bluuebunny/crossref_metadata_2025 huggingface dataset:
Lack of B10 and MZ-like B cells in liver-transplant patients with PCH
Authors: Hasan Md Mahmudul , Thompson-Snipes LuAnn , Klintmalm Göran B. , Oh Sangkon , Joo HyeMee
Published: January 2023

Human CD24hiCD27+ B cells subset, also known as B10, is a mixture of marginal zone (MZ)-like B cells (IgD+CD27+) and memory B cells (IgD−CD27+), and can protect against autoimmune/inflammatory damage, in part, by producing IL-10, an anti-inflammatory cytokine. However, the role of B10 and MZ-like B cells in solid organ transplantation is poorly understood. In this study, we examined IL-10-producing human B cells subsets including MZ-like B cells, memory B cells and transitional B cells (CD24hiCD38hi) in the blood of healthy controls compared to liver transplant patients with plasma cell hepatitis (PCH), a variant of late onset rejection. First, we phenotypically and functionally characterized different human B cells subsets that produce cytokine IL-10 in several human tissues including tonsil and spleen, and PBMC. Then, we demonstrated that CD24hiCD27+ B cell is the major source of IL-10 producing cells and capable to suppress alloantigen stimulated T cell proliferation and cytokine production in IL-10 dependent manner. We also found that the frequency of CD24hiCD27+ B, as well as IgD+CD27+MZ-like B cells is significantly lower in PCH+ liver transplanted patients than healthy controls. In line with this, the frequency of IL-10+CD19+ B cells is negligible in PCH+ liver transplant patients compared with healthy controls upon CpG B stimulation. Our findings suggest that a lack of immunosuppressive IL-10 producing B cell subsets could be associated with the rejection in the PCH+ patients.
Antigens Associated with Non-HLA Cardiac Transplant Rejection
Authors: Gates Katherine , Griffiths Leigh
Published: January 2023

Despite advances in management of acute rejection episodes, rejection incidence remain largely unchanged in heart transplant patients. While Human Leukocyte Antigen (HLA) is a common initiator of graft-specific immune responses, cases of non-HLA antibody mediated rejection (AMR) also commonly occur. Identification of non-HLA antigens is therefore critical to increasing understanding of heart transplant rejection. We hypothesize that cardiac non-HLA antigens contribute to AMR and novel proteomic techniques can be utilized to identify multiple antigens simultaneously. Serum from heart transplant patients was collected longitudinally at pre-operation (T1), acute rejection (T2), and post-rejection (T3) time points. Protein was extracted from biopsies of each patient’s donor heart. Human IgG antibodies were isolated with Protein G HP SpinTraps. Resultant human anti-human heart affinity columns were utilized to capture antigenic proteins from individual patient’s heart protein extracts. Proteins eluted from columns were subjected to LC-MS/MS analysis. Proteins were considered antigenic if they significantly increased relative to T1 samples using Gaussian linearized modeling. Non-HLA graft specific antibodies were found to increase at T2 and T3 in all patients. At T2, 82 antigens were identified with a population frequency of 33 to 83%. Over 50% of antigenic proteins were associated with action potential, contractile function, endothelial dysfunction, and other causes of cardiac allograph vasculopathy. The cardiac non-HLA antigens identified in this study further understanding of the immunological mechanisms of transplant rejection and have potential to be utilized as biomarkers for cardiac allograft dysfunction.
Display of CD47 protein on pancreatic islet grafts improves engraftment following intraportal transplantation
Authors: Shrestha Pradeep , Bowen William S. , Batra Lalit , Tan Min , Yolcu Esma S. , Shirwan Haval
Published: January 2023

Intraportal islet transplantation is a curative therapy for type 1 diabetes. However, early islet engraftment is hampered by a thrombotic and inflammatory reaction defined as an instant blood-mediated inflammatory reaction (IBMIR). The CD47 molecule is an important regulator of IBMIR, by blocking phagocytosis. We hypothesized that the transient display of the CD47 protein on the surface of islet grafts may prevent early islet destruction by IBMIR following intraportal transplantation. We generated a chimeric CD47 fused with a modified streptavidin (SA-CD47). SA-CD47 was transiently displayed on the surface of rat splenocytes that had been modified with biotin taking advantage of the strong affinity between biotin and streptavidin (SA). SA-CD47 protein was also effectively displayed on the surface of biotinylated islets. Importantly, in a syngeneic marginal mass intraportal islet transplantation model, 7/8 streptozotocin-diabetic C57BL/6 mice transplanted with 125 SA-CD47 engineered islets established euglycemia long term (>80 days). In marked contrast, only 1/7 mice achieved euglycemia when transplanted with SA-engineered islet grafts. The engrafted islets in the SA-CD47 group had a comparable response to naïve mice in the intraperitoneal glucose tolerance test. Flow cytometric and qRT-PCR analyses of liver tissues did not reveal significant changes in cellular and molecular immune responses between SA-CD47 and SA-engineered control groups, suggesting that inhibition of islet phagocytosis as underlying mechanism of islet engraftment and survival. Collectively, these results demonstrate the utility of SA-CD47 as a regulator of IBMIR for prevention of early islet graft loss following intraportal transplantation
Bioengineering localized and controlled drug delivery to actuate Enhanced Costimulation Blockade and promote long-term transplant survival
Authors: Raimondi Giorgio , Majumder Poulami , Ivanova Vera , Calderon-Colon Xiomara , Mirdad Amr , Lozano Marcos Iglesias , Lee WP. Andrew , Brandacher Gerald , Schneider Joel , Patrone Julia
Published: January 2023

We recently demonstrated that a transient daily administration of the Jak-inhibitor Tofacitinib (Tofa) combined with CTLA4-Ig exerts what we call “enhanced costimulation blockade” and promotes long-term transplant survival in a mouse heart transplant model. To minimize the systemic exposure to Tofa and maximize its immune-modulatory effect, we explored two innovative drug delivery methods: a peptide-based hydrogel (Tofa-Hydro), and solid lipid nanoparticles (Tofa-SLN).

Tofa-Hydro is a safe injectable hydrogel. A one-time application of Tofa-Hydro in the area surrounding the transplanted heart (combined with systemic CTLA4-Ig) rendered long-term graft survival (MST=127d Vs ‘CTLA4-Ig only’ MST=36, P<0.05). Importantly, application of Tofa-Hydro to a distal site ablated this synergistic improvement of survival, demonstrating the importance of the site of delivery. Tofa-SLN showed the unique property of accumulating in the lymphatics, reaching draining lymphoid tissues, and being taken up by immune cells. Tofa-SLN-conditioned dendritic cells were profoundly inhibited in their maturative response to LPS. These cells were less stimulatory for alloreactive T-cell and worked in concert with CTLA4-Ig to minimize T cell activation.

Our results highlight two promising strategies for controlled and localized immune modulation. Injectable hydrogels allow a sustained localized delivery that requires a fraction of the dose of Tofa required when delivered systemic. Tofa-SLN can be passively directed to lymphoid tissues and effectively target and modulate antigen presenting cells. Overall, these strategies can maximize the immunomodulatory impact of Tofa while minimizing its toxic side-effects.
Alloreactive T cells develop in the very early period following human stem cell transplantation and are characterised by a range of novel features
Authors: Moss P. , Verma Kriti , Zuo Jianmin , Malladi Ram
Published: January 2023

Allogeneic stem cell transplantation can cure some patients with hematopoietic malignancy but relies on development of a donor T cell alloreactive immune response which develops very early but is difficult to study due to intense lymphopenia. We characterised the phenotype and functional profile of T cells at day 14 in patients undergoing transplantation. T cells were present at very low number but displayed intense proliferation with strong cytokine production. Oligoclonal expansions at day 14 came to represent a substantial fraction of the established T cell pool and were recruited into tissues affected by graft-versus-host disease. Transcriptional analysis uncovered a range of potential targets for immune manipulation including OX40L, TWEAK and CD70. These findings reveal that recognition of alloantigen rapidly drives naïve T cells towards a unique phenotype characterised by intense functional activation. Moreover they demonstrate that early clonal T cell responses are recruited to sites of subsequent tissue damage and provide a range of novel targets for potential therapeutic immunomodulation.
Soy-enriched diet limits tumor progression and reduces myeloid derived suppressor cells in the murine TRAMP model of prostate cancer.
Authors: Mace Thomas A. , Loftus Shannon , King Samantha , Young Gregory , Geraghy Connor , Fareen Matthew R. , Ware Brandon , Olson Brian , Thomas-Ahner Jennifer , Clinton Steven K. , Lesinski Gregory B.
Published: January 2023

Epidemiological studies suggest diets enriched with soy confer a lower risk of prostate cancer. Data from our group and others indicate that soy has immunomodulatory properties which may contribute to their health promoting effects. Prior results from our group in a randomized phase II clinical trial revealed that men with asymptomatic prostate cancer on a soy-enriched diet had significantly reduced systemic inflammatory cytokines and myeloid-derived suppressor cells (MDSCs). We hypothesized that a soy-enriched diet limits systemic immunosuppression by reducing MDSC expansion. To understand how dietary soy modulates immune biomarkers in prostate cancer, we administered TRAMP (transgenic adenocarcinoma of the mouse prostate) mice a soy-enriched (n=28) or control diet (n=32) for 16 weeks. TRAMP mice administered a soy-enriched diet had a reduced tumor burden compared to control fed mice (p=0.0419). Splenic CD11b+Ly6GloLy6C− granulocytic (p=0.0195), CD11b+Ly6C+Ly6G− monocytic (p=0.0005), and total MDSCs (p=0.0009) were significantly reduced in TRAMP mice on a soy-enriched diet compared to control diet. However, we did not observe differences in circulating CD4+CD25+Foxp3+ T-regulatory cells or other circulating immune subsets such as CD4+ T cells, phenotypically defined T helper-1 (CD4+CXCR3+CCR4−CCR6−)/T helper-2 (CD4+CCR4+CXCR3+CCR6−) T cells, CD8+ T cells, and NK cells. Ongoing studies are examining plasma cytokine levels and immunosuppressive cell populations in the tumor microenvironment of mice from this study. Together, these data indicate that soy as a dietary natural product may have a modulatory effect during chemopreventive or immunotherapy in the setting of prostate cancer.
Opposing roles of CD2 and 2B4 in iNKT cell cytotoxic responses
Authors: Das Rupali , Lee Hyun Hee , Gohl Trevor , Mack Ryan , Griffin Ryan
Published: January 2023

Invariant natural killer T cells (iNKTs) comprise a unique lineage of innate-type lipid-reactive T lymphocytes with important roles in host immunity, including defense against specific pathogens and cancers. iNKTs rapidly produce cytokines and mount potent cytotoxic responses following T cell receptor (TCR) engagement, but the mechanisms that control iNKT cell functions remain poorly understood. CD2 and 2B4 are receptors on iNKTs that both bind to CD48 on target cells and modulate immune activation by facilitating cell-cell interactions and transducing intracellular signals. To determine their roles during iNKT cell activation, we examined the TCR-induced killing activity of murine iNKTs that lacked expression of CD2 or 2B4. Through these studies, we observe that Cd2−/− iNKTs fail to kill target cells while 2b4−/− iNKTs exhibit significantly enhanced cytolysis, both in vitro as well as in vivo. These novel findings suggest that CD2 is a positive and 2B4 a negative regulator of TCR-induced iNKT cell functions. We are currently investigating whether 2B4 exerts its inhibitory role by directly competing with CD2 for CD48 binding and/or by inducing a negative signal. As CD1d (the ligand for the invariant TCR) and CD48 (the ligand for CD2 and 2B4) are both widely expressed on hematopoietic tumors, further elucidation of the mechanisms by which CD2 and 2B4 regulate TCR-induced iNKT cell anti-tumor activity is of significant scientific and clinical importance. The results obtained from these studies will have an impact by establishing new paradigms for iNKT signaling and offering insights into how iNKTs can be best activated to enhance host immunity and treat hematopoietic cancers such as leukemia and lymphoma.
Engineering Chimeric Antigen Receptor T-cells Against Immune Checkpoint Inhibitor PD1 and PD-L1 for Treating Pancreatic Cancer
Authors: Fan Ming huei , Fan Kai Fang , Liu Chao-Lien
Published: January 2023

Pancreatic ductal adenocarcinoma (PDA) is a serious and aggressive disease with 5-year survival rate of 6%, the lowest of any tumor type routinely checked. Immune checkpoint inhibitor pathway PD1 (Programmed-Death-1) /PD-L1 (Programmed-Death-1 Ligand-1) axis is responsible for inhibitory T cell signaling and contributes to the maintenance of immune homeostasis. PD1/PD-L1 pathway plays an essential role in the tumor microenvironment and blockade of PD1/PD-L1 by monoclonal antibodies has success in the treatment of several tumor types. However, even though PD-L1 is expressed on pancreatic cancer cells, current clinical trial using PD1/PD-L1 antibodies blockade failed to show therapeutic effects. In this study, we first generated both PD1ACR (activating chimeric receptor)-T and PDL1CAR (chimeric antigen receptor)-T cells, elucidate the molecule mechanisms leading to the development of effector cells as well as to test the cytotoxicity function of these CAR-T cells. CFPAC1, a high PDL1-expressed cell line, was checked among four pancreatic cancer cell lines including, CFPAC1, SU8686, Capan1 and ASPC1 using flow cytometry and western blot analysis. The in vitro cytotoxicity activities towards CFPAC1 were significantly in the PD1ACR-T (35%) and PDL1CAR-T (55%) at E/T (effector/tumor) ratio of 10 compared to the controls. Furthermore, in tumor xenografts of NSG mice, both PD1ACR-T and PDL1CAR-T treated mice groups showed significantly suppressed tumor growth at 59% and 77%, respectively. Therefore, we concluded that PD1/PDL1 mediated CARs possess dramatic cytotoxic activities towards PDL1-expressed pancreatic cancers both in vitro and in vivo, which strongly support the potential clinical immunotherapeutic applications.
Regulatory T cells modulate immune cells and promote tumor growth in prostatic tumor microenvironment
Authors: Kumar Sanjay , Stokes James , Malik Shalie , Singh Udai P. , Singh Rajesh , Ponnazhagan Selvarangan , Manne Upender , Singh Santosh Kumar
Published: January 2023

Several tumors express antigens that can be recognized by T lymphocytes, suggesting that T cell-mediated immune response may play a critical role in anti-tumor immunity. Studies demonstrated that Regulatory T cells (TR) suppress the functions of immune cells, which are capable to recognized and eliminate tumor cells. However, there exists an utmost demand for further research how immune cells (CD8+ cells, NK cells, etc.) fail to suppress tumor growth and proliferation in the prostatic tumor microenvironment. To investigate this, tumor induction studies in C57/BL-6 mice using transgenic adenocarcinoma of mouse prostate (TRAMP cell lines: -C1, -C2 and -C3) as tumor models were performed. TRAMP-C3 did not form tumor while TRAMP-C1 and TRAMP-C2 cells are tumorigenic. For tumor induction, mice were administered with TRAMP (C1-3) cells. Mice were sacrificed by cervical dislocation when tumor sizes reached ~18–20mm in diameter. A single cell suspension (obtained from spleen, draining lymph node, and tumor) was stained with various cell surface/intracellular immune markers/cytokines to enumerate CD8+ T/TR/NK/Ly6G/Ly6C+ -MDSC cells. Additionally, the expression of TGF-β, IFN-γ, IL-4, IL-17, PD-1 and PDL-1 and cell cycle analysis for CD8+ T cells was also carried out. Our findings suggest that recruitment of suppressive TR and Ly6G/Ly6C+ -MDSC cells and high expression TGF-β and PD-1 and lower expression of NKG2D and IFN-γ and low number of NK and CD8+ cytotoxic T cells in spleen, draining LN and tumor of TRAMP-C1 and TRAMP-C2 (as compared to TRAMP-C3 mice) facilitated tumor growth. Therefore, the expression of PD-1 and TGF-β on TR suggest that TR might be playing a significant role in facilitating tumor growth and progression.
Uncoupling Therapeutic from Immunotherapy-related Adverse Effects for Safer and Effective Anti-CTLA-4 Antibodies in CTLA4 Humanized Mice
Authors: DU XUEXIANG , Tang Fei , Liu Mingyue , Liu Yang , Zheng Pan
Published: January 2023

Anti-CTLA-4 monoclonal antibodies (mAbs) confer cancer immunotherapeutic effect (CITE) but cause severe immunotherapy-related adverse effect (irAE). The irAE greatly limited application of immunotherapy targeting the CTLA-4 molecules. However, targeting CTLA-4 has shown remarkable long-term benefit and thus remains a valuable tool for cancer immunotherapy, especially if the irAE can be brought under control. A major obstacle to this goal is lack of animal model that recapitulate clinical irAE. Here we report that mice with the humanized CTLA4 gene recapitulated cardinal features of clinically observed irAE and CITE in humans. Of two anti-human CTLA-4 mAbs with comparable CITE, Ipilimumab but not L3D10 induced severe irAE, when used either alone or in combination with anti-PD-1. The irAE, but not CITE, corresponds to systemic T cell activation and reduced Treg/Teff ratios among autoreactive T cells. While Ipilimumab+anti-PD-1 induces irAE and CITE in mice homozygous for human CTLA4, the same treatments induced CITE without irAE in mice heterozygous for human and mouse CTLA4 alleles, thus revealing a distinct genetic requirement for irAE and CITE. Humanization of L3D10 that leads to loss of blocking activity further increased safety without affecting the therapeutic effect. Taken together, our data demonstrate that complete CTLA-4 occupation, systemic T cell activation and preferential expansion of self-reactive T cells are dispensable for tumor rejection but correlate with irAE, while blocking B7-CTLA-4 interaction impacts neither safety nor efficacy of anti-CTLA-4 antibodies. These data provides important insights for clinical development of CTLA-4 targeting immunotherapy.
Identification and characterization of novel mediators of tumor-induced T-cell dysfunction
Authors: Markowitz Geoffrey J. , Philip Mary , Wong Stephen , Schietinger Andrea , Mittal Vivek
Published: January 2023

Immunotherapies targeting T-cell functionality, such as anti-PD-1, have been shown to have efficacy in numerous cancers, including non-small cell lung cancer (NSCLC). However, treatment with anti-PD-1 induces responses in only a subset of patients. This raises the possibility that other effectors of T-cell dysfunction remain to be elucidated. To evaluate possible novel mediators of T-cell dysfunction, we isolated CD8+ T-cells from orthotopic KrasG12D/p53−/− murine lung adenocarcinomas (HKP-1) that exhibited either stable or progressive disease as a function of tumor growth, both in the presence and absence of anti-PD-1 therapy. RNAseq analysis revealed increased expression of numerous genes including Tim3, PD-1, Lag3, 2B4, and CISH, and decreased expression of GzmB and Eomes in CD8+ T-cells isolated from progressive tumors indicative of a dysfunctional state, as well as multiple novel genes not previously associated with T-cell dysfunction. To explore the functional role of the novel candidate genes, we have developed in vitro and in vivo assays evaluating cytotoxicity, cytokine production, proliferation, and tumor growth, leveraging Ova expression in our HKP-1 cell line (KP1-Ova). We have begun to target this unique set of candidate genes associated with dysfunction in tumor-specific T-cells through RNAi/shRNA and adoptive transfer approaches to facilitate therapeutic reprogramming and enhanced T-cell-mediated immunity in lung cancer. We expect to provide novel strategies for designing rational immunotherapies targeting dysfunctional tumor-specific CD8+ T-cells to generate durable anti-tumor immune responses for the treatment of lung adenocarcinoma.
Manipulating the epigenetic framework of T cells with histone deacetylase inhibitors for more robust and durable anti-tumor responses
Authors: McCaw Tyler R. , Liu Mingyong , Li Mei , Starenki Dmytro , Cooper Sara , Arend Rebecca , Forero Andres , Buchsbaum Donald , Randall Troy
Published: January 2023

Malignant cells often harbor an imbalance in histone acetyltransferase and histone deacetylase (HDAC) activity, which can be disrupted by HDAC inhibitors. We hypothesized that HDAC inhibition can boost anti-tumor T cell responses by modifying tumor cell phenotypes as well as directly altering the transcriptional framework of tumor-specific T cells. To test this, we treated two murine breast cancer cell lines, TS/A and 4T1, with class I (entinostat), or pan-specific (panobinostat), HDAC inhibitors. Culture of tumor cells with either inhibitor increased surface expression of MHC and costimulatory molecules as well as T cell chemoattractants. Tumor growth was significantly impaired by HDAC inhibition in a manner absolutely dependent on adaptive immunity, specifically CD8 T cells and IFNγ production, but not CD4 T cells or B cells. Indeed, CD8 T cell production of effector cytokines was dramatically augmented. This enhanced effector function was paralleled by an increase in T-bet and a decrease in Eomes, consistent with a transcriptional program that is less susceptible to exhaustion. We also found that simply adjusting timing of HDAC inhibition relative to T cell activation could abolish anti-tumor effects or lead to rejection in nearly 50% of mice. These findings corresponded with a significant shift in the responding T cell repertoire. Collectively, our data suggests that HDAC inhibition reprograms the transcriptional profile of T cells in ways that impact priming, improve effector functions, and reduce susceptibility to exhaustion. Thus, appropriately timed HDAC inhibition may synergistically potentiate existing tumor immunotherapies, like T cell reinvigoration strategies.
Immunosuppressive effects of sMIC abrogate immunotherapy efficacy in a mouse model of breast cancer
Authors: Berkley Amy M. , Toy Evangeline , Cook Ryan , Ye Zhengmao , Grogan Jane , Schartner Jill , Kim Jeong
Published: January 2023

Expression of the NKG2D activating receptor on CD8+ T cells and NK cells is thought to play an important role in detection and clearance of tumor cells. The soluble version of an NKG2D ligand in humans, MHC I chain-related molecule (sMIC), has been correlated with poor prognosis in multiple human cancers and hypothesized to serve as a tumor immune evasion strategy. Yet, it is unclear whether sMIC impairs the activity of T cell directed immunotherapies including checkpoint inhibitors. We utilized a syngeneic mouse model of breast cancer, EMT6, as well as EMT6 engineered to express high levels of sMIC to examine the impact of sMIC on responses to anti-PDL1/anti-TIGIT combination immunotherapy. Following treatment with anti-PDL1/anti-TIGIT combination immunotherapy, WT EMT6 tumors were rejected and tumor associated T cells and NK cells isolated from these tumors showed increased IFNγ secretion. In contrast, mice bearing EMT6 tumors expressing sMIC and treated with the same regimen were unable to reject tumors. Consistent with this observation, CD8+ and NK cell TILs from EMT6. sMIC tumors showed decreased ability to produce IFNγ compared to those from WT EMT6 tumors. Taken together these results suggest the importance of targeting the MIC-NKG2D pathway to further improve combination immunotherapy efficacy in patients.
Canonical Wnt signaling in Treg contributes to colon cancer
Authors: Osman Abu , Saadalla Abdulrahman , Quand Jasmin , Gounari Fotini , Pavelko Kevin , Khazaie Khashayarsha
Published: January 2023

Regulatory T-cells (Tregs) play a dual role in colon cancer, promoting immune tolerance and suppressing inflammation. Two types of Tregs infiltrate the gut. Thymic derived Helios+CD4+Foxp3+ Tregs have high affinity for self-antigens, including tumor-antigens and protect the tumor, while Helios−CD4+Foxp3+ extra-thymic Tregs that are generated in the gut have microbial specificity and suppress harmful inflammation. In colon cancer, there is a preferential expansion of a population of Helios−CD4+Foxp3+RORγt+ Tregs with enhanced T-cell suppressive properties and pro-inflammatory properties that promote tumor growth. We showed that this population of Tregs in cancer patients expresses elevated levels of β-catenin. To test the role of canonical Wnt signaling in the generation of pro-inflammatory Tregs, we stabilized β-catenin or deleted its DNA binding partner TCF-1 in Tregs. In both instances expression of RORγt was upregulated. While the stabilization of β-catenin led to loss of Treg functions, ablation of TCF-1 reproduced the phenotype and functional characteristics of Tregs in colon cancer. The TCF-1 deficient Tregs were pro-inflammatory, potent T-cell suppressive, and tumor promoting.
IL-21 selectively protects CD62L+ NKTs from activation-induced cell death during ex vivo expansion and enhances antitumor activity of NKT cell therapy in vivo
Authors: Ngai Ho , Tian Gengwen , Courtney Amy N. , Marinova Ekaterina , Huang Wei , Guo Linjie , Liu Bin , Metelitsa Leonid S.
Published: January 2023

Vα24-invariant natural killer T cells (NKTs) have potent antitumor properties that can be exploited for use in cancer immunotherapy. This requires rigorous protocols for ex vivo expansion of primary NKTs that preserve their longevity and function. We recently reported that CD62L+ NKTs persist longer and have better antitumor activity in vivo than CD62L− counterparts. However, the conditions governing preservation of CD62L expression in NKTs during expansion remain largely unknown. Comparative gene expression analysis of CD62L+ and CD62L− NKTs revealed significantly higher IL-21R expression in the former subset, which was confirmed at the protein level by flow cytometry. Hence, we hypothesized that IL-21 preferentially supports CD62L+ NKTs. We expanded primary human peripheral blood NKTs by stimulating in vitro with cognate antigen, α-galactosylceramide, and supplemented the culture with IL-2, IL-21, or both cytokines. In contrast to IL-2, IL-21 alone failed to support NKT cell expansion, but combined treatment with IL-2 and IL-21 produced more NKTs and preserved a significantly higher frequency of CD62L+ cells. We further demonstrated that IL-21 selectively downregulates BIM in CD62L+ NKTs and protects the subset from activation-induced cell death. Functionally, IL-2/IL-21-expanded NKTs secreted more TH1 cytokines and were more cytotoxic against lymphoma cells, which was associated with granzyme B upregulation by IL-21. Following adoptive transfer to NSG mice, IL-2/IL-21-expanded NKTs persisted significantly longer and had higher therapeutic efficacy in a lymphoma model compared with IL-2-expanded NKTs. Our results instruct inclusion of IL-21 in NKT-cell expansion protocols for cancer immunotherapy applications.
Drugging drug resistance with bystander-assisted immunotherapy.
Authors: Mancini Rock , Nielsen Amy E. , Hantho Joseph D. , Burt Anthony J.
Published: January 2023

Acquired drug resistance is a longstanding challenge that reduces the efficacy of chemotherapeutic drugs. In contrast, the efficacies of modern immunotherapies do not directly correlate to the prevalence of chemo-resistant phenotypes. Here we improve upon this paradigm by targeting the action of immunotherapeutics to two general mechanisms of acquired drug resistance: irregular metabolism and drug efflux mediated by the ABC superfamily of transport proteins. In chemo-resistant prostate cancer cells, we demonstrate that these two mechanisms act in concert to selectively metabolize our newly developed class of enzyme-directed prodrug to the Toll-Like Receptor 7 immunotherapeutic Imiquimod. Following metabolism, liberated Imiquimod undergoes drug efflux to the extracellular space where it activates bystander immune cells in local proximity. In-vitro, we characterize this process of Bystander-Assisted ImmunoTherapy (BAIT) using an AT3B-1 chemo-resistant prostate cancer model with RAW-Blue and JAWSII reporter immune cell lines. Co-culture of AT3B-1 cancer cells with reporter immune cells results in immunogenicity mediated selectively by cancer cells. This results in enhanced NF-κB transcription, expression of cell surface markers, and secreted cytokines indicative of a cell-mediated immune response. Our prodrug is non-immunogenic with healthy cells alone and the enzyme-directing groups are stable for over 3 days in serum. Taken together, our results demonstrate that BAIT co-opts common mechanisms of drug resistance to elicit immunogenicity mediated by cancer cells themselves. We anticipate that BAIT will find use as a new mechanism of action that exploits drug resistant phenotypes to generate an immune response.
Non-cellular mechanism of peripheral immunosuppression as a novel feature in experimental glioblastoma
Authors: Khadka Roman H. , Ayasoufi Katayoun , Jin Fang , Malo Courtney Sarah , Desai Nidhi , Johnson Aaron J.
Published: January 2023

Peripheral immune suppression is a hallmark of highly malignant Glioblastoma multiforme (GBM). Given that effective immunotherapy for GBM relies on a functional immune system, it is crucial to define the underlying mechanism of peripheral immunosuppression. We therefore evaluated immunosuppression using the GL261 model of GBM. We first examined cytokine production by microglia and brain-infiltrating lymphocytes for immune suppressing factors. We observed relatively unchanged levels of IL-10 and TGF-β in glioma bearing mice compared to controls. Therefore, we next investigated cellular components of the blood for evidence of suppression. However, we did not observe enhanced frequencies of Tregs (Foxp3+ CD4+) in tumor bearing mice. Finally, we analyzed cytokine levels in the serum of mice harboring gliomas compared to glioma negative animals. This assay revealed fluctuations in chemokine and cytokine levels between these two groups, prompting the analysis of a potential serum derived factor in promoting immunosuppression. We therefore evaluated T cell activation and proliferation in the presence of serum isolated from glioma harboring mice and healthy controls. CFSE-labeled T cells stimulated with CD3/CD28 beads in the presence of glioma negative mouse serum had normal proliferation. In contrast, CD4+ and CD8+ T cell proliferation was completely inhibited when cultured with serum from glioma bearing mice. These studies demonstrate the importance of a serum derived factor in mediating immunosuppression. Defining and inhibiting this circulating factor could be the first step towards improving immunotherapy strategies for GBM.
Identification of dysfunctional CD8+ T-cell subsets rescued by PD-L1 blockade in the tumor microenvironment
Authors: Yamauchi Takayoshi , Hoki Toshifumi , Odunsi Kunle , Ito Fumito
Published: January 2023

While depletion studies using mice clearly show that CD8+ T cells are of crucial importance in anti-tumor efficacy, gaps in understanding the exact mechanisms that regulate CD8+ T cell responses in the tumor microenvironment (TME) are a major impediment in efforts to improve immunotherapy. We have evaluated phenotypical and functional heterogeneity of tumor-infiltrating lymphocytes (TILs) after adoptive transfer of ex vivo primed Pmel-1 T-cell receptor transgenic CD8+ T cells specific for the gp100 antigen expressed on B16 melanoma. We found that the chemokine receptor, CX3CR1 identifies distinct effector CD8+ T-cell subsets, CX3CR1 negative (−), intermediate (int), and high (hi) in the TME. A CX3CR1hi subset contained terminally-differentiated CD8+ T cells that express higher levels of killer-cell lectin like receptor G1 (KLRG1), TNF-related apoptosis-inducing ligand (TRAIL), perforin, and granzyme A. Significantly more CX3CR1int CD8+ T cells expressed CD25 compared to other subsets, suggesting this is the subset that is rapidly proliferate and generate terminally-differentiated T cells. Unexpectedly, despite their terminally differentiated status, a CX3CR1hi subset expressed significantly lower levels of co-inhibitory receptors, PD-1, LAG3, and TIGIT compared to CX3CR1− and CX3CR1int subsets in the TME. In line with this, proliferation and cytokine production of CX3CR1− and CX3CR1int subsets were significantly decreased in the TME compared to a CX3CR1hi subset. Importantly, functional burst after PD-L1 blockade came almost exclusively from CX3CR1− and CX3CR1int subsets. These findings provide a better understanding of dysfunctional CD8+ TILs and can be exploited to develop more effective cancer immunotherapy.
Inhibition of intrinsic coagulation improves immune checkpoint-targeted gene delivery of cationic solid lipid nanoparticles
Authors: Sun Tianmeng , Wu Xuan , Yang Yong Guang
Published: January 2023

Tumor-targeted delivery of CRISPR/Cas9 plasmid or siRNA to inhibit immune checkpoint protein expression in tumor cells has the potential to improve anticancer immune responses. Cationic solid lipid nanoparticles (cSLNs) are promising carrier for the delivery of negatively charged nucleic acids. cSLNs with high surface charge and low PEG density have been demonstrated to enhance transfection efficiency in vitro, but for unknown reasons fail to improve gene delivery in vivo. We prepared 9 kinds of cSLNs with varying surface charge and PEG density and detected the interactions of these cSLNs with blood cells. We found that increasing surface charge and/or reducing PEG density present a risk for systemic platelet aggregation and activation. Thrombosis was clearly detected in liver, lung and kidney from mice treated with cSLNs with high surface charge and low PEG density. Mechanistic studies revealed that the intrinsic coagulation pathway was responsible for cSLN-induced thrombus formation. Furthermore, pretreatment of recipient mice with heparin, a clinically-approved intrinsic coagulation inhibitor, was highly effective in preventing toxicity, prolonging the circulation time, improving tumor accumulation and enhancing the tumor cell uptake in tumor-bearing mice. These results indicate that the thrombotic toxicity of cSLNs is an important factor affecting their drug delivery efficiency in vivo, and that heparin treatment offers a useful strategy for improving cSLN-based cancer immunotherapies.
Porcine SIRPA binds to human CD47 to inhibit phagocytosis: Implications for human hematopoietic stem cell transplantation into severe combined immunodeficient (SCID) pigs
Authors: Boettcher Adeline N. , Cunnick Joan E. , Powell Ellis J. , Egner Timothy K. , Charley Sara E. , Loving Crystal L. , Tuggle Christopher K.
Published: January 2023

Xenotransplantation success with human hematopoietic stem cells into non-obese diabetic (NOD)-derived severe combined immunodeficient (SCID) mice is dependent on the ability of NOD mouse signal regulatory protein alpha (SIRPA) to bind human CD47 (hCD47). Interaction between NOD mouse SIRPA and hCD47 results in higher phagocytic tolerance compared to other SCID mouse strains. In the context of developing a humanized SCID pig, we investigated whether porcine SIRPA binds hCD47 and subsequently inhibit phagocytosis of human cells. Porcine SIRPA bound to hCD47 in vitro. Porcine monocytes phagocytosed human and porcine red blood cells (RBCs) at significantly lower levels than mouse RBCs. When human RBCs were pre-incubated with CD47 blocking antibody B6H12 or non-blocking antibody 2D3, phagocytosis was induced only after B6H12 incubation, indicating the lower phagocytic activity requires interaction between porcine SIRPA and hCD47. Collectively, we provide the first evidence that porcine monocytes may be phagocytically tolerant towards human cells, suggesting that porcine SCID models have the potential to support engraftment of human stem cells.
Analysis of T cell antigen receptor expression by porcine natural killer T cells
Authors: Yang Guan , Artiaga Bianca Libanori , Jayaprakash Anitha , Sachidanandam Ravi , Driver John
Published: January 2023

Invariant natural killer T (iNKT) cells are an innate-like T cell subset expressed by some, but not all mammals. Unlike conventional T cells, iNKT cells express a semi-invariant T cell receptor (TCR) (Vα24-Jα18/Vβ11 in human and Vα14-Jα18/Vβ8.2, Vβ2, or Vβ7 chains in mice) that recognizes glycolipid antigens presented by the non-polymorphic molecule CD1d. Once activated, iNKT cells generate an extensive array of immune responses that may be useful for treating cancer, autoimmunity and infectious disease in humans. In addition, there may be potential to target iNKT cells for veterinary applications. Pigs express iNKT cells that can be detected using mouse or human CD1d-tetramer reagents loaded with the prototypic iNKT cell ligand, α-galactosylceramide (α-GalCer). We exploited this phenomenon to define the TCR repertoire of iNKT cells (CD3α+CD1d tetramer+) compared to conventional T cells (CD3α+CD1d tetramer−) sorted from the peripheral blood of commercial mixed-breed pigs. We found that iNKT cells are highly enriched for the α-chain and CDR3α loop from Vα87*-Jα60* that possess a high level of amino acid sequence homology with human Vα24 (85%) and Jα18 (86%) as well as mouse Vα14 (74%) and Jα18 (79%). Particularly striking was that the most abundant CDR3α sequence was a near perfect match for the human invariant CDR3α. Porcine iNKT cells were also biased for Vβ84* which is homologous to Vβ11 (74%) in humans, as well as Vβ8.2 (71%) and Vβ2 (80%) in mice. Our findings indicate that the porcine iNKT cell TCR is highly homologous to mouse and particularly human iNKT cell TCR, which suggests that iNKT cells among these species play a similar role in immunity and disease.
Characterization of the immune reagent chicken IL-16
Authors: Grant Ar’Quette , Kim Woohyun , Lillehoj Hyun S.
Published: January 2023

Interleukin-16 has been characterized as a pro-inflammatory cytokine that mediates an immune response in human and mouse monocytes and peripheral blood mononuclear cells (PBMC), and it plays a role in proliferating B-cells and mylomas. The function of chicken IL-16 ortholog (ch-IL-16) is far less understood and only shares 52% and 49% sequence homology with human and mouse, respectively. Although it has previously shown chemotaxis abilities in chicken splenocytes, the low sequence homology to their mammalian counterparts, the current study was undertaken to characterize the function of yeast-expressed ch-IL-16 as an immune reagent in pro-inflammatory cytokine production and cell proliferation. In a dose dependent manner, ch-IL-16 was applied to 5 × 106 cells/mL PBMC and HD11 chicken monocytes, and the extracted RNA was used to determine the pro-inflammatory cytokine production. Also in a dose dependent manner, ch-IL-16 was applied to primary bursal lymphocytes, PBMC, and RP-9 chicken myeloma cells. Ch-IL-16 produced pro-inflammatory cytokines: IL-6, IL-1β, and TL1A which is the chicken equivalent to TN1-α at an optimal concentration between 40 and 50 ng/mL. We are still in the process of analyzing the proliferation effects of ch-IL-16 on bursal lymphocytes, PBMC and RP-9. Future directions also include the production of monoclonal antibodies to neutralize the function of ch-IL-16. These results would be useful in the further characterization of the chicken immune system, and the role of ch-IL-16 as a mediatory cytokine.
Resistance of field-isolated porcine epidemic diarrhea virus to interferon and neutralizing antibody
Authors: Park Jung-Eun , Shin Hyun-Jin
Published: January 2023

Porcine epidemic diarrhea virus (PEDV) infects pigs and causes enteric disease characterized by watery diarrhea and dehydration. Despite the extensive vaccination, PEDV strains with variations in the spike gene have been reported in several countries, including Korea, China, and the United States. Variant PEDV belonging to genogroup G2 has higher pathogenicity and morbidity than conventional PEDV belonging to genogroup G1. To understand the pathogenesis of the variant PEDV, we examined the susceptibility of the variant PEDV to interferon and PEDV specific neutralizing antibodies, which are known to important for the control of PEDV infection. We found that variant PEDV (WG strain) showed higher resistance to interferon than conventional PEDV (KPEDV-9 strain). Similarly, KPEDV-9 strain grew well in interferon-deficient Vero cells but did not grow in interferon-releasing porcine alveolar macrophages, whereas WG strain showed similar infectivity in both cells. We also found that WG strain is not blocked by the neutralizing antibodies against KPEDV-9, suggesting the differences in the antigenicity of two strains. To elucidate the possible mechanism of resistance to interferon and neutralizing antibody in variant PEDV, sequencing analysis was performed. We found huge variations in spike and ORF3 proteins. Our results indicate that the recently introduced variant PEDV exhibits higher resistance to the interferon than the existing PEDV and avoids the neutralizing antibody against the existing PEDV. These results also provide understanding of the occurrence of variant PEDV and its pathogenesis.
Identification of a DRB3*1101-restricted CD4 T cell response against bovine respiratory syncytial virus
Authors: Hofstetter Amelia R. , Yadav Akshay , Norimine Junzo , Sacco Randy E.
Published: January 2023

A better understanding of the immune response elicited by bovine respiratory syncytial virus (BRSV) vaccines is needed for vaccine improvement. Although killed-BRSV vaccines are available as part of multivalent products, their efficacy is controversial. We screened for BRSV-specific T cell responses in cows with a history of annual vaccinations. We defined the minimum BRSV F peptide 261–270 (INDMPITNDQ), which elicits IFN-γ from CD4 T cells in four of fourteen cows tested. The response is restricted by the bovine class II MHC molecule DRB3. All four cows share the DRB3*1101 allele. We are currently developing a specific DRB3*1101:INDMPITNDQ tetramer to facilitate further characterization of this response.
Host antigen modulated anti-influenza immunity with antigen level dependent distinct mechanisms of severe influenza
Authors: Huang Ching-Tai , Dutta Avijit , Huang Chi-Wei , Chen Tse-Ching , Lin Chun-Yen , Lin Yung-Chang , Chang Chia-Shiang , He Yueh-Chia , Huang Yu-Lin , Chen Ting-An
Published: January 2023

Some healthy individuals suffer from severe disease and even mortality with the same strain of influenza virus that causes self-limited illnesses in the majority of people. This fact exemplifies the importance of host factors in influenza disease manifestation. Our C3-HA transgenic mice express hemagglutinin (HA) as a self-antigen that is identical to the HA of PR8 influenza virus. Compared to wild type mice, C3-HA mice incurred severe disease with PR8, but not with HA-mismatched influenza virus. Self-HA in C3-HA mice altered the immune response to the infection and impaired clearance of the influenza virus. However, different mechanisms contribute for exacerbated disease in C3-HA mice with different level of HA. In C3-HALow mice, low-level self-HA modulated HA specific immunity upon infection with less clonotypic expansion, T-bet induction and effector cytokines IFN-γ and TNF-α production of both HA-specific CD4+ and CD8+ T cells on day 4- post infection. The T cell activation status contracted further on day 7- post infection. Impaired immunity resulted in delayed virus clearance leading to severe disease and death. In C3-HAHigh mice, high-level self-HA also modulated impaired immunity and virus clearance on day 4- post infection. In contrast to further reduction on day 7- post infection in C3-HALow mice, the HA specific T cell immunity was higher on day 7- than day 4- post infection in C3-HAHigh mice. The boosted HA specific immunity resulted in severe disease with autoimmunity as a predominant feature in pathology. Our animal model exemplified the complicated interaction between cross-reactive self-antigen and influenza virus with severe disease as the results of antigen level dependent distinct mechanisms.
Single-cycle respiratory syncytial virus infection induces robust adaptive immune responses and reduces disease severity in mice
Authors: Schmidt Megan E. , Oomens Antonius G.P. , Varga Steven M.
Published: January 2023

Respiratory syncytial virus (RSV) is the leading cause of severe respiratory tract infection in infants and young children. However, despite extensive efforts, there remains no licensed RSV vaccine. Recently, a recombinant infectious RSV strain lacking the gene for the matrix (M) protein (M-null RSV) was developed as a potential vaccine candidate. The M protein is required for reassembly of the virion following infection and gene expression within a host cell. Infection with M-null RSV produces viral proteins but does not result in the generation of infectious virus progeny resulting in a single-cycle infection. To determine its immunogenicity in vivo, mice were infected with either M-null RSV or a wild-type (WT) RSV. M-null RSV-infected mice exhibited significantly reduced lung viral titers, weight loss, and pulmonary dysfunction compared to mice given WT RSV. Remarkably, M-null RSV infection induced CD4 and CD8 T cell responses in the lung and airways of similar magnitude to that of a WT RSV infection at acute time-points. Germinal center B cell numbers and serum antibody levels were also similar between M-null and WT RSV-infected mice. M-null RSV infection also generated robust memory immune responses. A substantial proportion of B cells, CD4 T cells, and CD8 T cells remained within the lung tissue of M-null RSV-infected mice 30 days post-infection. Importantly, RSV-specific memory CD8 T cells expressing CD69 and CD103, the canonical markers of tissue resident memory, were also generated by M-null RSV infection. Given the reduced disease severity and robust immune responses developed following single-cycle infection, M-null RSV may be an effective vaccine candidate.
Intranasal infection by MCMV reveals key roles for viral evasion of MHC-I to enable viral spreading and CD4 T cell help to promote functional CD8 T cell responses
Authors: Zhang Shunchuan , Snyder Christopher M.
Published: January 2023

Natural Cytomegalovirus (CMV) infection occurs primarily through the oral/nasal mucosa. Recent evidence from murine (M)CMV infection suggested that MCMV is spread to salivary gland (SG) by dendritic cells after intranasal (i.n.) inoculation. In agreement with this, a recombinant MCMV, which cannot replicate in hematopoietic cells, could not spread to SG after i.n. infection. However, the mechanisms by which infected cells evade immune control while spreading the virus are unclear. In fact, previous work suggested that deleting all three genes blocking MHC-I antigen presentation results in only a subtle defect in total latent viral loads and SG replication in vivo. Surprisingly, we found that viral evasion of MHC-I antigen presentation was critical for MCMV to replicate persistently in nasal mucosa and spread to SG after i.n. inoculation, in contrast to intraperitoneal (i.p.) infection. Remarkably, depletion of CD4 T cells reversed this immune restriction on viral spreading, enabling even MCMV lacking all evasion genes to replicate in nasal mucosa and spread to SG after i.n. infection. This was not due to a direct anti-viral effect of CD4 T cells as delaying CD4 T cell depletion until day 7 post infection restored immune control of MCMV at later times. Instead, priming and expansion of functional CD8 T cells was impaired by lack of CD4 T cells after i.n. infection, in contrast to i.p. infection. Thus, intranasal inoculation revealed a critical role for CD4 T cell help in the promotion of protective CD8 T cell responses. Moreover, our data suggest since MCMV spreads only in infected hematopoietic cells after i.n. infection, viral evasion of MHC-I is necessary to protect CD8 T cells from killing infected cells carrying MCMV.