bluuebunny/crossref_metadata_2025 huggingface dataset:
Low avidity CD8+ T cells are less exhausted than their high avidity counterparts and provide superior anti-tumor immunity
Authors: Singhaviranon Summit , Dempsey Joseph , Hagymasi Adam , Mandoiu Ion , Srivastava Pramod K
Published: August 2023

The interaction of T cell receptors (TCRs) on T cells with MHC I-peptide complexes on cancer cells elicits the cytotoxic activity of CD8+ T cells. Here we analyze how the strength of this interaction, or T cell avidity, shapes the exhaustion of CD8+ TILs and dictates anti-tumor immunity. We developed a novel tetramer decay assay to isolate T cells based on their TCR avidities. We used this method to study low and high avidity CD8+ TILs responding to the tumor neoantigen PDPRMUT of the murine sarcoma Meth A. Single cell sequencing reveals that high avidity CD8+ TILs are more terminally exhausted (51.41% vs 27.21%) and less effector-like (28.39% vs. 60.0%) than low avidity CD8+ TILs. We used flow cytometry to analyze PDPRMUT-specific CD8+ TILs with low or high avidity from 28 day-old Meth A tumors. There were significantly more TIM3+PD-1+ cells (Paired t-test; P < 0.0001) and TOX+PD-1+ cells (Paired t-test; P < 0.05) in the high avidity CD8+ TILs than the low avidity CD8+ TILs. We adoptively transferred 1,000 PDPRMUT-specific low or high avidity CD8+ T cells, or control CD8+ T cells into tumor-bearing mice 10 days after tumor challenge. Low avidity T cells significantly improved the survival of mice (Mantel-Cox; P = 0.0044), while high avidity t cells did not do so (Mantel-Cox; P = 0.7081). These results establish a novel correlation between avidity, exhaustion and T cell-mediated tumor control in vivo.
Temporal requirements for TGF-β and IL-7 in generating resident memory T cell responses to melanoma
Authors: Ramirez Delaney E , Hawkes Aaron R , Searles Tyler G , Wang Chen-Yu , Nemeth Alexander N , Pioli Patricia A , Turk Mary Jo
Published: August 2023

Resident memory CD8+ (TRM) cells persist in tissues and are critical mediators of the host anti-tumor response. We previously demonstrated that protective, melanoma/melanocyte antigen-specific CD8+ TRM cells are generated in skin and B16 tumor-draining lymph nodes (LNs) of mice following CD4+ T cell depletion and tumor excision surgery. Whereas TGF-β and IL-7 have previously been shown to play important roles in generating viral Ag-specific TRM cells, a role for these cytokines in generating TRM responses against tumor antigens had not been studied. We employed antibody-mediated blockade of TGF-β (mAb clone 1D11.16.8) or IL-7 receptor (IL-7R; mAb clone A7R34) during primary tumor growth or after tumor excision to define a temporal role for these cytokines in generating the TRM response. Sentinel gp100-specific pmel CD8+ T cells were used to monitor priming and memory generation. TGF-β blockade during tumor growth did not impair primary responses but significantly reduced CD103+ CD69+ TRM formation in skin and draining LNs one month later. TGF-β signaling was only required during priming, as delayed blockade after tumor excision had no effect. Conversely, IL-7R blockade during priming did not impair TRM generation in skin or LN. However, its blockade after tumor excision significantly impaired TRM formation in both locations. These studies demonstrate temporally distinct requirements for TGF-β and IL-7 in promoting TRM responses against tumor antigens.

Supported by grants from the NIH (R01CA22502, T32-AI007363), The Knights of the York Cross of Honour and O. Ross McIntyre, M.D. Endowment to MJT
PD-1 in balancing expansion of effector T cells and attrition of memory T cell during tumor-reactive T cell responses
Authors: Mao Zhiming , Hirdler Jacob , Harrington Susan , Liu Xin , Gicobi Joanina , Dellacecca Emilia , Hsu Michelle A , Zhang Wenjing , Park Sean , Dong Haidong
Published: August 2023

Though anti-PD-1/PD-L1 immune checkpoint blockade therapy has demonstrated robust anti-tumor immunity in human patients with advanced cancers such as melanoma, only a fraction of patients achieved such durable anti-tumor responses. Among all the non-responders, some patients responded initially but developed resistance to the therapy in the later treatments. This reflects an urgent need to understand the role of PD-1 signals in CD8+ T cells at priming and at effector stages during tumor development for the benefit of immunotherapy. The controversy of rapid tumor progression in PD-1 conditional knockout in regulatory T cells and tumor regression in PD-1 targeted deletion in regulatory T cells indicated germline PD-1 knockout mice might not be sophisticated enough to study the role of PD-1 in CD8+ T cells during tumor development. Thus, we produced a CD8 T cell-specific PD-1 knockout mouse model (CD8 E8ICre-PD-1fl/fl, also called CD8-PD-1 cKO mice) in which PD-1 is deleted in CD8+ T cells in the peripheral tissues. When CD8-PD-1 cKO mice were challenged with B16-OVA mouse melanoma cells, we found a delayed tumor growth and prolonged survival of CD8-PD-1 cKO compared to control mice. However, after 20 days of tumor challenge, the delayed tumor growth rebound in CD8-PD-1 cKO mice. In an OVA antigen immunization model, we found increased effector cells and tissue resident cells in the spleen of CD8-PD-1 cKO mice compared with control mice on day 7 after immunization. Taken together, our results suggest that PD-1 may restrain the expansion of effector T cells that contribute to the early phase of tumor control, but the augmented effector T cell response may cause an attrition of memory T cell pool that will be needed for long term protection.

R01CA256927
Thymoquinone treatment enhances T cell-mediated anti-tumor response
Authors: Pandey Rachana , Sirbu Cristian , Hankins Gerald , Fandy Tamer , Du Wei
Published: August 2023

Oil from Nigella sativa seeds (“black seed oil”) has been used for health care and medical purposes for over 2000 years. The major active compound thymoquinone (TQ) has been shown to have immune regulatory effects in various diseases. Tumor microenvironment is critical for tumor development. Immune cells, particularly T cells, are central players to kill tumor cells and affect tumor progression and metastasis. The impact of TQ on human T cell-mediated anti-tumor response was investigated. Using human PBMCs, we activated T cells with anti-CD3/CD28 in vitro, and treated cells with TQ. First, we tested the impact of TQ on T cell activation, proliferation and apoptosis; second, the anti-tumor effect of T cells after treatment with TQ; last, we determined alterations in T cells gene expression at the RNA level after TQ treatment. TQ treatment increased activation and proliferation of T cells at days 2–4 and more activation-induced cell death at day 3. After 7–11 days, TQ-treated cells showed less survival than cells treated with control medium. At low TQ concentration (10uM), treatment induced more CD3+ T cell proliferation; while at 20uM and 50uM TQ showed more cytotoxic effect to T cells, and more cell death. When co-cultured with multiple types of cancer cells, TQ-treated T cells exhibited significantly enhanced anti-tumor activity, indicated by more tumor cell apoptosis and less cell expansion. Gene expression study showed upregulation of NFAT signaling pathway, including 10+ fold up-regulation of IL-2, which might explain the enhanced anti-tumor effect after TQ treatment. In summary, TQ treatment can enhance human T cell-mediated anti-tumor effect in vitro. This immunoregulatory function could benefit patients with different cancers.

Supported by grants from NIH/ NIGMS (2U54GM104942-02, 5U54GM104942-03).
Loss of OGT enhances type I interferon-dependent antitumor immunity
Authors: Wen Haitao , Chen Jianwen
Published: August 2023

The O-GlcNAc transferase (OGT) is an essential enzyme that mediates protein O-GlcNAcylation, a unique form of posttranslational modification of many nuclear and cytosolic proteins. Recent studies observed increased OGT and O-GlcNAcylation level in a broad range of human cancer tissues compared to adjacent normal tissues, indicating a universal effect of OGT in promoting tumorigenesis. Here, we show that OGT is essential for tumor growth in immunocompetent hosts via repressing cyclic GMP-AMP synthase (cGAS)-dependent DNA sensing pathway. We found that deletion of OGT (Ogt−/−) caused a markedly reduction in tumor growth in both syngeneic tumor models and genetic colorectal cancer (CRC) model induced by mutation of the Apc gene (Apcmin). Pharmacological inhibition or genetic deletion of OGT induced a robust genomic instability (GIN), leading to cGAS-dependent production of type I interferon (IFN-I) and IFN-stimulated genes (ISGs). As a result, deletion of Cgas from Ogt−/− MC38 CRC cells restored tumor growth, which correlated with impaired CD8+ T cell antitumor immunity. Mechanistically, we found that OGT-dependent cleavage of host cell factor C1 (HCF-1) is required for the avoidance of GIN and IFN-I production. In sum, our results identify OGT-mediated genomic stability as an important tumor cell-intrinsic mechanism to repress antitumor immunity.

Supported by the National Institutes of Health R01GM120496 (H.W.)
Enforced expression of c-Myc and T58A-mutant c-Myc in adult thymic epithelial cell subsets
Authors: Swanbery Nathan J
Published: August 2023

Thymic epithelial cells (TEC) are essential for T lymphopoiesis. Cortical (c)TEC support T lineage commitment and positive selection, whereas medullary (m)TEC delete self-reactive T cells to enforce central tolerance. Both cTEC and mTEC contain several distinct subtypes that were only recently characterized, and precursor-progeny relationships among adult TEC remains incompletely understood. Prior work in our lab demonstrated that enforced expression of c-Myc broadly in TEC under a constitutive FoxN1-Cre increased thymus size and TEC numbers, while maintaining morphology and function. To elucidate which TEC subtypes require enforced c-Myc expression to drive thymic expansion in adults, we’ve developed and acquired Cre-ERT2 systems under the control of TEC-specific genes FoxN1, Aire, and Ccl21a. This allows us to target expression of c-Myc or stabilized T58A-mutant c-Myc to specific subsets of TEC in adult mice. Analysis of these systems is currently ongoing and results will be presented. We hypothesize that enforced expression of c-Myc or stabilized T58A-mutant c-Myc in specific adult TEC subsets will expand the targeted population and may eventually result in transformation. Regardless of whether our hypothesis is correct, our experiments will contribute to better understanding of c-Myc-driven thymic hyperplasia and may provide a mouse model for human thymoma.
CEACAM marks intratumoral Tregs that exhibited highly suppressive and activated phenotypes
Authors: Chung Youseung , Jeon Seung Hyuck , Jeon Minwoo , Kim A Reum , Lee Yong Joon , Kim Eui-soon , Shin Eui-Cheol , Park Su-Hyung
Published: August 2023

Regulatory T cells (Tregs) exert immunosuppressive functions and hamper anti-tumor immune responses in the tumor microenvironment. Understanding the heterogeneity of intratumoral Tregs, and how it changes with tumor progression, will provide clues regarding novel target molecules of Treg-directed therapies. Here we characterized the phenotypes and suppressive function of Tregs from tumor tissues of 42 patients with renal cell carcinoma using multicolor flow cytometry, RNA sequencing, and in vitro functional assay. We found that carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) was selectively expressed on intratumoral Tregs, while its expression on peripheral Tregs or other immune cells was minimal. CEACAM1+ intratumoral Tregs accumulated with tumor progression, while the CEACAM1− subset did not. Notably, we found that CEACAM1 marked intratumoral Tregs that exhibited highly suppressive and activated phenotypes with substantial clonal expansion. Depletion of CEACAM1-expressing cells from tumor-infiltrating leukocytes led to increased effector functions of exhausted T cells. Moreover, CEACAM1+ cell depletion further enhanced anti-PD-1-–mediated reinvigoration of exhausted CD8+ T cells. Collectively, our results demonstrate that CEACAM1 delineates highly suppressive subset of intratumoral Tregs, and can be a target for selective depletion of intratumoral Tregs.
Biodegradable Nanoparticles Immunotherapy for the Treatment of Carcinogen-Induced Bladder Cancer
Authors: Podojil Joseph R , Cogswell Andrew C , Chiang Ming-Yi , Neef Tobias , Yu Yanni , Murthy Tushar , Boyne Michael T , Elhofy Adam , Meeks Joshua J , Miller Stephen D
Published: August 2023

Patients with locally advanced and metastatic urothelial carcinoma have a low survival rate (median 15.7 months, 13.1–17.8), with a 23% response rate to monotherapy treatment with anti-PD-L1 immunotherapy. We recently reported the potent effects of i.v. infusions of immune modifying poly(lactic-co-glycolic acid) (PLGA) nanoparticles (IMPs; named ONP-302) to both decrease B16.F10, MC-38, LLC, and 4T1 tumor growth and allow for B16.F10 tumor responsiveness to anti-PD-1 treatment. We next sought to determine if ONP-302 treatment could significantly decrease the more clinically relevant BBN-carcinogen induced bladder cancer. The present data show that ONP-302 treatment significantly decreases bladder cancer stage and overall bladder weight. While minor epithelial hyperplasia remained, ONP-302 treatment inhibited widespread carcinoma in situ and muscle invasive tumor development, as compared to saline treated mice. Mechanistically, ONP-302 infusion decreased tumor growth via the activation of the cGAS/STING pathway within myeloid cells, and subsequently increased CD8+ T cell and NK cell activation via IL-15. In subsequent studies to assess the cellular mechanisms involved in ONP-302 function, we found that in vitro treatment with ONP-302 rapidly induced oxidized DNA within the cells that took up PLGA nanoparticles and oxidized DNA was present within the supernatant as well. Furthermore, co-treatment of ONP-302 with DNase inhibited the STING mediated cytokine production. These findings indicate that ONP-302 allows for tumor control via reprogramming myeloid cells and inducing a STING pathway activation, increases anti-PD-1 response rates, and significantly decreases BBN-carcinogen induced bladder cancer.
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

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

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

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

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

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

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

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

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<0.05). Furthermore, we found that allo-HCT recipient mice transplanted with S1PR1 KO T cells demonstrate significantly improved overall survival (p<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

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>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<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<0.05) and GFAP (non-myelinating Schwann cells) were reduced (P<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

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>α5) and immunity (α4<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

Lymph node (LN) stromal cell-derived laminins α4 and α5 are differentially regulated in tolerance (α4>α5) and immunity (α4<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).
Monocytic myeloid-derived suppressor cells mediate the resolution of lung ischemia-reperfusion injury via MerTK-dependent efferocytosis
Authors: Leroy Victoria , Kollareth Denny Joseph Manual , Tu Zhenxiao , Atkinson Carl , Upchurch Gilbert R , Sharma Ashish K
Published: August 2023

Ischemia-reperfusion injury (IRI) after lung transplantation results in primary graft dysfunction leading to acute and chronic graft rejection. We investigated the role of monocytic myeloid-derived suppressor cells (M-MDSCs) to mediate inflammation-resolution of lung IRI. A murine model of lung hilar ligation was used in Balb/c (WT) and cebpβ−/− (M-MDSC deficient) mice. Lung function, inflammation and M-MDSC infiltration were assessed after 6- or 24hrs in mice undergoing sham control or IRI surgeries. CD11b+Ly6G−Ly6C+iNOS+ M-MDSC lung infiltration in WT mice was significantly increased after 24hrs of IRI compared to 6hrs of IRI (9.9×103±1.2×103 vs. 6.5×103±0.4×103; p=0.02; n=8/group), The upregulation of M-MDSC infiltration at 24hrs was accompanied by increased pulmonary compliance (5.3±0.2 vs. 2.9±0.1 μl/cm H2O; p<0.0001; n=12/group), decreased neutrophil infiltration as well as decreased pro-inflammatory IL-17, TNF-α, and CXCL1 secretion compared to 6hrs of lung IRI. The resolution of lung IRI at 24hrs was blocked in cebpβ−/− mice compared to Balb/c mice as demonstrated by decreased pulmonary compliance (3.1±0.1 vs. 5.3±0.2 μl/cm H2O; p<0.0001; n=7–12/group), that was accompanied by a decrease in M-MDSC lung infiltration (3.3×103±0.20×103 vs. 9.9×103±1.2×103; p=0.003; n=5–8/group). In vitro analysis using a hypoxia-reoxygenation model of lung IRI displayed an increase in efferocytosis as shown by M-MDSC-dependent uptake of apoptotic neutrophils as well as alveolar epithelial cells via a MerTK-dependent mechanism, Collectively, our results indicate that lung IRI entails dysregulated inflammation-resolution which can be mitigated by M-MDSC-mediated MerTK-dependent efferocytosis.
Assessment of donor compatibility in human solid organ transplantation and evaluation of allogeneic responses using a novel humanized mouse model
Authors: Ajith Ashwin , Musa Abu , Horuzsko Daniel David , Horuzsko Anatolij
Published: August 2023

The end result of solid organ transplantation is dictated by selection of a well-matched donor, leading to allograft survival. An allogeneic immune response is the main immunological barrier for successful organ transplantation. Donor and recipient human leukocyte antigen (HLA) mismatching results in poor graft acceptance, inflammation and rejection. The current evaluations for donor-recipient HLA incompatibility does not provide adequate information on the immunogenicity of individual HLA mismatches and impact of non-HLA-related alloantigens, especially in vivo. Here we demonstrate a novel method for analysis of alloimmune responsiveness between donor and recipient in vivo by introducing a humanized mouse model. Using molecular, cellular, and genomic analyses, we demonstrated that a recipient’s personalized humanized mouse provided the most sensitive assessment of allogeneic responsiveness to potential donors. In our study, HLA typing and mixed lymphocyte reaction (MLR) for assessment of allogeneic response was indistinguishable between 2 related donors of the recipient. Contrastingly in recipient’s humanized mouse model, the donor selected by HLA typing induced the strongest allogeneic response with increased infiltration of cytotoxic GzmB+CD8+ T cells. Moreover, the same donor induced upregulation of genes involved in the allograft rejection identified by transcriptomic analysis of graft infiltrating CD45+ cells. Humanized mouse model determined the lowest degree of recipient-donor alloimmune response, allowing for better selection of donor and minimized immunological risk of allograft rejection in transplantation. This approach can be used to evaluate alloresponses in cell based therapies as well.

This study was supported by research funding from the Carlos and Marguerite Mason Trust, and by U.S. National Institutes of Health, National Cancer Institute Grant CA 172230
Engineering islet surface with a novel form of thrombomodulin protein to mitigate instant blood-mediated inflammatory reaction following intraportal transplantation
Authors: Turan Ali , Zhang Lei , Tarique Mohammad , Ulker Vahap , Arguc Feyza , Yolcu Esma , Shirwan Haval
Published: August 2023

Intraportal infusion of islets initiates an innate immune response termed instant blood-mediated inflammatory reaction (IBMIR) that is responsible for significant islet mass peritransplant. We herein used thrombomodulin (TM) as a multifaceted innate immune regulator to mitigate IBMIR to prevent the destruction of islets in a minimal mass syngeneic intraportal transplantation model.

A novel form of TM molecule chimeric with streptavidin (SA-TM) was produced in insect cells and characterized for structure and attachment to biotinylated cell surface taking advantage of the high affinity interaction of biotin with streptavidin (SA). Islets were engineered with SA-TM and assessed for the ability to mitigate IBMIR in an in vitro blood loop assay and survival following intraportal transplantation (200 IEQ/mouse) in STZ diabetic mice (n=7). SA-engineered islets served as control (n=6). Animals were monitored for islet engraftment and subjected to various mechanistic studies.

SA-TM prevented phagocytosis of xenogeneic cells by macrophages and inhibited neutrophil NETs formation. Engineering islets with SA-TM did not impact the viability and function of islets and prevented damage in a blood loop assay. SA-TM-engineered islets showed enhanced engraftment compared to SA-engineered islets (83% vs 29%) following transplantation. Enhanced engraftment was associated with decreased frequency of intragraft myeloid cells and expression of various inflammatory mediators, such as HMGB-1, tissue factor, and IL-1β.

Engineering islets with the SA-TM protein provides a facile and clinically applicable approach to modulate innate immune responses for prevention of IBMIR with potential applications to allo/auto islet transplantation.

Funded in parts by NIH U01AI132817 and R01AI121281 grants.
CD154:CD11b blockade increases MPEC differentiation of virus-specific CD8+ T cells
Authors: Alexander Katie , Liu Danya , Ford Mandy L
Published: August 2023

CD154 pathway blockade has been shown to significantly improve graft survival in transplantation. Recently, CD11b was identified as an alternate receptor for CD154. CD154:CD11b interactions promote rejection by increasing the recruitment of innate and adaptive immune cells into the allograft. However, the effects of CD154:CD11b blockade on the protective immune response to infection during immunosuppression has not been elucidated. To address this, MHV68, a murine homolog of EBV, was used to assess the antigen-specific CD8+ T cell response. Naïve B6 mice were infected with MHV68 and treated on days 0, 2, 4, and 6 with a peptide inhibitor of CD154:CD11b binding (cM7). cM7 treatment resulted in an increase in tetramer+ antigen-specific CD8+ T cells 10 days post-infection that exhibited an increase in gene expression associated with activation and antigen processing. To determine if this increase was a result of enhanced proliferation, CTV-labeled OT-I T cells were stimulated in vitro in the presence or absence of CD154:CD11b blockade. Results indicated that CD154:CD11b blockade did not impact proliferation. Instead, cM7 treatment in vitro resulted in an increase in the expression of CD69, CD25, and CD44, and in vivo resulted in an increase in CD127hiKLRG1lo MPECs. In conclusion, these data demonstrate that CD154:CD11b blockade enhances the quantity and quality of virus-specific CD8+ T cells during protective immunity. We speculate that this could be due to the inhibition of terminal differentiation of antigen-specific T cells resulting in enhanced differentiation of high quality memory precursors, or could be due to an altered balance of CD154:CD40 vs CD154:CD11b binding, resulting in an increase in CD40-driven costimulation.

Supported by grants from NIH (R01 AI152516)
Oral administration of the commensal Alistipes onderdonkii prolongs allograft survival
Authors: Kwan Montserrat , Li Zhipeng , Rasic Mladen , Sepulveda Martin , McIntosh Christine , Shastry Vivaswat , Chen Luqiu , Finn Patricia , Perkins David , Alegre Maria-Luisa
Published: August 2023

Intestinal commensals can exert immunomodulatory effects on the host, with beneficial or detrimental consequences depending on underlying diseases. We have previously identified a specific commensal, Alistipes onderdonkii, as mediator of prolonged allograft survival and reduced alloimmunity. Here, we investigated its sufficiency and mechanism of action and found that oral administration of a beneficial A. onderdonkii strain DSM19147 but not a neutral strain, DSM108265, was sufficient to delay minor mismatched skin graft rejection in a TNF-dependent manner. Specifically, DSM19147 decreased TNF production in T cells and antigen-presenting cells, and TNF neutralization prolonged graft survival. Metagenomics and metabolomics analyses between DSM19147 versus strains that did not prolong graft survival identified unique genes and metabolites correlated with the anti-inflammatory effect of DSM19147. Candidate metabolic pathways unique to DSM19147 pointed to sialic acid and KDO2-lipid A modifications. Our findings identify A. onderdonkii DSM19147 as an anti-inflammatory probiotic that improves transplant survival.

Supported by NIAID grant 2R01AI115716 to MLA, NIAID grant 1U01AI132898 to MLD and DP, NIDDK grant TL1DK132769 to MR, NIAID grant 5T32AI007090 to MS and MK, and by the Growth, Development, and Disabilities Training Program grant (T32 HD007009) to CM.
Tregs are important to maintain CD4+ Tconv hypofunction in allograft tolerance
Authors: Cassano Alexandra , Mora-Cartin Ricardo , Wang Peter , Wang Ying , McIntosh Christine , Chong Anita S F , Alegre Maria-Luisa
Published: August 2023

Regulatory T cells (Tregs) are necessary for the induction of murine cardiac transplantation tolerance via costimulation blockade. In this model, peri-transplant Treg depletion prevents induction of graft-reactive Tconv hypofunction. Whether Tregs are necessary to maintain Tconv dysfunction long-term after tolerance has been achieved remains to be seen. Our preliminary data indicate that complete depletion of Tregs using diphtheria toxin (DT) in Foxp3-DTR mice at the maintenance phase of tolerance precipitated rejection of previously tolerized cardiac allografts and resulted in the de novo production of graft-specific alloantibodies – potentially implying a reinvigoration of Tconvs which then provide help to B cells. Indeed, these Tconvs showed a trend toward renewed cytokine production after Treg depletion and were transcriptionally distinct by RNAseq from Tconvs in tolerant mice. Conversely, Tconvs from tolerant mice infected with Listeria monocytogenes, which precipitates rejection, were transcriptionally similar to Tconvs from unmanipulated tolerant mice. Gene set enrichment analysis revealed that B cell-mediated immune response related pathways were enriched in differentially expressed genes between graft-specific Tconvs from tolerant and Treg-depleted mice. Tconvs from Treg-depleted mice expressed significantly higher levels of genes associated with Tfh cell differentiation compared to both tolerant and naïve graft-reactive Tconvs. This indicates a unique state of potential reinvigoration and implicates Tregs in actively maintaining dysfunction of graft-specific Tconvs, and also suggests that hypofunctional graft-reactive Tconvs may become Tfh cells when reinvigorated by Treg depletion.

Supported by the T32 Immunology Training Grant.