bluuebunny/crossref_metadata_2025 huggingface dataset:
Goblet cell Hypoxia-Inducible Factor (HIF) is necessary for mucin processing and maintenance of mucosal integrity
Authors: Whitney Alyssa K , Schwisow Kayla D , Bayless Amanda J , Golden-Mason Lucy , Mehta Gaurav , Kuhn Kristine A , Colgan Sean P , Campbell Eric L
Published: January 2023

Secreted mucus is essential for intestinal homeostasis, separating luminal bacteria from the colonic epithelium and preventing inappropriate inflammation. Perturbations in goblet cell mucus secretion have been shown to lead to spontaneous colitis in mice. Treatment of mice with a prolyl hydroxylase inhibitor (AKB-4924) leads to stabilization of hypoxia inducible factor-1 alpha (HIF-1α) and regulation of hypoxia-responsive genes that promote mucosal protection. Interestingly, goblet cell hyperplasia was observed following in vivo administration of AKB-4924, which may account for the mucosal protection observed. Thus, we hypothesized epithelial stabilization of HIF-1α leads to goblet cell differentiation.

Utilizing 3D murine colonic organoid system, treated with a gamma secretase inhibitor (DBZ), AKB-4924 or hypoxia, we analyzed changes in differentiation markers by qPCR. Contrary to our hypothesis, neither AKB-4924 nor hypoxia resulted in the induction of goblet cell differentiation factors. Furthermore, mice lacking HIF-1α in the intestinal epithelium (HIF-1αfl/fl-VillinCRE) exhibited normal colonic goblet cell numbers. However, these goblet cells appeared less organized with diffuse mucin granules. Fluorescent in situ hybridization with a eubacterial probe revealed diminished thickness, with reduced proximity to the microbiome in HIF−/− mice. Finally, profiling the composition of the mucus layer revealed significantly altered expression profile of both secreted and adherent mucins.

We conclude that epithelial HIF-1α alters goblet cell function, regulating intestinal mucus, which may contribute to the mucosal-protective role of HIF.
CX3CR1 mononuclear phagocytes utilize the microbiota to promote balanced intestinal immune responses
Authors: Diehl Gretchen , Hill-McAlester Andrea , Ochoa Karina , Galan Carolina
Published: January 2023

The development and proper functioning of the mammalian immune system relies on a reciprocal relationship between the host and the microbiota. These interactions shape both the composition of the microbiota along with the development and responsiveness of the immune system. Such a relationship requires mechanisms to reduce inflammatory immune responses within the intestine, which can damage the epithelial barrier and lead to the development of disorders such as inflammatory bowel disease. We find that CX3CR1 expressing mononuclear phagocytes (MNPs) are critical for maintaining this intestinal homeostasis. Using mice where we can selectively deplete CX3CR1+ MNPs in vivo, we demonstrate that CX3CR1+ MNPs in the lamina propria promote epithelial repair and, in models of colitis, limit TH1 responses against the microbiota itself. We also find they have functions in immune inductive sites such as the MLN where they limit the generation of inflammatory TH1 immune responses against pathogenic bacteria. Finally, we find they are required for the induction of antigen specific TH17 responses against members of the microbiota such as segmented filamentous bacteria (SFB). We find that the intact microbiota is critical for these homeostatic roles. After disruption of the microbiota with antibiotics, CX3CR1+ MNPs now promote inflammatory immune responses and are unable to promote epithelial barrier repair. Understanding this crucial relationship between the microbiota and intestinal immune cells will allow for selective targeting to modulate responsiveness of the intestinal immune system and promote homeostasis.
Intestinal resident CX3CR1+ cells support gut immune system by helping production of IgA from B cells
Authors: Kim Young-In , Lee Eun-Je , Chang Sun-Young
Published: January 2023

Intestinal resident CX3CR1+ cells are known to sample neighboring or circulatory antigens and present them to CD8+ T cells via cross-presentation. Here we show that intestinal CX3CR1+ cells co-cultured with B cells can induce production of IgA. Unlike intestinal CD103+ DCs, CX3CR1+ cells are independent of retinoic acid and signals from commensal microbiota to induce production of IgA. CX3CR1+ cells express BAFF, APRIL and TNF-α which is required for IgA class-switching of B cells. Intravenous injection of soluble OVA in mice can promote production of IgG and IgA in the serum and feces. Intestinal CD103+ DCs are known to present to CD4+ T cells, however CX3CR1+cells present antigens to CD8+ T cells via cross-presentation. Addition of OT-I cells further enhances production of IgA of B cells co-cultured with CX3CR1+cells. Intestinal CX3CR1+cells are tissue resident cells and do not migrate to the MLN. CCR7 deficiency or FTY720 treatment does not affect the production of intestinal IgA after intravenous injection of OVA in mice. These results suggested that intestinal CX3CR1+cells have an important role in maintaining homeostasis of the intestinal immune system.
A murine model of spontaneous self-reactivity to the ileum
Authors: Jeschke Jonathan C , Mayne Christopher G , Salzman Nita H , Williams Calvin B
Published: January 2023

Inflammatory lesions of Crohn’s disease are commonly found in the ileum yet few mouse models of enterocolitis manifest inflammation at this site. In order to study inflammation targeted to the ileum, we developed a transgenic mouse line (HD5-mHEL/Hb) that expressed a model antigen (mHEL/Hb) using a Paneth cell promoter (HD5). The abundance of Paneth cells is unique to the ileum and variations in their antimicrobial-secreting function are associated with Crohn’s disease. HEL-reactive (3A9) transgenic TH cells accumulate in the ileum of lymphopenic HD5-mHEL/Hb mice following their adoptive transfer. To study spontaneous TH cell reactivity to the ileum, we bred HD5-mHEL/Hb lines to mHEL/Hb-reactive transgenic TH cell lines. A subset of progeny with both the mHEL/Hb antigen and the 3A9 transgene (Bigenic3A9) develop a spontaneous wasting phenotype that is associated with colitis. Bigenic3A9 mice show TH cell activation and accumulate increased numbers of IL-17A-producing TH cells in the ileum. To study the necessity of effector cytokines for the spontaneous disease of Bigenic3A9 mice, Bigenic3A9 mice were bred on IFNγ and IL17A knockout lines. IL17A−/− Bigenic3A9 mice do not show an altered predisposition to the wasting phenotype of IL17A+/+ Bigenic3A9 mice. Unexpectedly, IFNγ−/− Bigenic3A9 mice exhibit an increased incidence of wasting disease which is associated with a skew of TH cell populations towards an IL-17A-producing phenotype. These results identify a significant protective role for IFNγ in Bigenic3A9 mice and suggest protection may result from inhibitory effects on the polarization of IL-17A-producing TH cells.
Discovery of selective RORγ inverse agonists and demonstration of efficacy in inflammatory disease models
Authors: Damarla Ravi Krishna Babu , Ujjinamatada Ravi K , Nellore Kavitha , Dodheri Samiulla S , Chelur Shekar , Charamanna K.B , Mukherjee Subhendu , Reddy CH Sandeep , Balasubramanian Wesley Roy , Rathod Sanjay B , Lakshminarasimhan Anirudha , Rao K Narasimha , Ramanathan Anuradha , Mahalingam Natarajan , Girish AR , Samajdar Susanta , Pandit Chetan , Ramachandra Murali
Published: January 2023

The nuclear hormone receptor RORγ controls the differentiation of Th17 cells that plays a key pro-inflammatory role in a variety of autoimmune diseases. RORγ inverse agonists from multiple structural classes were discovered and screened in a RORγ radio-ligand binding assay as well as in a cell based reporter assay and functional effect was evaluated using primary mouse/human CD4+ve T-cells differentiation to Th17 cells. Pharmacokinetic profile of potent compounds was determined to support evaluation of efficacy as well as safety profile in rodents.

Lead compounds have demonstrated good activity (< 100 nM) in reporter assay. Crystal structure of RORγ in complex with known inverse agonists as well as novel compounds, were solved to support lead optimization. Selected compounds demonstrated >100 fold selectivity against RORα as well as other nuclear receptors with significant inhibition of IL-17 release in Th17 differentiation assay. Lead compounds have shown optimal physicochemical profile including good solubility and high free fraction, leading to excellent pharmacokinetic profile in mice and rats. Efficacy of lead compounds has been demonstrated in multiple disease models including asthma, psoriasis, Experimental auto immune encephalomyelitis and collagen induced arthritis. Potential lead candidates have been identified with excellent in-vitro safety profile including CEREP-44 safety panel, Ames test and cytotoxicity. One of the lead candidates has also demonstrated good therapeutic window as determined by a 7 day rat toxicity study.

Conclusions

We have identified structurally diverse small molecule inverse agonists of RORγ and have demonstrated efficacy in relevant disease models as well as good therapeutic window.
Targeting the PDE8A-Raf-1 kinase signaling complex to treat autoimmune inflammation
Authors: Basole Chaitali P , Nguyen Rebecca , Lamothe Katie , Vang Amanda , Clark Robert , Epstein Paul , Baillie George , Brocke Stefan
Published: January 2023

The levels of cAMP are regulated by phosphodiesterases (PDE), which are targets for the treatment of inflammatory disorders. We previously showed that PDE8A regulates T cell motility. We report here that PDE8A exerts its control of T cell function through the Raf-1 kinase signaling pathway. To mimic T cell migration under physiologic conditions, we performed flow chamber assays. The highly PDE8-selective enzymatic inhibitor PF-04957325 significantly suppresses rolling and adhesion of in vivo MOG35-55 activated inflammatory CD4+ T effector (Teff) cells while interacting with inflamed brain endothelial cells under shear flow conditions. Recently, PDE8A was shown to associate with Raf-1 creating a compartment of low cAMP around Raf-1 thereby protecting it from protein kinase A mediated inhibitory phosphorylation. To test this in Teff cells, we used a cell permeable peptide (DP) that disrupts the PDE8A-Raf-1 signaling complex. Disruption of the PDE8A-Raf-1 complex by DP significantly reduces adhesion of Teff cells to endothelial cells. We further observed that disrupting PDE8A-Raf-1 through DP specifically reduces adhesion, spreading and locomotion of Teff cells while interacting with the vascular adhesion molecule ICAM-1. Our investigation of the effect of PDE8 inhibitor on chronic and relapsing-remitting experimental autoimmune encephalomyelitis (EAE) in vivo indicate suppression of clinical and histopathological signs of disease. Collectively, our studies demonstrate that PDE8A inhibition by enzymatic inhibitors or PDE8A-Raf-1 kinase signaling complex disruptors significantly decreases Teff cell adhesion and migration on endothelial cells, and represents a novel approach to treat autoimmune inflammation in vivo.
CD27/CD70 mediated negative regulation of inflammatory T cell response
Authors: ONeill Rachel , Leigh Nicholas , Du Wei , Kumar Sandeep , Chen Chuan , Qiu Jingxin , Chen George , Borst Jannie , Ashwell Jonathan D , McCarthy Philip , Cao Xuefang
Published: January 2023

Costimulatory pathways are involved in T cell activation and function. The costimulatory molecule CD27 is a TNF receptor family member expressed on T cells and its ligand, CD70, is known to be expressed on activated antigen-presenting cells, T-, B- and NK cells. The CD27/CD70 pathway has been shown to be critical for T cell activation, differentiation and survival. In this study we have used murine models to study the roles of CD27/CD70 in allogeneic graft-versus-host disease (GVHD) and syngeneic inflammatory bowel disease (IBD). Our results reveal a novel and negative regulatory role played by this pathway as specified in 3 aspects: 1) Allogeneic hematopoietic cell transplantation (allo-HCT) shows that both CD27−/− and CD70−/− donor T cells caused more severe GVHD than WT donor T cells, suggesting that CD27/CD70 signaling in donor T cells inhibits allogeneic T cell response. 2) When transplanted into syngeneic RAG1−/− hosts in an autoimmune IBD model, both CD27−/− and CD70−/− donor CD4+ CD25− T cells caused more severe IBD than WT T cells, suggesting that CD27/CD70 signaling in T cells inhibits autoimmune T cell response. 3) When used as hosts for allo-HCT, both CD27−/− and CD70−/− mice exhibited more severe GVHD compared to WT mice, suggesting that CD27/CD70 signaling in the host inhibits allogeneic T cell response. Mechanistic analyses reveal that this pathway executes immune suppression by limiting T cell expansion and effector function (e.g., TNFa and IFNg production) via a regulatory T cell-independent mechanism that at least partially involves activation-induced T cell death. Overall, our study demonstrates that CD27/CD70 signaling plays a novel role in suppressing allogeneic and autoimmune inflammatory T cell responses.
T-cell depleted haploidentical hematopoietic stem cell transplantation is the optimal graft source for patients with familial hemophagocytic lymphohistiocytosis who do not have matched family donors.
Authors: DENNISON Jayakumar David , Al Rawas Abdulhakeem , Al Ghaithi Hamed , Al Khabori Murtadha , Tauro Melanie , Al Mamari Sahimah , Al Zadjali Shoaib , Al Said Bushra , Nazir Hanan , Tony Surekha , Saad Ashraf , Al Abri Qamariah , Al Farsi Khalil , Al Tamemi Salem , Wali Yasser , Al Kindi Salam , El Shinawy Mohamed , Zachariah Mathew , Mathew Leni , Al Huneini Mohammed
Published: January 2023

Familial hemophagocytic lymphohistiocytosis (HLH) is a fatal disorder of immune regulation. When managed appropriately with systemic chemotherapy, cure can be achieved by subsequent allogeneic hematopoietic stem cell transplantation (HSCT). Although the best results are obtained with matched-family donors (MFD), alternative donor transplants may be necessary when such donors are not available. As there is urgency to proceed with curative therapy, T-cell depleted haploidentical HSCT could be a viable option as a parent or other haploidentical family member would be available instantly. Between August 2010 and September 2015 we performed haploidentical HSCT for six patients with HLH in this institution. These patients did not have MFDs, and in our center it was logistically easier to perform an haploidentical HSCT rather than to pursue with an unrelated donor or cord blood search from the international registries. The median age at transplant was 6 months (5–31 months) and all had perforin gene mutations. The first two patients had CD3/CD19 depleted procedures while the rest underwent TCR alpha/beta and CD19 depletion for faster immune reconstitution. All six patients engrafted following the transplant with minimal graft versus hot disease. Four patients are cured from the disease with a median follow-up of 3 years (7mths to 5 years). Two patients (siblings) who were transplanted in partial remission expired 2 months following transplant from multi-organ failure after successful engraftment. In the presence of an appropriate transplant laboratory facility, T-cell depleted haploidentical HSCT should be considered as the graft of choice for patients with HLH who do not have HLA-matched family donors.
A novel anti-CD40 antibody prevents activation of alloreactive T cells and prolongs skin allograft survival
Authors: Jangalwe Sonal N , Ralph Kerry , Ramanujam Meera , Greiner Dale L , Brehm Michael A
Published: January 2023

Targeting the CD40-CD154 pathway to induce transplantation tolerance has been of interest to transplantation immunologists for over two decades. The efficacy of anti-CD154 antibody (Ab) observed in murine and non-human primate models for preventing allograft rejection and suppressing autoimmunity, generated great interest in the development of this antibody for clinical use. However, clinical trials with anti-CD154 Ab resulted in thromboembolic complications in patients as a result of CD154 expression by human platelets and limited the clinical application. To develop an alternative approach to anti-CD154 Ab, recent efforts have focused on targeting its receptor, CD40. In this study we evaluated the effects of a novel murine antagonistic antibody to CD40 (BI CD40-1) on alloreactive immune responses. Costimulation blockade consisting of donor splenocytes (DST) and BI CD40-1 reduced activation of alloreactive CD8 T cells as shown by inhibition of expansion, decreased cytokine production and decreased killing of allogeneic targets using an in vivo cytotoxicity assay. Moreover DST/BI CD40-1 treatment significantly prolonged skin graft survival in a BALB/c to B6 MHC mismatch model. The efficacy of BI CD40-1 in preventing alloreactive T cell responses was similar to anti-CD154 Ab. These results indicate that anti-CD40 Ab is an attractive candidate for translation to clinical application.
The increase in thymopoiesis following T cell progenitor therapy is dependent upon the input population and continued interaction between developing T cells and the thymic microenvironment.
Authors: Smith Michelle J , Reichenbach Dawn K , Parker Sarah L. , Mohtashami Mahmood , Stefanski Heather E. , Hogquist Kristin Ann , Holländer Georg A. , Zúñiga-Pflücker Juan Carlos , Blazar Bruce R
Published: January 2023

The inclusion of in vitro derived T cell progenitor (proT) therapy with hematopoietic stem cell transplant (HSCT) aids in the recovery of the thymus damaged by total body irradiation and improves de novo thymopoiesis. To understand the interaction between proTs and the thymic microenvironment, wildtype (WT) mice were lethally irradiated and given T cell deficient donor (Rag1−/−) marrow along with in vitro generated proT from WT donors, limiting mature T cell development to infused proT. Donor proTs within the host thymus led to a significant increase in thymic epithelial cell (TEC) numbers by day 21 post-transplant, and increased actively cycling TECs as measured by Ki67 expression and BrdU uptake. However, that gain was temporary and lost by day 28, suggesting that continued signaling from proT cells is required to sustain TEC cycling and mass. We also find a significant improvement in total thymocyte number by day 21 followed by a significant increase in the total mature T cell number in the secondary lymphoid organs by day 28. This protective surge is also temporary, receding by day 60. In this time period, infused DN2 proTs selectively increased thymocyte number while DN3 proTs preferentially led to a greater TEC numbers. Interestingly, an exception in persistence occurs when DN3 proTs are used and the increase in mature T cells in the spleen persists at day 60. As a result of the lack of competition for thymic niches by cells from the Rag1−/− graft, a subpopulation of the infused proT persisted in the thymus in an immature state at day 60. These findings highlight the importance of the interaction between developing T cells and TECs in the proliferation and survival of these critical components of the thymic microenvironment.
Peripheral blood natural killer cell precursors arising in early post transplant period have a mixed myeloid and lymphoid phenotype
Authors: Schafer Jolie Rae , Ciurea Stefan , Lee Dean
Published: January 2023

Immune cell reconstitution after hematopoietic stem cell transplantation (HSCT) has a major impact on patient survival and cancer relapse. In studying immune reconstitution after haploidentical HSCT we observed that some patients had high NK-cell-like activity despite systemic immunosuppression and low numbers of mature CD3-CD16+CD56+ NK cells. Using high-parameter mass cytometry we identified a unique subset of immune cells with both myeloid and lymphoid phenotype and NK cell like function. These CD3-negative cells are the dominant cell type in the lymphocyte FSC/SSC region one month after haplo transplant. They are uniquely identified by the expression of CD4, CD14, and KIR2DL2/3, and also express CD16, HLA-DR, CD11a, CD80, CD86, and NKG2A. To determine their proliferative capacity, we purified myeloid-like NK-like cells by sorting CD3−/4+/14+/KIR2DL2+ cells from peripheral blood of patients and expanded these cells in the presence of the K562 clone9.mbIL21 feeder cells. The average expansion was 32 fold after 2 weeks. After expansion, the myeloid-like NK-like cells lost expression of CD4 and reduced expression of CD14 and KIR2DL2/3, but gained expression of CD56. Our data is consistent with recent reports suggesting that some NK cells may differentiate from myeloid progenitors. Additional phenotyping and functional analysis is being investigated, as is assessing their frequency in normal peripheral blood, cord blood, or after HSCT from other donor types.
NKT cells in the liver are important for periferal tolerance induction
Authors: peng wang zhan , hongyu liu , zhuonan li , Reyes Jorge D. , Perkins James D. , wei li
Published: January 2023

NKT cells are mainly located in the liver in mice. The role of liver inherent NKT cells are still unclear.

Method

In this study, we employed mouse orthotopic liver transplantation and heterotopic heart transplantation models to critical examine the role of liver NKT cells in liver and peripheral tolerance induction.

Results

NKT cells were increased in the tolerated liver grafts which expressed higher levels of CD95L and PD-L1, while the CD4+CD25+Foxp3+ regulatory T cells (Treg) were markedly increased in the both liver and spleen at day 7 post transplantation. The heart allograft survival were prolonged significantly in the recipient which accepted donor spleen cells by portal vein (p.v.) injection in contract to the recipient which received the donor spleen cells by tail vein (i.v.) injection. In vitro immunological assay revealed that the number of NKT cells in the liver was increased and Treg were increased in both liver and spleen from the p.v. treated mice significantly. The IL-2 expression was decreased, IL-4 and IL-10 were increased. Further, significantly increased IL-4, IL-10, and IFN-γ production was detected from liver NPCs under aGalCer stimulation.

Conclusion

NKT cells are important to liver transplant tolerance induction, appear to play a key role in down regulation of peripheral immune responses and facilitate CD4+CD25+Foxp3+ Treg induction.
Non-hepatic Islet transplantation: Local Delivery of Reagents to Aid in Islet Engraftment
Authors: Gebe John A , Gooden Michel D , Vernon Robert B
Published: January 2023

Islet transplantation offers a potential cell replacement treatment for type 1 diabetes, but faces a number of significant challenges, including: 1) poor short-term islet survival and 2) long term islet loss by alloimmunity and recurring autoimmunity.

Method

We have developed a Bioengineered Islet Implant (BII) device that incorporates local biological reagent delivery to promote islet immune tolerance and survival. The BII consists of two integrated components: a poly-vinyl-alcohol (PVA) scaffold to support islets within a collagen hydrogel, and a local drug delivery mechanism located within the implant device through incorporating drug-alginate delivery system.

Results

The implant device is capable of reversing STZ-induced diabetes when implanted in mesentery or subcutaneous sites. Short-term islet survival measured at 24 hours is enhanced in recipient transplants utilizing BII incorporating local delivery of VEGF. As proof-of-concept for delivery of local immunomodulators together with transplanted islets, we incorporated anti-CD3e MAb (2C11) or anti-CD95 MAb (Jo-2) within the BII, and respectively found dramatically reduced lymphocyte infiltration into islets and a delay in the onset of diabetes in the DO11/RIP-Ova murine model of T1D. In addition, local delivery within the BII of the extracellular component hyaluronic acid led to the generation of enhanced antigen-specific IL-10 responses.

Conclusion

These data demonstrate the potential for a novel strategy of islet cytoprotection during transplantation, providing a suitable matrix and local immunomodulation capable of supporting islet cell engraftment, survival, and function in non-hepatic sites without encapsulation.
Decreased frequency and impaired function of circulating Tregs from renal transplant patients under long-term Belatacept treatment
Authors: ALVAREZ EVELYN KATY , CORTÉS ARIMELEK , ALEMÁN GERMÁN , ALBERÚ JOSEFINA , GARCÍA EDUARDO , SOLDEVILA MARIA GLORIA
Published: January 2023

Regulatory T cells are cells with immunosuppressive capacity that are important in tolerance maintenance after organ transplantation. Belatacept, is an effective alternative to CNI that appears to preserve graft survival and function. However, the impact of this drug in the homeostasis of Tregs in transplanted patients remains controversial. Here, we report a detailed phenotypic and functional characterization of Tregs from kidney recipients undergoing 7 years of treatment with Belatacept or Cyclosporine A. We found a significant reduction in the proportion of CD4+ CD25hiCD127lo/−FOXP3+ T cells in all patients compared to controls. Interestingly, only Belatacept treated patients displayed an enrichment of the CD45RA+ “naïve” Tregs, while the percentage of Helios+ Tregs remained unaffected. Functional analysis demonstrated that Tregs from transplanted patients displayed a significant reduction in their suppressive capacity compared to Tregs from controls, which correlated with decreased levels of FOXP3 and CD25. Finally, methylation studies at the Treg-specific demethylation region (TSDR) of the FOXP3 gene showed that Belatacept treatment may result in methylation of CpG islands within the TSDR, which correlated with the impaired Treg suppression function. Our data indicate that Belatacept can have deleterious effect on the frequency and function of Tregs cells, suggesting that the beneficial effects reported by Belatacept cannot be explained by circulating Tregs.
CD62L+ NKT cells have superior in vivo persistence and anti-tumor activity
Authors: Tian Gengwen , Courtney Amy N , Jena Bipulendu , Heczey Andras , Liu Daofeng , Marinova Ekaterina , Guo Linjie , Xu Xin , Dotti Gianpietro , Cooper Laurence J , Metelitsa Leonid S
Published: January 2023

Va24-invariant Natural Killer T cells (NKTs) have potent antitumor properties and are being developed for cellular immunotherapy of cancer. Such therapy requires extensive ex vivo expansion of primary NKTs while preserving their longevity and function. However, the mechanism responsible for NKT-cell maintenance remains poorly understood. In this study we analyzed the proliferative potential of human NKT-cell subsets and found that antigen-induced in vitro expansion of primary NKTs is associated with the accumulation of a CD62L-positive subset. Following magnetic sorting, only CD62L-positive cells survived and proliferated in response to TCR-stimulation in vitro. After transfer to NSG mice, CD62L-positive NKTs persisted 5 times longer and had higher therapeutic efficacy in a lymphoma model compared with CD62L-negative NKTs. Proliferating CD62L-positive cells downregulated or maintained CD62L expression when they were activated via TCR alone or in combination with co-stimulatory receptors, respectively. After testing 161 clones of K562 cells, genetically modified to express CD1d and various combinations of co-stimulatory molecules, we selected the B-8-2 clone (HLAnullCD1dmedCD86high4-1BBmedOX40Lmed) which induced the highest rate of NKT-cell expansion with the preservation of CD62L-positive cells. Compared with NKTs expanded with autologous PBMC, those expanded with B-8-2 exhibited a prolonged in vivo persistence and superior antitumor activity. Thus, our results reveal a previously unanticipated functional hierarchy in human NKTs that can be exploited for cancer immunotherapy.
Crytotanshinone Induces the Maturation of Human Dendritic Cells through Activation of NF-kB, p38 and JNK MAPKs
Authors: Liu Shuo , Xin Haiyun , Nie Yingjie , Trivett Anna , Oppenheim Joost J. , Yang De
Published: January 2023

Danshen or Salvia miltiorrhiza Bunge is often used in Traditional Chinese Medicine (TCM) for the treatment of a variety of diseases including cancer. Cryptotanshinone and tanshinone II are two major tanshinones isolated from the roots of Danshen. In this study, we found that cryptotanshinone, but not tanshinone II, possesses the capacity to induce maturation of human dendritic cells (DCs). Treatment of human monocyte-derived DCs with cryptotanshinone upregulated DC surface expression of costimulatory molecules (CD80, CD86) and HLA-DR, as well as DC production of proinflammatory cytokines. In addition, DCs treated with cryptotanshinone acquired the capacity to migrate in response to CCL21/SLC, suggesting the upregulation of CCR7. Cryptotanshinone treatment reduced the level of I-kBa in DCs, indicative of NF-kB activation. Cryptotanshinone also increased the levels of phosphorylated p38 and JNK mitogen-activated protein kinases (MAPKs), but not phosphorylated Erks. Thus, cryptotanshinone can induce the maturation of human DCs through activation of NF-kB, p38 and JNK MAPKs, and may have the capacity to promote adaptive immune responses.
Reciprocal regulation of neutrophils and B cells elicits autoantibody-associated protumorigenic effects in human hepatocellular carcinoma
Authors: Liu Rui-Xian , Kuang Dong-Ming
Published: January 2023

Despite recent advances in identifying autoantibody-mediated carcinogenesis in animal models, direct evidence supporting the important role of autoantibodies in the immunopathogenesis of human cancers is still lacking. We recently found that neutrophils are accumulated in hepatocellular carcinoma (HCC), and their levels positively correlated with poor survival of patients. Here we detected high titers of proteinase 3 (PR3)-specific anti-neutrophil cytoplasmic autoantibodies (ANCA) in plasma from patients with advanced stage HCCs, and that increased titers of anti-PR3 autoantibodies also correlated with poor clinical outcome of patients. Neutrophils, B cells, plasma cells all predominantly accumulated in the peritumoral stroma of HCC tumors. The neutrophils isolated from HCC tumors, but not paired blood, significantly and effectively secreted PR3 proteins; accordingly, only B cells/plasma cells derived from HCC tumors markedly produced anti-PR3 autoantibodies. Further kinetic experiments revealed addition of conditioned medium from HCC tumor neutrophils to the ex vivo induction system of plasma cells led to sequential B cell activation, somatic hypermutation, and anti-PR3 autoantibody secretion. Reciprocally, we also demonstrated that, in the presence of anti-PR3 autoantibodies, the neutrophils are more potent to produce protumorigenic factors oncostatin M and matrix metalloproteinase-9. Thus, the secretion of PR3 by tumor neutrophils may represent a novel mechanism that links the autoimmunity to disease progression in the tumor milieu.
EGFR antibodies in immunotherapy: complement optimized antibody enhances polymorphonuclear cell mediated killing
Authors: Schwanbeck Ralf , Derer Stefanie , Roesner Thies , Kellner Christian , Beurskens Frank , Lohse Stefan , Peipp Matthias , Valerius Thomas
Published: January 2023

Antibodies against epidermal growth factor receptor (EGFR) are used in therapy for solid cancers such as head and neck cancer, colorectal and non-small lung carcinoma. Though complement-dependent cytotoxicity (CDC) is thought to be an important mechanism involved in immunotherapy, single unmodified EGFR IgG1 antibodies fail to trigger efficient CDC. Here, we generated a CDC-optimized variant of the EGFR antibody matuzumab (H425-wt) by introducing amino acid substitutions K326A/E333A (H425-mt). This antibody was used to investigate the impact of complement activation on the capacity of effector cells such as mononuclear cells (MNC) and polymorphonuclear cells (PMN) to exert antibody-dependent cell-mediated cytotoxicity (ADCC). H425-mt but not H425-wt significantly induced complement deposition, release of anaphylatoxins and CDC against distinct tumor cell lines, while no differences in ADCC by isolated MNC or PMN were detected. Notably, compared to H425-wt stronger cytotoxicity was induced by H425-mt in whole blood assays and in experiments using MNC or PMN combined with serum. While MNC-ADCC was not affected by C5 cleavage, PMN’s cytotoxic activity in the presence of serum strongly depended on C5 cleavage, suggesting a direct interaction between complement and PMN-ADCC. PMN, which strongly express C5a receptors, were stimulated with C5a resulting in an up-regulation of activated complement receptor 3 (CR3) and an enhanced CR3-dependent PMN-ADCC against tumor cells. In conclusion, complement-optimized EGFR antibodies may constitute a promising strategy to improve tumor cell killing by enhancing the interaction between humoral and cellular effector functions in antibody-based tumor therapy.
A high-throughput image cytometry-based screening method for the detection of IL2-induced peripheral blood mononuclear cell-mediated cytotoxicity
Authors: Chan Leo , McCulley Kelsey , Banerjee Pinaki
Published: January 2023

Cell-mediated cytotoxicity assays have been an important functional test for investigating the cytotoxic effect of immune effector on target cancer cells. Cytotoxicity assays have traditionally been performed using the 51Chromium (51Cr) release assay, which involves labeling the tumor cells (target) with radioisotopes. The 51Chromium release assay is highly hazardous, time-consuming, and can only acquire end point readout. Furthermore, it can generate inconsistent results due to batch variation, ability of the target cell for 51Cr uptake, and low sensitivity. In this work, we demonstrate a novel high-throughput cytotoxicity screening assay using the Celigo imaging-cytometry method. First the live K562 target cells were stained with Calcein AM, and were co-cultured with PBMCs from two healthy donors in a flat- bottom 96-well plate in the presence or absence of IL-2. Direct cell counting of Calcein positive cells was implied to calculate the changes in number of live target cells in the same well from T = 0 to T = 4 hours. Thus, accurate cell-mediated killing was determined using the “self-referencing” calculation, which could be a more direct method for assessing cytotoxicity than 51Cr release. The donor PBMCs were added to each well with different Calcein-stained target cells at Effector-to-Target (E:T) ratios. The 96-well microplate was scanned and analyzed using the Celigo image cytometer at different time points to measure the % lysis of target cell. The proposed image cytometry method can scan and analyze in 7 min, which can be utilized to perform high-throughput screening of potential antibody- or chemical-based cancer drug, which could be a more efficient method for academic, industry, and clinical research.
Optimizing adoptive cancer immunotherapy with Vγ2Vδ2 T cells through zoledronate pulse stimulation
Authors: Al-Janabi Mohanad Hameed Nada , Wang Hong , Workalemahu Grefachew , Tanaka Yoshimasa , Morita Craig T
Published: January 2023

Human γδ T cells expressing Vγ2Vδ2 TCRs monitor foreign- and self-prenyl pyrophosphate metabolites in isoprenoid biosynthesis to mediate immunity to microbes and tumors. Vγ2Vδ2 cells have been used for adoptive cancer immunotherapy with some partial and complete remissions. Most trials have used continuous zoledronate exposure to expand Vγ2Vδ2 cells. Zoledronate inhibits farnesyl pyrophosphate synthase causing isopentenyl pyrophosphate to accumulate that then stimulates Vγ2Vδ2 cells. Because zoledronate exposure is toxic, we hypothesized that a short period of exposure would reduce T cell toxicity but still be sufficient for monocytes uptake. Supporting this, pulse zoledronate exposure with IL-2 resulted in more uniform expansion of Vγ2Vδ2 cells with higher purity and cell numbers as compared with continuous exposure. These Vγ2Vδ2 cells also had higher levels of CD107a and perforin and slightly increased tumor cytotoxicity. Importantly, adoptive immunotherapy with Vγ2Vδ2 cells derived by pulse stimulation controlled human PC-3 prostate carcinoma cells in immunodeficient NSG mice significantly better than those derived by continuous stimulation. Zoledronate pulse stimulation of Vγ2Vδ2 cells in IL-15 also resulted in higher purity and cell numbers. Like with CD8 αβ T cells, IL-15 preserved early memory Vγ2Vδ2 T cell subsets better than IL-2. However, despite this fact, adoptive immunotherapy with Vγ2Vδ2 cells derived in IL-15 showed similar inhibition of PC-3 tumors as those derived in IL-2. Thus, zoledronate pulse stimulation maximizes the purity, quantity, and quality of expanded Vγ2Vδ2 cells. This simple modification to existing protocols could enhance the effectiveness of adoptively transferred Vγ2Vδ2 T cells.
Studying the Effect of β-estradiol on HCC Antigen-Specific T Cells
Authors: Navarro Flor C
Published: January 2023

Hepatocellular carcinoma (HCC) has a 4:1 male-to-female overall incidence ratio. These statistics lead to the belief that there are gender specific mechanisms that affect the development of this cancer. Previous research studies demonstrated that the gender differences regarding HCC may be related to a protective effect from estrogen (E2). However, the antitumor immune response following cancer development is not defined. To test specific immune responses to HCC we are utilizing a known hepatitis C virus (HCV) antigen to generate Ag-specific T cells that target HCC expressing the HCV antigen since the chronic infection of accounts for approximately 50% of HCC. With this model, we tested the effect of E2 on the immune response during immunotherapy. We have identified a novel effect of E2 treatment of TCR-transduced Ag-specific T cells. E2 treatment induced an increase in their cytotoxicity and effector function. Treatment at super physiological concentrations of β-estradiol resulted in an up-regulation of CD107a expression, demonstrating degranulation and an increase in TNFα secretion. On the other hand, E2 treatment also caused a reduction in the number of cytokines produced simultaneously demonstrated in a polyfunctional analysis of the T cells. Polycytokine profiles of the T cells showed that as E2 increases, the number of T cells producing 3 or 4 cytokines simultaneously decreases while the number of T cells displaying a reduced number of functions increases. Previous studies on memory T cell development show that memory T cells result from cells with greater polyfunctional abilities. Our results suggest that while exposure to E2 increases rapid effector function, it is at the expense of the generation of memory T cells.
Optical control of Calcium influx improves intratumoral cytotoxic T lymphocyte effector functions.
Authors: Kim Kyun Do , Bae Seyeon , Nedelkovska Hristina , Capece Tara , Kim Minsoo
Published: January 2023

Regulatory T cells (Tregs) have critical roles in the maintenance of immunological self-tolerance and in the control of antitumor immune responses. In tumor microenvironment, Tregs use several mechanisms to suppress cytotoxic T cell responses, including suppression of TCR-induced Ca2+-NFAT signaling, secretion of inhibitory cytokines and inhibition of the stimulatory capacity of antigen presenting cells. Particularly, suppression of Ca2+ signals in cytotoxic T cell is a significant challenge because Ca2+ plays pivotal roles in tumor-specific CD8+ cytotoxic T lymphocytes (CTLs) function by regulating the degranulation of CTLs, their expression of Fas ligand and production of TNF-α and IFN-γ.

Here we developed a strategy for optically controlling calcium signal using Ca2+ translocating channelrhodopsin (CatCh). The Ca2+/NFAT pathway is activated upon blue light (480nm) illumination via increase in intracellular calcium in CatCh expressing CTLs. In vitro, light stimulation enhances CatCh expressing CTLs activation, IFN-γ production and tumor cytotoxicity. Importantly, CatCh expressing CTLs restore against Treg-induce immunosuppression by light stimulation. In vivo, blue LED illumination onto the melanoma improved the efficacy of adoptive T-cell transfer immunotherapy, with a significant reduction in tumor growth. Together, our findings indicate that strong intracellular calcium signals can boost T cell functions by overcoming immunosuppressive Treg responses at tumor sites.
Fatty acid metabolism is essential to maintain functions of CD8+T cells within a hypoxic and hypoglycemic tumor microenvironment
Authors: Zhang Ying , Liu Ling , Kurupati Raj , Zhou Xiangyang , Rabinowitz Joshua , Ertl Hildegund
Published: January 2023

The efficacy of immunotherapy for solid tumors is dampened by functional declines of tumor-infiltrating T lymphocytes (TILs), which is generally viewed as the result of their exhaustion due to persistent antigenic stimulation. Disputing this notion, we observed in a mouse melanoma model that bystander CD8+TILs increase expression of co-inhibitors and lose effector functions, suggesting alternative mechanisms. Our data show that metabolic stresses within tumor microenvironment (TME) profoundly affect differentiation and functions of TILs. Hypoxia through HIF-1α drives increases of co-inhibitor LAG-3 while lack of glucose (Glu) enhances PD-1 levels on activated CD8+T cells. Both conditions impair functions of CD8+T cells. When deprived of Glu, activated CD8+T cells enhance fatty acid (FA) catabolism and this is further increased under hypoxia. Using 13C-stable isotope tracing in vivo and liquid chromatography-mass spectrometry (LC-MS) analysis, our study show that CD8+TILs in late stage tumors increasingly depend on catabolism of FAs including ketone bodies fueled by exogenous FA uptake and triacylglycerol (TG) turnover to meet their energy demand. Promoting FA catabolism of CD8+TILs increases their PD-1 expression but preserves some effector functions and improves their anti-tumor efficacy. Our findings show that lack of Glu and oxygen plays a critical role in driving the metabolic reprograming and functional exhaustion of CD8+TILs. They further indicate that metabolic interventions that promote FA catabolism of adoptively transferred or vaccine-induced CD8+T cells may improve the efficacy of cancer immunotherapy.
Toll-like receptor agonists targeted to cancer cell metabolism
Authors: Mancini Rock , Nielsen Amy , Hantho Joseph
Published: January 2023

Imidazoquinoline Toll-Like Receptor (TLR) agonists such as Imiquimod are effective immunotherapeutics in the treatment of basal skin cell carcinoma, and more recently, melanoma. However, direct injection of imidazoquinoline TLR agonists causes systemic inflammation and autoimmune side-effects thereby limiting clinical use to topical applications. Here we use a metabolic caging strategy to confine the immunostimulatory effects of imidazoquinoline TLR agonists to tumor microenvironment. We have covalently attached a galactopyranoside metabolic cage to the primary amino group, critical for activity, of the TLR7 agonist Imiquimod. Addition of the metabolic cage abrogates TLR agonism as measured by NFκB transcription in RAW-Blue cells or inflammatory cytokines produced by primary murine Bone Marrow Derived Dendritic Cells (BMDCS). The galactopyranoside cage is efficiently removed via enzymatic cleavage from exogenously added β-galactosidase resulting in activation of immune cells that is comparable to native agonist. Enzymatic cleavage also occurs using β-galactosidase that is overexpressed in B16 melanoma cells. This results in increased immunostimulatory effects in co-cultures of BMDCs and B16 melanoma cells relative to BMDCs co-cultured with healthy melanocytes. We anticipate this will provide a general framework upon which TLR agonists can be targeted to the tumor microenvironment.
Increased leptin during obesity abrogates antitumor immunity
Authors: Murphy Katherine A , James Britnie R , Brincks Erik L , Kucaba Tamara A , Griffith Thomas S
Published: January 2023

Cancer immunotherapy offers the promise of long–lived protection against a variety of tumors; yet, the efficacy seen in laboratory models has not always translated into clinical success. One reason may be the preclinical use of young, healthy mice lacking common co-morbidities found in adult humans. Obesity is associated with an increased risk of developing cancer and correlates with a poor prognosis. Further, obesity related factors, such as leptin, can alter the behavior of tumor and immune cells. Renal cell carcinoma (RCC) more closely correlates with obesity than cancer at any other anatomical site and obesity-related cytokines play important roles in the progression of metastatic RCC. We investigated the therapeutic efficacy of a recombinant adenovirus encoding TRAIL in combination with CpG in an orthotopic mouse model of RCC. Ad5-TRAIL/CpG induced systemic T cell-mediated antitumor immunity capable of reducing primary and metastatic tumor burden, as well as reduced MDSC within the tumor-bearing kidney. In contrast, Ad5-TRAIL/CpG failed to induce antitumor immunity or reduce tumor burden in diet-induced obese (DIO) mice and MDSC levels remained high in the tumor-bearing kidney. Leptin reduced Ad infection of the tumor cells, decreased TRAIL receptor (DR5) expression, and Ad5-TRAIL-induced death of RCC cells. DC activation by CpG was also decreased in the presence of leptin. Leptin neutralization in DIO mice restored Ad5-TRAIL/CpG efficacy, and leptin-deficient (ob/ob) obese mice had reduced tumor burden. These data suggest the elevated leptin present during diet-induced obesity affects both tumor and immune cells that leads to reduced antitumor immunity.
Targeting of a novel signaling program in Philadelphia chromosome-like (Ph-like) acute lymphoblastic leukemia (ALL)
Authors: Thomas Kerri R , Allenspach Eric J , James Richard , Tasian Sarah , Teachey David , Rawlings David J
Published: January 2023

Molecular crosstalk between the interleukin-7 receptor (IL-7R) and the pre-B cell receptor (pre-BCR) represents a key developmental checkpoint regulating early B cell proliferation and differentiation. Dysregulation of these pathways has been linked to early lymphoid malignancies. While Philadelphia chromosome-like acute lymphoblastic leukemias (Ph-like ALL) lack the canonical chromosome translocation present in BCR-ABL1+ (Ph+) ALL, these tumors share a similar gene expression profile. Notably, however, Ph-like ALL exhibits a poor clinical prognosis and is refractory to most available therapies. A subset of Ph-like ALL results from alterations in the IL-7R signaling pathway, with gain-of-function mutations in IL-7Rα and loss-of-function mutations in the adaptor, SH2B3, in numerous samples. In this study we directly assess the signaling program downstream of the IL-7R and SH2B3 and its role in disease pathogenesis. Primary human Ph-like ALL tumors display constitutive STAT5 activation, a phenotype recapitulated by in vitro analysis of transduced cell lines expressing an activating IL-7Rα mutation or loss of SH2B3. Our results suggest that both IL-7Rα and SH2B3 mutations can individually contribute to this altered signaling program and may be capable of inducing oncogenic transformation. We have also developed a mouse model that allows for inducible expression of an activating IL-7Rα mutation for future in vivo analysis. Current work is focused on using mass spectrometry and phosphoproteomics to determine downstream signaling aberrations in both primary tumors and our stably transduced cell lines. The results from these studies may lead to new therapeutic targets for high-risk ALL patients.