foxp3+ regulatory T cells (Tregs) while depleting FRC-laminin α5 increased Tregs. We hypothesized that clinically available immunosuppressants regulate immunity by altering LN structure.
B6 mice received tacrolimus, rapamycin, prednisolone, mycophenolate mofetil (MMF), or FTY720 for 3, 7, and 30 days. Peripheral LNs (pLN) and mesenteric LNs (mLN) were analyzed by immunohistochemistry. Intestinal permeability (IP) was analyzed by FITC-dextran gavage.
Both tacrolimus and rapamycin resulted in early, transient, mixed pro and anti-inflammatory effects on LN laminin α4:α5 ratios, but resulted in pro-tolerant LN Treg increases. Prednisolone had a pro-tolerant effect by increasing LN Tregs. MMF showed early, transient, pro-tolerant effects by increasing LN Tregs and laminin ratios. FTY720 resulted in early, transient, pro-inflammatory changes and increased IP after 7 days, but resulted in a pro-tolerant increase in LN Tregs after 30 days.
Tacrolimus and rapamycin induced early, pro-inflammatory changes, yet both exerted later, pro-tolerant effects. Prednisolone displayed consistent pro-tolerant effects across all time points. MMF showed transient, pro-tolerant effects after 3 days, which disappeared by day 7. FTY720 resulted in early, transient, pro-inflammatory changes, later becoming pro-tolerant. Overall, immunosuppressants affect LN stromal laminin remodeling and Treg distribution. These results suggest a novel approach for evaluating the global effects of immunosuppressants in vivo.
This study was supported by grants from the NIH (R01AI114496 and T32AI95190-10).