bluuebunny/crossref_metadata_2025 huggingface dataset:
Immunosuppressive activity of cyclosporin A in vivo via NFAT-independent inhibition of TCR signaling
Authors: Otsuka Shizuka , Melis Nicolas , Dutta Debjani , Weigert Roberto , Ashwell Jonathan D
Published: January 2023

Calcineurin inhibitors (CNIs) such as cyclosporin A (CsA) and FK506 are widely used in treatment of autoimmune diseases and tissue transplant rejection. It is well known that CNIs inhibit NFAT-dependent gene expression, which is thought to be the major mechanism of their therapeutic effectiveness. We recently reported that CNIs inhibit TCR-proximal signaling and the upregulation of LFA-1/ICAM1 binding by preventing calcineurin’s dephosphorylation of Lck Ser-59 (LckS59), an inhibitory modification. This raises the possibility that CsA’s inhibition of TCR signaling may contribute to immunosuppression in clinical settings. Here we find that LckS59A (which cannot accept a phosphate at Ala59) knock-in T cells are resistant to CsA’s inhibition of TCR-proximal signaling. Correspondingly, whereas CsA prevented LckWT T cells from antigen-induced and LFA-1/ICAM-mediated binding to APCs in vitro, LckS59A T cells were resistant. Moreover, CsA was able to reverse existing LckWT T:APC interactions within minutes whereas LckS59A T:DC binding remained stable. To determine if disruption of T:DC binding occurs in vivo, we performed 2-photon imaging of lymph nodes from mice in which labeled antigen-specific T cells and antigen-pulsed DC had been injected. Antigen-specific T:DC formed as early as 3 hr after T cell injection. Notably, CsA markedly reduced the formation of antigen-specific LckWT T:DC but not LckS59A T:DC clusters in lymph nodes. Given that T:DC adhesion is an early and necessary step in the generation of T cell immunity, these data demonstrate a new and perhaps major immunosuppressive mechanism for CNIs immunosuppression.
Recipient LAG3 deficiency results in antibody-mediated rejection of mouse renal allografts
Authors: Nicosia Michael , Lee Juyeun , Fan Ran , Gorbacheva Victoria , Beavers Ashley , Dvorina Nina , Baldwin William , Fairchild Robert L , Min Booki , Valujskikh Anna
Published: January 2023

The functions of coinhibitory receptor lymphocyte activation gene-3 (LAG3) in T cells are well studied, however its role in humoral immune responses remains poorly characterized. The goal of this study was to test the role of recipient LAG3 in a mouse model of renal allograft rejection

Compared to WT animals, naïve B6.LAG3−/− mice have increased splenic cellularity and higher frequencies of CD44hi memory T cells, CXCR5hi follicular T cells, and B220+CD138+plasma cells yet do not develop spontaneous autoimmunity. Furthermore, naïve B6.LAG3−/−(H-2Db) mice have increased frequencies of memory T cells against H-2Dd, H-2Ds, H-2Dq and H-2Dk alloantigens. C3H (H-2Dk) kidney allografts were transplanted into B6.WT or B6.LAG3−/− recipients after bilateral nephrectomy. Whereas 4/4 WT recipients accepted C3H allografts for longer than 60 d, recipient LAG3 deficiency led to rapid allograft rejection (MST of 14 d, n=5) with serum creatinine levels of 0.1 and 1.35 mg/dl respectively at d14 posttransplant. Graft histology at rejection revealed minimal T cell infiltration, diffuse C4d staining, atrophic peritubular capillaries, endothelial swelling and edema characteristic of antibody mediated rejection (AMR). Compared to WT, LAG3−/− recipients had elevated frequencies of anti-donor IFNg producing T cells and increased levels of anti-donor MHC-II antibodies. Recipient CD8 T cell depletion did not alter the rejection kinetics in LAG3−/− recipients (MST of 16 d), while B cell depletion significantly extended C3H kidney allograft survival (MST of >30 d).

These results suggest the predominant role of alloantibody rather than T cells in renal allograft injury and identify LAG3 is a potential therapeutic target for AMR prevention and treatment.
Formation and function of resident memory t cells in renal allografts
Authors: Daya Khodor Abou , Zhao Daqiang , Tieu Roger , Rammal Rayan , Oberbarnscheidt Martin H. , Lakkis Fadi G
Published: January 2023

The role of TRM in transplantation is unknown. In this study, we investigated the formation and function of TRM in a mouse kidney transplantation model.

(B6xBALB/c) F1.ova kidneys were transplanted to B6 recipients and 1m OT-I Teff cells transferred on day 2. Graft, blood, bone marrow, SLO, and liver were harvested 4 and 8 wks after transplantation. TRM were identified as CD44hiCD62LlowCD69+ CD103+/− cells after excluding in vivo labeled T cells. OT-I and polyclonal TRM were transcriptionally characterized using scRNAseq. TRM residency was tested by parabiosis and re-transplantation. To further establish a causal relationship between the TRM OT-Is and rejection we depleted them at wk 4 post adoptive transfer using anti-Thy1.1 (250 ug IV per day for 7d).

Mean serum creatinine (mg/dl) was significantly higher in allo vs syn group at wk 8 (0.7 vs 0.2, p<0.05). Graft histology showed mixed acute and chronic rejection in the allo group. Flow analysis of allograft cells demonstrated TRM cells among OT-I and endogenous T cell populations at 4 & 8 wks. The OT-I population was exclusively TRM phenotype by flow and scRNAseq, rapidly produced IFNγ upon re-stimulation, and was not detected anywhere else. There was no significant difference in mean number of OT-Is between wk 4 and wk 8 (277K vs 234k, p=0.74). OT-I T cells could not be detected in the parabiont kidney graft or tissues or in the secondary host outside the re-transplanted kidney, indicating that the TRM are indeed resident in the graft and do not re-circulate. Depleting the OT-Is preserved kidney function at wk 8 compared to the non-depleted group (Cr= 0.7 vs 0.2, p<0.05).

Our findings show that donor-specific TRM form in kidney allografts, are functional, and contribute to rejection.
The alarmin HMGB1 in cell cytoplasm can modulate the apoptosis to autophagy switch of pancreatic beta cells under stress
Authors: Chung Hyunwoo , Seo Sol , Hong Sung Ji , Choi So Won , Park Chung-Gyu
Published: January 2023

High mobility group box 1 (HMGB1) is an infamous alarmin which is known to be harmful to pancreatic beta cells and associated with diabetes mellitus pathogenesis and pancreatic islet graft failure. However, recent studies have indicated that cytosolic HMGB1 could function as a modulator to relieve cells from apoptotic stress by inducing autophagy. Particularly, pancreatic beta cells have been well-known to demonstrate the apoptosis to autophagy switch when exposed to hypoxia or lipoxiticy. In this study, we have investigated the beta cells under hypoxic and lipotoxic stress with CCK8 assay and flow cytometry while utilizing a small molecule inhibitor of HMGB1 which can suppress the cytosolic accumulation of HMGB1. The results demonstrated that the cytoplasmic HMGB1 blockade decreased the viability of mouse beta cell line MIN6 and primary islets. Moreover, the cytoplasmic HMGB1 blockade decreased the autophagic flux and increased the ratio of apoptotic MIN6 cells. Also, it was noteworthy that the MIN6 cells have shown high autophagic flux even in basal state. We believe this study linked the role of cytoplasmic HMGB1 in apoptosis to autophagy switch and the importance of autophagy in beta cells under apoptotic stress. We expect this study to open up a novel field of HMGB1 utilization within diabetes- and islet transplantation-related therapeutics, where the current field is mainly focused on the exhaustive blockade of HMGB1.
Autophagy Induced due to Moderate Endoplasmic Reticulum Stress in T Cells Enhances Immunotherapeutic Tumor Control
Authors: Chakraborty Paramita , Chatterjee Shilpak , Tran Danh T. , Pytel Dariusz , Kim Dosung , Nadig Satish N. , Atkinson Carl , Wang Hongjun , Diehl J. Alan , Gangaraju Vamsi , Palanisamy Viswanathan , Mehrotra Shikhar
Published: January 2023

A number of external or internal stimuli cause endoplasmic reticulum (ER) stress conditions. In response to ER stress, stressed cells activate well-orchestrated cellular signaling processes to resolve the stress or remove itself by triggering apoptosis. In the tumor microenvironment, induction of sustained ER stress is known to dampen the antitumor activity of T cells. However, how T cells respond to suboptimal ER stimuli is unknown. Using melanoma antigen gp100 reactive T cells, we found that T cells transiently activate protein kinase R-like endoplasmic reticulum kinase (PERK) dependent signaling network to ameliorate low level of ER stress. While transient PERK activation in T cell enhances stemness, promotes mitochondrial biogenesis, increases T cells anti-tumor immunity in B16 melanoma bearing mouse, overexpression of PERK triggers T cell dysfunctionality and death. Mechanistically, we discovered that transient activation of PERK decreases overall protein translation and induces protective autophagy to prevent cell damage. Conversely, knockdown of PERK abrogates autophagy activation, hampers mitochondrial biogenesis in response to suboptimal ER stress, which in turn compromises antitumor function. Furthermore, blocking autophagy in T cells hampers T cell anti-tumor activity. Lastly, using gp100 reactive LC3-GFP expressing T cells we demonstrated that, T cells that initiate autophagy due to suboptimal ER stress show better anti-tumor potential compared to those, that fail to enter into the process. Overall, these preclinical data highlight that, both transient ER stress and autophagy pathways are important for healthy T cell function and therapeutically, can be manipulated to regulate their antitumor potential.
Exploiting cancer cell metabolism with stimuli-responsive toll-like receptor agonists
Authors: Mancini Rock , Ryan Austin , Pulukri Anunay , Davaritouchaee Maryam , Abbasi Armina , Hendricksen Aaron , Opp Larissa , Burt Anthony , Nielsen Amy
Published: January 2023

Methods

We report stimuli-responsive Toll-Like Receptor (TLR) agonist prodrugs that rely upon the irregular metabolism (Warburg Effect and efflux potential) common among different types of cancers to activate immune cells. Beginning with Coley’s Toxins in the late 1800’s, TLR agonists have exhibited anticancer properties, and more recently have been shown to act synergistically with anti-CTLA-4 or anti-PD-L1 checkpoint blockades. Specifically, TLR7/8 agonists deplete MDSCs, activate CD8+ T-cells, M1 macrophages and pDCs; and enhance abscopal effects across animal models for melanoma, breast, and prostate cancers. Despite these profound effects, the severe systemic inflammatory toxicity elicited by TLR agonists presents a significant barrier to using them as drugs. To address this, we developed stimuli-responsive TLR agonist prodrugs targeted to cancer cell metabolism.

Results

Our prodrugs are inactive until conversion by complementary stimuli selected from α-mannosidase, β-galactosidase, or β-glucuronidase with concomitant efflux via MDR1 and comparable processes. We observe conversion and efflux in several cancer types, including B16 melanoma, 4T1 breast, and TRAMP prostate cancers, resulting in immunogenicity as measured by activation of primary BMDCs. In-vivo, prodrugs do not induce systemic inflammation and perform differently than parent TLR agonist in a 4T1 neoadjuvant resection metastasis model.

Conclusion

These results suggest that stimuli-responsive TLR agonist prodrugs target the action of TLR agonists to cancer cell metabolism and mitigate systemic inflammatory effects relative to the parent TLR agonist.
Innate immune populations contribute to effective immune responses against B lymphoblastic leukemia cells
Authors: Hunter Rae , Henry Curtis J. , Porter Christopher C.
Published: January 2023

Pediatric patients diagnosed with acute lymphoblastic leukemia (ALL) have a 15–20% mortality rate, in spite of current advances in therapies, including immunotherapies. Despite the use of immunotherapies in patients with relapsed or refractory ALL, relapse rates post-treatment remains over 50 percent. These observations highlight our lack of understanding of immune evasion mechanisms used by leukemia cells.

Our group has previously published that IL-12 promotes a very effective immune response to B-cell ALL cells in vivo. While T cells are required to maintain remission, we observed prolonged survival in IL-12 treated Rag1−/− mice with leukemia as compared to untreated mice, suggesting a major role of the innate immune response in ALL cells. Depletion of NK cells with anti-NK1.1 did not abrogate the elimination of an immunogenic model of leukemia, in which calcineurin has been knocked down by shRNA. However, depletion of myeloid cells from immunocompetent mice using colony-stimulating factor-1 receptor (CSF1R) depleting antibodies promoted the progression of this leukemia, which was eliminated in all isotype treated, immune-competent recipients, indicating myeloid cells play a critical role in the immune response to ALL cells. Ongoing studies suggest dendritic cells (DCs), specifically CD11b+CD11c+ DCs, are reduced in mice transplanted with parental relative to immunogenic, Cn-deficient ALL cells. Furthermore, macrophages cultured in vitro with parental cells expressed significantly lower surface levels of CD80, CD40 and CD86 compared to levels found on the surface of cells cultured with Cn-deficient leukemia cells. Overall, our data suggest that ALL cells may compromise the T-cell priming capacity of innate immune cells.
Tumor-associated myeloid cells provide critical support for T-ALL in vivo
Authors: Lyu Aram , Triplett Todd A. , Godfrey Wesley H. , Nam Seo Hee , Ames Rachel Y. , Hu Zicheng , Sarang Adviti , Selden Hilary J. , Lee Chang-Han , Georgiou George , Horton Terzah M. , Ehrlich Lauren I. R.
Published: January 2023

T cell acute lymphoblastic leukemia (T-ALL) is a malignancy of immature T cells. Despite harboring multiple genetic alterations that drive disease, primary T-ALL cells require exogenous signals to survive. We previously reported that tumor-associated myeloid cells support survival of primary mouse T-ALL cells in vitro. However, the contribution of myeloid cells to T-ALL progression in vivo remains unclear. Here, we depleted phagocytic myeloid cells from leukemic mice, resulting in a significant reduction in tumor burden in multiple organs and prolonged survival. T-ALL burden also diminished following myeloid depletion in lymphodeficient mice, indicating that adaptive immunity was not required to eliminate T-ALL cells, thus suggesting tumor-associated myeloid cells could directly support T-ALL growth. Consistent with this possibility, multiple subsets of tumor-associated myeloid cells support survival of mouse and primary patient T-ALL cells in vitro. Acute myeloid depletion reduced activation of several pathways regulating cell survival in T-ALL cells, implicating these pathways in promoting T-ALL progression in vivo. Computational analysis of published datasets shows an association between enriched macrophage gene signatures and poor prognosis in T-ALL patients, further implicating a pro-tumor role for myeloid cells. Together, our results indicate that an altered myeloid compartment directly promotes T-ALL progression at divergent T-ALL sites in vivo.
A novel Cullin 4A/BCereblon E3 Ligase Modulator, FPFT-2216, offers protective effect against Myelodysplastic syndrome
Authors: Hailu Yohannes Gemechu , Kishimoto Tadamitsu
Published: January 2023

Thalidomide is an immunomodulatory drug (IMiD) having a therapeutic action in hematological malignancies and inflammatory diseases. Despite the therapeutic benefits, thalidomide is known for its high toxicity and responsible for a number of children born with anomalies when taken by pregnant women. Thalidomide’s high toxicity resulted in the development of more powerful and less toxic analogs.

The mouse is resistant to IMiDs effect and for this reason, we developed mice carrying human cereblon to study IMiDs mechanism. We investigated the potential IMiDs effect on T cell stimulation/suppression. We found that T-lymphocytes from humanized cereblon mice treated with a novel IMiD, FPFT-2216, result in higher production of IL-22, IL-2 and IL-9 whereas wild type mice were resistant. Furthermore, IMiDs showed potential degradative effects towards Cereblon substrates like Aiolos and interferon regulatory factor 4 among humanized cereblon mice but not in wild type. These IMiD-mediated T-cell stimulations might augment anti-tumor immunity.

Next, we assessed the potential therapeutic effects of FPFT-2216 in Myelodysplastic syndrome (MDS). NSG mice were engrafted with MDS patient derived cell line, MDS-L, and treated by DMSO, or FPFT-2216. Mice treated with FPFT-2216 showed a longer survival rate and less infiltration of MDS-L into their bone marrow as compared to their control. Moreover, In vitro experiments confirmed that FPFT-2216 offers a significant anti-MDS effect. In conclusion, FPFT-2216’s strong preclinical effect in MDS-L cell provides a hint that it can help in treating MDS patients and it supports further safety-check in humanized cereblon mice, other primates and clinical trials.

Conflict of interest: None
Modified culture protocol for differentiation of human promyelocytic leukaemia PLB-985 cell-line into mature neutrophil-like granulocytes
Authors: Shehu Shaayau , Edwards Steven W. , Wright Helen L.
Published: January 2023

Neutrophils are considered to be mature and terminally differentiated. They are short-lived, lack proliferation capacity and are impossible to transfect in vitro to express exogenous genes or proteins. These properties have made their ex vivo experimental manipulation and biochemical studies of their signal activation mechanisms and genetic makeup, challenging. Establishment of cell line models capable of differentiating along the myeloid lineage into mature neutrophil-like granulocytes, with similar morphological and functional properties to blood neutrophils would, therefore, be an important tool to enable transfection of key genes and determine the effects of such manipulations on neutrophil functions. PLB-985 is a recently established cell line that has been demonstrated to show greater levels of differentiation into granulocyte in the presence of appropriate differentiation-inducing agents, but the efficiency of differentiation reported so far was very low, and the differentiated cells only partly resembled mature blood neutrophils. This study developed a modified differentiation protocol and an optimised culture conditions that induced the PLB-985 cell line to differentiate into mature, neutrophil-like granulocytes, Terminally-differentiated, neutrophil-like phenotypes of PLB-985 cells that resembled mature blood neutrophil morphology, and which had an appreciably delayed apoptosis have been consistently cultured. This was evident by the acquisition of multi-lobed nucleus, granulated cytoplasm and delayed apoptosis. The differentiated PLB-985 cells may, therefore, provide an excellent neutrophil-model system for in vitro study of neutrophil differentiation, signalling and functions.
Neutral ceramidase regulates ferroptosis in myeloid-derived suppressor cells
Authors: zhu huabin , Lu Chunwan , Yang Dafeng , Redd Priscilla S , Ibrahim Mohammed L. , Klement John D. , Smith Alyssa D. , Albers Thomas , Lebedyeva Iryna , Coant Nicolas , Hannun Yusuf , Liu Kebin
Published: January 2023

Myeloid-derived suppressor cells (MDSCs) are key targets in cancer immunotherapy. Neutral ceramidase (nCDase), a key enzyme that hydrolyzes ceramide into sphingosine, is highly expressed in colon carcinoma and regulates apoptosis and autophagy in colon carcinoma cells. We report here that nCDase is also highly expressed in tumor-infiltrating immune cells in mouse and human colon carcinoma in vivo. To target nCDase, we performed molecular docking based on human nCDase structure aganist a virtual chemical library. In vitro enzymatic inhibition analysis of top hits identified nCDC06 as a potent nCDase inhibitor. Chemical and NMR analysis determined nCDC06 structure and molecular weight (325.1 Dalton). nCDC06 has an IC50 of 10.16 to 25.91 mM for human nCDase and 18.6–30.2 mM for mouse nCDase. nCDC06 induces MDSC cell death in a dose-dependent mechanism. Inhibition of ferroptosis decreased nCDC06-induced MDSC cell death. Analysis of ultracellular structure, ROS accumulation, and glutathione revealed that nCDC06 suppresses MDSCs at least in part through a ferroptosis-dependent mechanism. Gene expression profiling revealed that nCDC06 activates the p53 pathway in MDSCs. Furthermore, nCDC06 therapy significantly inhibited tumor growth in an experimental lung metastasis mouse model. We determined that nCDase regulates MDSC ferroptosis and nCDC06 is potent in activating the p53 pathway to induce MDSC ferroptosis to suppress tumor growth in vivo.
Metabolic barriers imposed on tumor associated macrophages
Authors: Roy Sohini , Young Kim
Published: January 2023

Macrophages comprise a broad spectrum of heterogeneous cell population with diverse functions in immune responses in pathological conditions including cancer, inflammation and infection. Macrophages are extremely plastic in nature and highly specialized in sensing their microenvironment which governs their functional phenotype. Simplistically, macrophages can be categorized into classical inflammatory or M1 type and anti-inflammatory or M2 type. While the M1 macrophages play a crucial role in activating immune responses and release a wide array of pro-inflammatory and cytotoxic molecules, M2 macrophages facilitate resolution of inflammation by secreting immunosuppressive anti-inflammatory factors, phagocytosing and clearing apoptotic debris and promoting sustainability of regulatory T cells while suppressing effector T cells. Emerging evidences indicate an intimate connection between a robust switch in metabolic adaptations and different classes of macrophages. Tumor associated macrophages (TAM) are abundantly present in tumor microenvironments and can account for up to 50% of myeloid infiltrate in some types of human solid malignancies and contribute to disease progression, therapy resistance and clinical outcome. However, the metabolic circuitries in TAM underlying their ability to sustain tumor evolution and influence their own functional polarization is complicated and less characterized in many tumor types. In the current study, using multi-prong approaches like bulk myeloid as well as single cell RNA sequencing as well as functional assays, we highlight the metabolic pattern of TAMs in head and neck cancer and melanoma and propose immunometabolic strategies to harness TAM for controlling tumor growth.
Dendritic cells dysfunction in tumor-draining lymph nodes
Authors: Bandola-Simon Joanna Marta , Wong Nathan , Cam Maggie , Roche Paul A
Published: January 2023

An effective anti-tumor response requires activation of both CD4 helper T cells (through MHC-II molecules) and CD8 cytotoxic T cells (through MHC-I molecules). Activation of tumor-specific T cells depends on tumor antigen presentation by DCs. However, it is known that the tumor microenvironment largely suppresses immune responses. The focus of our study is to characterize the functional status of DCs in tumor-draining lymph nodes and to identify the mechanisms that lead to defective presentation of tumor antigens. We transplanted tumors into the flank of mice and analyzed DCs from both tumor-draining lymph node (TDLN) and non-draining lymph nodes (NDLN) as our model system. Cross-presentation of OVA to CD8 T cells was not suppressed in TDLN in comparison to the NDLN either in vivo and ex vivo. However, both in vivo and ex vivo CD4 T cell activation by DCs from TDLN was defective. Subset analysis revealed that both migratory and resident DCs in TDLN show a less mature phenotype as compared to their counterparts isolated from NDLN. Functional assays revealed defects in antigen uptake and processing in DCs from TDLN. We also identified differences in the proportion of MHC-II molecules containing the Invariant chain CLIP peptide in DCs from TDLN in comparison to NDLN. These features of DC function, supported by RNA-Seq analysis, contribute to defective antigen presentation to CD4 T cells and may explain the dysfunction of DCs in tumor-bearing mice. Further analysis should enable moving towards “correcting” DC defects that would allow CD4 T cell priming to provide help to anti-tumor CD8 T cells.
CD4 T cell phenotypes differentially modulate the CD8 T cell response in kidney cancer
Authors: Cardenas Maria A , Prokhnevska Nataliya , Jansen Caroline S , Master Viraj A , Kissick Haydn
Published: January 2023

CD8 T cell infiltration can independently predict survival and response to immunotherapy in kidney cancer patients. Given the importance of the CD8 T cell response in cancer, it is crucial to understand what signals promote their infiltration and support the maintenance of the anti-tumor response. The proportion of tumor infiltrating CD8 T cells, as measured by flow cytometry, was found to significantly correlate (R = 0.8, p<0.0001) with tumor infiltrating CD4 T cells in 160 renal cell carcinoma patients, suggesting that CD4 T cells may promote the tumor-specific CD8 T cell response. We hypothesize that the phenotype of tumor infiltrating CD4 T cells affects CD8 T cell differentiation and may contribute to the efficiency of the anti-tumor response. We performed flow cytometry analysis on a subset of kidney cancer patients to further characterize the CD4 populations in the tumor tissue. T-regulatory cells (Treg), defined by their master transcription factor FOXP3, accounted for 15.5 ± 7.2 % of the tumor infiltrating CD4 T cells. Importantly, the Treg population negatively correlated with CD8 differentiation and expression of effector-like molecules. Additionally, we found a Th1-like population expressing high levels of EOMES and GZMK that have previously been associated with cytolytic capacities. In conclusion, we found various subsets of CD4 T cells in the tumor that may differentially modulate CD8 T cell function and influence the anti-tumor response.
Unsuccessful checkpoint blockade and vaccination therapy despite expansion of tumor neoantigen-specific CD8 T cells in the Lewis Lung Carcinoma (LLC) model
Authors: Li Shamin , Simoni Yannick , Zhuang Summer , Ma Shaokang , Chee Jonathan , Redwood Alec , Robinson Bruce , Newell Evan
Published: January 2023

The most promising cancer immunotherapies to date target immune checkpoint receptors, such as PD-1 and CTLA-4. However, T cell checkpoint blockade has no inherent tumor specificity and shows uneven response, with up to 70–80% of patients resistant to these therapies. Here, we use a spontaneous mouse model resistant to immunotherapy to explore how immune checkpoint blockade (ICB) affects tumor-specific T cells and understand why these cells fail to eradicate tumor cells after treatment. Using combinatorial tetramer staining by CyTOF, we identified a new tumor-specific neoantigen in Lewis Lung Carcinoma (LLC). These tumor-specific CD8+ tumor-infiltrating lymphocytes (TILs) strongly exhibit an activation phenotype and can also be detected in different peripheral compartments at lower frequency. Surprisingly, despite an absence of tumor regression, we observed a remarkable expansion of tumor-specific CD8+ TILs after ICB with anti-PD1, anti-CTLA4, or neoantigen peptide vaccination. This expansion is associated with phenotypic changes, including enrichment of exhaustion markers and CD39 expression indicating chronic TCR activation and confirming recognition of tumor cells. Current single-cell RNA sequencing and TCR profiling analysis will correlate transcriptomics and clonality of these tumor-specific T cells after cancer therapies with anti-tumor response and elucidate their functionalities within the tumor microenvironment. Overall this work provides a new model for studying neoantigens in tumors and revisits the role of neoantigen-specific T cells in the context of ICB, which will have important implications in designing future immunotherapeutic strategies.
Lymphoma mutations in HVEM (TNFRSF14) disrupt bi-directional activation of BTLA and CD160 singaling within the tumor microenvironment
Authors: Sedy John R , Lin Wai , Huang Sarah , Balmert M. Olivia , Nguyen Jennifer , Moshayedi Aref , Ware Carl
Published: January 2023

Herpesvirus entry mediator (HVEM; TNFRSF14) is frequently mutated in human cancer with a high degree of mutations in follicular and diffuse large B cell lymphoma. In lymphocytes, HVEM receptors are activated by its ligands LIGHT (TNFSF14) and Lymphotoxin-α, and engage in bi-directional signaling with the receptors B and T lymphocyte attenuator (BTLA) and CD160. We hypothesized that mutations in HVEM disrupted binding to either of its ligands resulting in altered cell-intrinsic HVEM signaling in lymphoma, or in altered activation of BTLA or CD160 in tumor infiltrating lymphocytes. Here, we show that point mutations identified in human lymphoma were localized to the extracellular domain and specifically target ligand binding, resulting in preferential loss of HVEM interactions with CD160 and BTLA compared to LIGHT (TNFSF14). Disrupted binding correlated with loss of receptor functionality resulting in abrogated cell-intrinsic HVEM activation of NF-κB, as well as reduced CD160 activation of ERK and AKT phosphorylation by HVEM ligation. We next identified HVEM-activated CD160 and BTLA signaling pathways through AP/MS and phospho-proteomic analysis in NK and T cells. Activation of CD160 versus BTLA resulted in contrasting phosphorylation status in a number of phospho-proteins, illustrating opposing functions of these receptors. In a mouse model of lymphoma we tested the impact of BTLA versus CD160 in regulating antigen-specific anti-tumor responses, demonstrating regulatory effects of these receptors in vivo. Together these data illustrate how selective pressures within the tumor microenvironment may drive cancer mutations that alter HVEM network signaling in vivo, and indicate potential targets for onco-immunotherapy.
Upregulated 4-1BB (CD137) expression on skin T cells from a mouse model of precancerous skin
Authors: Leggatt Graham R , Salim Nazhifah , Mattarollo Stephen
Published: January 2023

Non-melanoma skin cancer (NMSC), including both squamous cell carcinoma (SCC) and basal cell carcinoma (BCC), is a commonly diagnosed cancer in Australia. The role of the immune system in the development of NMSC is still poorly understood. Using a mouse model of precancerous, hyperplastic skin, we have investigated the expression and targeting of the T cell co-stimulatory molecule, 4-1BB (CD137). Our data shows that 4-1BB is expressed on both CD8 and non-CD8 T cells isolated from precancerous mouse skin with limited expression on T cells in normal skin or skin-draining lymph nodes. In contrast, the ligand for 4-1BB, 4-1BBL, was poorly expressed in skin and draining lymph nodes from precancerous or normal skin. Intradermal injection of antibodies against 4-1BB was more effective at labelling target cells within the hyperplastic skin than intravenous injection. Agonist antibodies against 4-1BB showed some enhancement of skin T cell function in vitro. The data suggests that 4-1BB expression is upregulated on T cells within precancerous mouse skin and may represent a promising target for immunotherapy against precancerous skin lesions.
Inhibition of PI3Kδ improves tumor specific T cell immunity and metabolic fitness
Authors: Rangel Rivera Guillermo O , Dwyer Connor , Arthontoulis Dimitrios , Knochelmann Hannah , Smith Aubrey S , Reyes Amalia Rivera , Wyatt Megan , Paulos Chrystal M
Published: January 2023

Durable responses have been observed with adoptive T cell therapy (ACT) in some patients. However, current protocols used to expand T cells often exhibit suboptimal tumor control. Failure in these therapies has been attributed to premature differentiation and impaired metabolism of the infused T cells. Previous work done in our lab showed that reduced PI3Kδ signaling improved ACT. Because PI3Kγ and PI3Kδ have critical regulatory roles in T cell differentiation and function, we tested whether inhibiting PI3Kγ could recapitulate or synergize PI3Kδ blockade. To test this, we primed melanoma specific CD8+ pmel-1 T cells in the presence of PI3Kγ (IPI-459), PI3Kδ (CAL101 or TGR-1202) or PI3Kγ/δ (IPI-145) inhibitors and infused them into B16F10 tumor bearing mice. Sole inhibition of PI3Kδ or PI3Kγ in vitro promoted greater tumor immunity and survival compared to dual inhibition. To understand how PI3Kδ or PI3Kγ blockade improved T cell therapy, we assessed their phenotype. CAL101 treatment produced more CD62Lhi CD44lo T cells compared to IPI-459, while TGR-1202 enriched mostly CD62LhiCD44hi T cells. Because decreased T cell differentiation is associated with mitochondrial metabolism, we focused on CAL101 treated T cells to study their metabolism. We found that CAL101 decreased glucose uptake and increased mitochondrial respiration in vitro, indicating augmented mitochondrial function. These findings indicate that blocking PI3Kδ is sufficient to mediate lasting tumor immunity of adoptively transferred T cells by preventing premature differentiation and improving mitochondrial fitness. Our data suggest that addition of CAL101 to ACT expansion protocols could greatly improve T cell therapies for solid tumors.
Illuminating the onco-GPCRome: targeting the prostaglandin receptor-Gαs signaling axis as a novel immune checkpoint in cancer
Authors: Wu Victoria H , Wenzel Alexander , Yung Bryan , Wang Zhiyong , Mesirov Jill P. , Gutkind J. Silvio
Published: January 2023

Recent advances in checkpoint blockade immunotherapy (CBI) have revolutionized cancer treatment, but the limited response rates in most cancers suggest that new approaches and targets are clearly needed to fully elucidate the underlying biology of dysfunctional CD8 T cells and achieve durable responses. G-protein coupled receptors (GPCRs) are the most intensively studied drug targets as they play key roles in many physiological processes, and they have remained longstanding favorable pharmacological targets. Using novel computational approaches integrating multiple single cell RNAseq databases, we first deconvoluted tumor-infiltrating T cell heterogeneity to generate a transcriptomic GPCR signature based on coupling specificity and receptor family. Preliminary data shows an upregulation of prostaglandin receptors, PTGER2 and PTGER4, on exhausted T cells, suggesting that these GPCRs, coupled to the Gαs G protein α subunit, may contribute to diminishing anti-tumor cytotoxicity. Expression and stimulation of EP2 and EP4 on highly activated CD8 T cells and subsequent Gαs signaling decreases cytokine secretion and CXCR3-mediated migration. Additionally, blockade of EP2 and EP4 significantly decreases tumor growth in multiple immune competent, syngeneic tumor models. Altogether, we hypothesize that activation of Gαs downstream signaling cascades may be dampening the anti-tumor cytotoxicity of CD8 T cells, thereby limiting the effectiveness of PD-1 and CTLA-4 blockade. EP2 and EP4 and Gαs signaling may represent promising candidates as immune checkpoints that can be targeted in combination with CBI as part of novel multimodality precision immunotherapy approaches to reactivate the immune system to destroy tumors.
Novel lipid A mimetics, BECC438 and BECC470, act as potent adjuvants in bacterial and viral subunit vaccines
Authors: Harberts Erin , Varisco David , Haupt Robert , Jain Akshay , Middaugh Charles R. , Ernst Robert K.
Published: January 2023

Infectious diseases continue to be the leading cause of morbidity and mortality worldwide, an urgent need exists to develop effective vaccines against a variety of pathogens. Effective vaccines contain target pathogen antigens and adjuvant(s) which effect the magnitude, duration and mechanism of action of the immune response. Bacterial enzyme combinatorial chemistry (BECC) was developed to synthesize custom bacterial lipid A that are partial agonists for innate immune receptor Toll-like receptor 4 (TLR4). BECC is a robust mechanism for design of lipid A mimetics that drive specific activating immunity while avoiding excessive adverse host reactions. Initial screenings identify two lead adjuvant candidates, BECC438 and BECC470, that are formulated with bacterial and viral antigens, including Yersinia pestis (rF1-V), Staphlococcus alpha toxin, and HPV (VLPs). Mice are immunized on two-week intervals with several squalene or aqueous based BECC formulations. Antigen-specific serum antibody titers measured by ELISA for total IgG, IgG1, and IgG2c(2a) for BECC formulations are significantly more balanced than those currently approved for clinical use. Pathogen challenge models also show increased levels of efficacy/survival. Both BECC compounds allow for durable antibody production, antigen sparing, dose sparing, adjuvant sparing, production of T follicular helper cells, and provide protection against a heterologous challenge in viral infection models. Additionally, BECC adjuvanted vaccines with the model antigen ovalbumin show balanced and durable antibody responses in multiple murine backgrounds C57BL6, BALBc, and CD-1. BECC molecules are effective components of next-generation infectious disease vaccines.
Vaccine-elicited T cells rely on a distinct transcriptional program from infection-elicited T cells
Authors: Thompson Scott Beckett , Ivanova Daria Leonidovna , Klarquist Jared , Scott-Browne James , Kedl Ross M.
Published: January 2023

Protection from pathogens relies on both humoral (antibody-mediated) and cellular (T cell-mediated) responses. While infections robustly elicit both of these types of immunity, currently approved vaccine adjuvants largely fail to induce significant T cell responses. However, recent work by our lab and others suggests that the mechanisms governing vaccine-elicited T cells (Tvac) may be substantially different than those governing infection-elicited T cells (Tinf). We have recently demonstrated that optimal subunit vaccine-elicited T cell responses rely on different cytokine signals (IL-27 and 15) and metabolic function (oxidative phosphorylation vs. aerobic glycolysis) leading to phenotypically and functionally different outcomes (memory vs. effector). Our goal was to investigate the transcriptional programming that promotes Tvac formation. Using a combined ATACseq, bulk RNA-seq and single-cell RNA-seq approach, we compared Tvac and Tinf at various time points post-immunization or infectious challenge. Our data indicate that the chromatin organization and gene expression of Tvac markedly diverges from Tinf even at early time points. Furthermore, single-cell analysis indicates that Tvac represent a unique subset of activated/memory T cells, distinct from Tinf. Remarkably, while displaying a gene signature similar to highly proliferative cells, Tvac also share transcriptional properties with recently identified stem cell memory T cells, such as oxidative phosphorylation and transcription factor TCF1 gene signatures. Taken together, our data have identified a novel transcriptional program that supports a robust vaccine-elicited T cell response with implications for future therapeutic vaccine design.
The quality of heat shock protein-mediated responses is determined by the antigen presenting cell
Authors: Sedlacek Abigail L , Kinner Lauren , Binder Robert
Published: January 2023

Immunization with Heat shock proteins (HSPs) elicits a spectrum of immune responses including non-antigen specific immunoregulatory responses and proinflammatory antigen specific responses. While some of the aspects of these mechanisms have been determined, many questions remain. HSPs interact with their receptor, CD91, which is expressed on the surface of antigen presenting cells (APCs). In response to HSPs, APCs upregulate expression of costimulatory molecules, release cytokines, and cross-present HSP-chaperoned peptides. This culminates in activation of T and NK cells, which are both required for an optimal Th1 response. We have uncovered unique signaling cascades in macrophages (MΦ), conventional dendritic cells (cDCs), and plasmacytoid dendritic cells (pDCs) which include NF-kB and p38 MAPK and result in different downstream immune responses. In MΦ, p38 MAPK mediates phosphorylation of STAT1 at both phosphorylation sites, Y701 and S737. Phosphorylation of STAT1 leads to the secretion of CXCL10 and downstream activation of NK cells. In contrast, cDCs fail to phosphorylate STAT1 at Y701, produce reduced amounts of CXCL10 and do not activate NK cells, thus yielding sub-optimal T cell responses. Alternatively, activation of pDCs by HSPs induces surface expression of NRP1. NRP1 expression allows for engagement and activation of regulatory T cells and downstream suppression of immune responses. Collectively, our data demonstrates that targeting HSPs to particular APCs is critical in elucidating the desired immune response and needs to be considered in future clinical applications.
Using lymph node slices to study vaccine adjuvants ex vivo
Authors: Ball Alexander , Catterton Megan A , Pompano Rebecca R
Published: January 2023

Despite the success of vaccines in preventing infectious diseases, there is a growing need for the development of new vaccine adjuvants. We recently demonstrated that live lymph node (LN) slices serve as a unique platform to study adaptive immunity ex vivo. These slices enable visualization of changes in the spatial organization and activation state of the tissue during an immune response. Here we hypothesized that different adjuvants induce unique cytokine and spatial profiles within tissue, and we examined the influence of three adjuvants (alum, CFA, & poly I:C) to test this. We transferred ovalbumin (OVA)-specific CD4+ T cells (OTII) into C57BL/6 mice before vaccinating the mice with OVA protein and adjuvant. LNs were collected on days 1, 4, and 7, sliced on a vibratome, and cultured overnight with either the intact OVA protein or PBS. The supernatant was analyzed for cytokine secretion by ELISA (IFNγ, TNFα, and IL-4) from cultured slices. Slices were immunostained for activation markers, CD69 and CD22, and analyzed with fluorescent imaging. Cytokine secretion was greatest in slices from vaccinated mice that had been co-cultured with the OVA protein in comparison to slices that were either cultured or immunized with PBS. The different adjuvants induced unique cytokine profiles in slices both in terms of timing and magnitude as TNFα, IFNγ, and IL-4 peaked on different days from one another within the same vaccination scheme as well as between adjuvants. The spatiotemporal distribution of T cell and B cell activation markers also differed between adjuvants. This study demonstrates that LN slices can analyze the unique influences of different adjuvants and highlights slices as a potential screen for novel vaccine adjuvants.
A microneedle vaccination with glycoprotein E of Varicella Zoster Virus elicits antibody production and polyfuctional T cells in mice
Authors: Cha Hye-Ran , Shim Doo-Hee , Lee Jaemyun
Published: January 2023

A microneedle has several benefits in that it is painless, easy to use and preventable from potential cross-contamination by using conventional needle. Besides these advantages, vaccination with microneedle may provide comparable or even higher immune response than vaccination through conventional intramuscular route. Here we have been developing herpes zoster vaccine using microneedle coated with recombinant glycoprotein E (gE) of VZV, which is essential in virus replication and cell-to-cell spread. Microneedles were coated by gE and three different adjuvants: double-mutant heat-labile toxin (dmLT), the combination of TLR1/2 and TLR3 ligands (Lpampo), or mutant cholera toxin A subunit (CTA). C57BL/6 mice were immunized with the coated microneedles two times at 2-week intervals. The overall increase in gE specific antibody titers was observed among adjuvanted groups compare to the non-adjuvanted group. Moreover, the humoral response was IgG1-dominated. Hence, a decreasing tendency of IgG1/IgG2c ratio was observed in gE adjuvanted with dmLT or Lpampo groups whereas relatively high ratio was detected in CTA group. This indicates that Th1 immune response complemented Th2 response in former groups. By conducting ELISPOT, we measured the amount of IFN-gamma secreting T cells as cell-mediated immune response is significant in immunogenicity of HZ vaccination. Moreover, polyfunctional CD4 T cells were increased after injection with gE +Lpampo coated microneedle. In consistent with humoral response, dmLT or Lpampo adjuvanted group exhibited enhanced cell-mediated immune response. Taken together, delivery of HZ subunit vaccine with microneedle may provide a potent immune response against reactivation of VZV.
Utilising nanoparticles to enhance the vaccine induced T cell immune response
Authors: Wilson Kirsty L , Powles Liam , Tsirikis Peter , Xiang Sue D , Selomulya Cordelia , Henderson Edward , Corrie Simon , Plebanski Magdalena
Published: January 2023

Vaccines are a powerful and cost-effective tool to limit the spread of disease, however, there are still some complex diseases for which we don’t yet have a successful and long-lasting vaccine, for example malaria and cancers. One reason for this is the difficult choice of target antigen, though there is also a need for potent adjuvants/delivery systems capable of inducing both antibody and T cell responses. Current licensed adjuvants, for example Alum, mainly elicit strong antibody responses, therefore, novel vaccine delivery systems need to be developed. One such system is by utilising nanoparticles to increase the vaccine induced immune response. Inorganic carboxylated polystyrene nanoparticles in the viral size range (40–50nm) are non-inflammatory, stable in solution, and target dendritic cells. Importantly, these nanoparticles elicit potent antibody, CD4+ T cell and CD8+ T cell responses to a range of antigens in murine studies, including to malaria and cancer peptides. Using fluorescent nanoparticles allows us to assess the interaction of these nanoparticles with specific cell subsets, such as antigen presenting cells, by flow cytometry. Furthermore, we can analyse the biodistribution of the nanoparticles using small animal imaging via the IVIS. Ideally, for maximum translation capacity into human vaccines these nanoformulations would be biodegradable. In addition to our model polystyrene nanovaccines we have been investigating biodegradable nanoparticles made from other materials, i.e. iron oxide. Comparable to polystyrene nanoparticles, biodegradable nanovaccines are also endocytosed by dendritic cells and are capable of inducing antibodies and cytokine secretion from both CD4+ T cells and CD8+ T cells.