bluuebunny/crossref_metadata_2025 huggingface dataset:
Profiling Mesenchymal Stem Cell Immunomodulation In Vitro
Authors: Bonnevier Jody L. , Finkel Dave , Galitz David , Andersen Marnelle , Klingenberg Sarah , Rivard Jim , Hammerbeck Christopher , Aho Joy
Published: January 2023

Mesenchymal stem cells (MSCs) are multipotent adult stem cells found in a variety of tissues, including bone marrow, adipose, and umbilical cord. Although MSCs have the ability to differentiate into a variety of cell types useful for regenerative medicine, their ability to modulate the immune system has been at the forefront in the clinic.

The mechanisms by which MSCs affect the immune system are not fully understood. This makes it difficult to design specific quality control measures for MSCs used for cell therapy as well as to understand what parameters are the most critical for their immunomodulatory abilities. This specific testing is important to determine batch-to-batch variability and to better forecast patient-to-patient efficacy. In this study, we demonstrate a workflow to investigate the effects of MSCs on immune cell subset populations. Human MSCs were co-cultured with human T cell subtypes (Th1, Th2, Th17, and Treg) that have been differentiated utilizing CellXVivo™ Differentiation Kits. Co-culture supernates are then analyzed via Proteome Profiler™ Antibody Arrays to screen for changes in cytokine levels, relative to MSCs cultured in the absence of T-cells. Analytes discovered to change in the screening arrays are then quantitated using Luminex® Assays. We hypothesize that these tools and techniques will allow for a better understanding of the mechanisms contributing to MSC immunomodulation and provide methods for routine quality control testing of MSC populations prior to clinical use.
Expression of Eos is critical for T regulatory cell (Treg) survival and function
Authors: Gokhale Ameya Sanjeev , Shevach Ethan M.
Published: January 2023

Eos is a transcription factor belonging to the Ikaros transcription factor family and is preferentially expressed in Treg and activated T-conventional (T conv) cells. Our previous studies had shown that young mice with a global deletion of Eos did not appear to have defective Treg. To fully characterize the role of Eos in Treg function, we generated Eosfl/flx Foxp3Cre (Eos cKO) mice. Beginning at 3–4 months of age, both CD4+ and CD8+ T cells, as well as Treg in the Eos cKO mice expressed an activated/memory phenotype. The percentage of Tregs in the Eos cKO mice was higher than controls, suggesting that the Eos deficient Tregs were attempting to inhibit the activated immune response. H&E staining revealed severe lymphoid infiltration of lungs, kidneys, liver, small intestine, salivary glands of Eos cKO mice. The autoimmune state in the Eos cKO mice was characterized by enhanced levels of IFN-g production by CD4+ Foxp3− T cells, elevated percentages of T-follicular helper cells, and elevated levels of serum anti-nuclear antibodies. Tregs from Eos cKO mice failed to protect in the IBD transfer model and cKO mice also demonstrated enhanced susceptibility to EAE. Nevertheless, the in vitro suppressive function of Treg from cKO was normal. We determined the phenotype of Eos deficient Tregs under non-inflammatory conditions in heterozygous Eosfl/fl Foxp3cre/+ female mice. Although the thymic development of Eos deficient Tregs was not impaired, a marked reduction in Eos deficient Tregs was present in secondary lymphoid tissues indicating a defect in Treg survival in a competitive environment. We conclude that Eos is not required for the development of Tregs, but plays a critical role in controlling many, but not all, of the suppressive functions of Treg.
Activation induced decrease and recovery of TCR 2D affinity and bond lifetime
Authors: Andargachew Rakieb , Kolawole Elizabeth Motunrayo , Evavold Brian D.
Published: January 2023

Shortly after encountering antigen, T cells are considered transiently unresponsive or refractory to subsequent simulation. To determine whether antigen stimulation also changes the cell surface biophysical interaction between TCR and pMHC, we assessed the 2D TCR affinity and bond lifetime under force between this receptor ligand pair. Virus (LCMV) specific SMARTA transgenic CD4+ T cells were stimulated with peptide in vitro and 2D TCR affinity and bond lifetime determined at various time points after stimulation using the 2D micropipette adhesion frequency assay and the biomembrane force probe (BFP) respectively. We found activated T cells downregulated 2D affinity at twelve and twenty-four hours after seeing antigen with affinity recovering to naïve levels by forty-eight hours. Unlike pMHC tetramer staining which was dependent on TCR expression levels, the decrease in affinity was independent of antigen dose and degree of TCR downregulation. TCR:pMHC bond lifetime under force was also reduced early after antigen encounter with a steady recovery observed with time. Our data demonstrate a decrease in bond lifetime and relative 2D affinity of TCR and pMHC result from early T cell activation events followed by a recovery phase with both changes occurring independent of activation induced modulation of TCR expression. Thus, TCR affinity for pMHC is dynamic in the 2D T cell membrane context with cellular events allowing the T cell to fine-tune the ability of TCR to interact with antigen.
Tetrandrine (TET) up-regulated mTNF expression on dendritic cells and consequently induces TNFR2 mediated proliferation of Tregs
Authors: He Tianzhen , Yang De , Chen Xin , Oppenheim Joost J.
Published: January 2023

Tetrandrine (TET) is a purified chemical compound with well-established anti-inflammatory and immunosuppressive properties. The underlying mechanism of its immunosuppressive effect remains incompletely understood. It is also reported that membrane-bound (mTNF) is more effective than soluble TNF to expand Tregs through TNFR2. Therefore, we examined the effect of TET on TNF expression on dendritic cells (DCs) and its effect on Tregs in wildtype and TNFR2 KO mice. We found that, TET didn’t directly expand Tregs in vitro. But TET increased mTNF expression on DC2.4 cells and human monocyte-derived DCs. In addition, these effects of TET weren’t due to LPS contamination. Intraperitoneal injection of TET into wildtype mice increased the number of Tregs by ~1.5-fold compared with control mice. TNFR2 expression was up-regulated on Tregs rather than Teffs by TET treatment. However, the number of Tregs wasn’t changed by TET in TNFR2 KO mice. Taken together, our study revealed that TET promoted mTNF expression on DCs and consequently expanded Tregs via mTNF-TNFR2 signaling. Therefore, this represents a novel mechanism underlying the anti-inflammatory effect of TET.
Chaperone mediated autophagy and T cell function
Authors: Reynolds Cara A. , Macian Fernando
Published: January 2023

Autophagy is a cellular quality control mechanism that degrades and recycles cellular components in the lysosome to maintain cellular homeostasis and to regulates protein levels in response to stimuli. Chaperone mediated autophagy (CMA) is a selective form of autophagy that targets soluble cytoplasmic proteins for degradation. In CMA, the chaperone Hsc70 recognizes and binds a CMA-targeting motif in the substrate and brings it to the LAMP-2A receptor on the lysosomal membrane. The receptor multimerizes and the substrate translocates into the lysosome to be degraded. We have previously reported that CMA is upregulated in activated CD4+ T cells, where it is required to support proliferation and cytokine production. However, the signaling pathways that regulate CMA activity during the immune response and cell type-specific functions of CMA are not fully characterized.

We have found that retinoic acid receptor alpha (RARα) signaling inhibits CMA, and that modulation of RARα signaling with chemically modified retinoic acid derivatives leads to CMA activation. We have used these novel CMA activators to identify target genes involved in the regulation of CMA in T cells. Furthermore, our data supports that LAMP-2A expression and CMA activity are down-regulated in aged T cells, which correlates with the age-associated decreases in T cell function. Interestingly, genetic restoration of LAMP-2A expression in aged cells enhances T cell responses. Therefore, we have explored how CMA activating compounds may restore CMA activity in aged T cells boosting T cell function.
Uracil DNA Glycosylase of Gammaherpesvirus alters somatic hypermutation through error-free DNA repair
Authors: Mu Yunxiang , Dong Qiwen , Zelazowska Monika Anna , Chen Zaowen , Plummer Joshua , Krug Laurie , McBride Kevin M.
Published: January 2023

The gammaherpesviruses (gHV) which include human Epstein Barr virus (EBV), Kaposi’s sarcoma-associated herpesvirus (KSHV) and murine gammaherpesvirus 68 (MHV68) are associated with B cell malignancy including Burkitt lymphoma. All herpesvirus encode a viral homolog of the mammalian Uracil DNA-glycosylase (UNG) gene. In B cells, mammalian UNG plays a critical role in the antibody diversification processes of class switch recombination (CSR) and somatic hypermutation (SHM). UNG processes U:G mismatches created by activation-induced cytidine deaminase (AID) in an error-prone fashion to accomplish CSR and SHM. During primary infection, gHV expands in the germinal center B cells which actively express AID and undergo CSR and SHM. However, the function of viral UNG (vUNG) on viral pathogenesis and host genome integrity during the mutagenic germinal center reaction is an open question. Using a MHV68 mouse infection model we find that germinal center cells are the major viral targets during acute infection. Analysis of infected cells demonstrates significant overlap of vUNG and AID expression. Using a cell based hypermutation reporter assay, we find that host UNG supports error-prone repair and mutagenesis while vUNG results in only high-fidelity error-free repair of AID lesions. In vitro biochemical characterization finds several differences between vUNG and host UNG, including the ability of vUNG to bind abasic sites. These results demonstrate a differential ability of UNGs to support error-prone versus error-free repair. The finding that vUNG is expressed in germinal center cells suggest a novel mechanism whereby gammaherpesvirus can affect antibody diversification and B lymphocyte function.
Role of E3 ubiquitin ligase GRAIL in B cell activation and tolerance
Authors: Nurieva Roza I. , Bieerkehazhi Shayahati , Waseem Tayab C. , Hoang Oanh N. , Sahoo Anupama , Alekseev Andrei , Galkina Elena
Published: January 2023

To date, B cells are believed to play a central role in pathogenesis of various autoimmune diseases as loss of B-cell tolerance with emergence of autoreactive B cells and pathogenic autoantibodies are the hallmark features of autoimmune disorders; however the intrinsic mechanisms that underlie initial breaks in B cell tolerance have not been completely defined. GRAIL, gene related to anergy in lymphocytes (encoded by Rnf128) is an E3 ubiquitin ligase associated with CD4+ and CD8+ T cell tolerance. Our data for first time show GRAIL expression in both mouse and human B cells, with higher expression in anergic B cells and that GRAIL deficiency in B cells leads to impaired B cell peripheral tolerance induction and greater susceptibility to autoimmune diseases. GRAIL deficient B cells exhibited hyperresponsivness in terms of proliferation and antibody production upon antigen stimulation in vitro and in vivo. Concomitantly, GRAIL-deficient B cells were less efficient in downregulation of IgM after B cell receptor (BCR) crosslinking and exhibited increased CD79a expression and activation leading to elevated activation of proximal BCR-signaling components and Ca2+mobilization; GRAIL expression promoted CD79a ubiquitination and degradation. Interestingly, GRAIL expression is also essential to control antigen-presenting property of B cells towards T helper 1 (Th1), Th2, Th17 and follicular helper T (Tfh) cells; since GRAIL deficiency in B cells leads to augmented development and function of Th1, Th2, Th17 and Tfh cells in vitro and in vivo. Thus, our results indicate that GRAIL is a crucial intrinsic factor controlling B cell activation and tolerance by targeting of BCR component CD79a for degradation.
Macrophages and the regulation of pulmonary fibrosis by Cadherin-11
Authors: To Sarah , Agarwal Sandeep K.
Published: January 2023

Pulmonary fibrosis is a chronic lung disease characterized by excessive deposition of extracellular matrix proteins by myofibroblasts leading to remodeling of the lung architecture. Alveolar macrophages play a key role in the development of pulmonary fibrosis and it has generally been associated with the polarization of these cells towards a profibrotic phenotype. Studies have shown the expression of the cell adhesion molecule Cadherin-11 (Cdh11) in alveolar macrophages from patients with pulmonary fibrosis and mice given bleomycin, and Cdh11-deficient mice are protected from bleomycin-induced pulmonary fibrosis suggesting an important role for Cdh11 in the pathogenesis of pulmonary fibrosis. In order to understand the role of Cdh11 in the development of pulmonary fibrosis, our study focuses on macrophages and aims to characterize the function and phenotype of Cdh11-deficient macrophages. We show that Cdh11-deficient mice have reduced numbers of tissue-resident alveolar macrophages compared to the wild type control mice and when given bleomycin, these mice have reduced numbers of CD11b+ monocyte-derived alveolar macrophages. Using BMDMs derived from Cdh11-deficient mice, we show that Cdh11-deficient macrophages have decreased expression of the alternative activation markers Arg1, Ym1, and CD206, and reduced secretion of TGF-β1, suggesting a reduced fibrotic phenotype. Furthermore, we show decreased phagocytosis, proliferation, and migration by Cdh11-deficient macrophages. These data suggest that Cdh11 may alter fibrotic responses by regulating the number and function of alveolar macrophages that are recruited to the lung.
Role of the KLRG1 pathway in the immune response
Authors: Tata Angela , Brossay Laurent
Published: January 2023

Killer cell lectin-like receptor G1 (KLRG1) is a C-type lectin inhibitory receptor that contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain. KLRG1 is well conserved in mammals and is mostly expressed on subsets of T cells and natural killer (NK) cells. Upon binding to its ligands, N-cadherin and E-cadherin, the phosphatases SHP-2 and SHIP-1 are recruited to the cytoplasmic tail of KLRG1. Some studies have proposed that KLRG1 is a marker of senescence, while other studies suggest that KLRG1 is a marker of recently activated cells. In addition, although KLRG1 inhibitory potential has been established in vitro, it is unclear whether KLRG1 inhibits NK cells and CD8+ T cells in vivo. To address its exact role, we have used CRISPR technology to generate mice deficient for KLRG1 and mice conditionally deficient for KLRG1 in the NK cell lineage. We found that NK cell frequency and number is not effected and that NK cell development is globally intact in KLRG1 deficient mice. The role of KLRG1 during viral infections and tumor development is currently being investigated.
Prostaglandin E2 threshold dictates susceptibility to skin infection in diabetic mice
Authors: Klopfenstein Nathan , Brandt Stephanie , Judge Allison , Serezani Carlos Henrique
Published: January 2023

Methicillin-resistant Staphylococcus aureus (MRSA) is the primary cause of skin infection in people with diabetes. MRSA skin infection is characterized by the migration of phagocytes and abscess formation to prevent deeper tissue infections. However, the events that drive poor host defense in diabetes is poorly understood. Here, we examined the role of the lipid mediator prostaglandin E2 (PGE2) and its impact on host defense during MRSA skin infection in diabetes. We have shown that diabetic mice are unable to control MRSA skin infection, along with excessive cytokine production and neutrophil migration. Our data indicate that PGE2 production is lower in diabetic mice than nondiabetic mice at both days 1 and 9. Daily topical treatment with a PGE2 analog decreased lesion size and improved bacterial clearance in the skin of infected diabetic mice. Reduced lesion size in PGE2-treated diabetic mice correlated with decreased production of the cytokines TNF-, IL-1, and IL-6 and neutrophil migration. While we did observe a well-defined abscess in infected nondiabetic mice, infection in diabetic mice resulted in poor abscess formation; and topical PGE2 treatment restores abscess formation. Poor skin defense in diabetic mice also correlated with increased numbers of dead cells at days 1 and 9 post-infection suggesting that dead cells are not eliminated. When we studied the expression of molecules involved in dead cell ingestion, we observed an increased abundance of inhibitory receptor SIRPa in infected diabetic mice and PGE2 ointment restored SIRPa levels back to those observed in infected nondiabetic mice. These data indicate that the threshold of PGE2 drives the inflammatory milieu during infection and effects skin homeostasis in diabetes.
Defect in neutrophil function accounts for impaired anti-fungal immunity in kidney dysfunction
Authors: Jawale Chetan , Ramani Kritika , Biswas Partha Sarathi
Published: January 2023

Chronic kidney disease is increasingly recognized as a major public health problem, and has a prevalence of 10% in the general population. Accumulation of uremic toxins results in systemic immunosuppression. Sepsis due to microbial infections accounts for 20% of deaths in patients with kidney diseases. Increased susceptibility of uremic patients to invasive fungal infections may attribute to impairment of innate immune defense. However, the molecular mechanisms of impaired anti-fungal immunity in kidney dysfunction are poorly understood. In this study, we have used the mouse model of aristolochic acid nephropathy to investigate the effect of uremia on antifungal immunity. A single IP injection of aristolochic acid I resulted in severe tubulointerstitial injuries accompanied by increased level blood urea nitrogen. Uremic mice showed increased Candida albicans burden in the internal organs following systemic infection. Increased fungal load was not attributed to defect in migration of neutrophils in the infected organs. Uremic condition impaired the ROS production and fungicidal activity of neutrophils. Interestingly, incubation of neutrophils with uremic serum led to impairment of their energy metabolism, indicated by reduced ATP content and lactate production. Neutrophils incubated with uremic serum showed reduced Glut1 protein expression and impaired glucose uptake capacity. Surprisingly, uremic serum inhibited PI3K/AKT pathway leading to aberrant activation of GSK3β in neutrophils. Furthermore, inhibition of GSK3β restored the glucose uptake in neutrophils incubated with uremic serum. These results indicate that uremia suppresses the anti-fungal activity of neutrophils by inhibiting their immunometabolism.
Myeloid Cells in the Immune Response to Giardia
Authors: Fink Marc Y. , Maloney Jenny G. , Singer Steven M.
Published: January 2023

Infection with the protozoan parasite, Giardia, produces symptoms associated with intestinal distress, but does not induce intestinal inflammation. Long-term sequellae of this infection include malnutrition, Irritable Bowel Syndrome and Chronic Fatigue Syndrome. Previous work indicates that T-cells are critical for clearance of this parasite; however, it is unclear which cells are responsible for the activation of T-cells. As such, our goal was to examine the contribution of myeloid cells to Giardia immunity as possible T-cell activators, effector cells and/or immune regulators.

Small intestinal lamina propria cells were isolated from Giardia-infected wild-type or CCR2 deficient mice. Flow cytometry was used to determine cell phenotypes and to monitor cytokine response. EdU labeling was used to examine in situ proliferation of resident macrophages. Lastly, wild-type mice were treated with an anti-CSF1R monoclonal antibody to deplete macrophage populations.

Flow cytometry demonstrated that CD11b+, F4/80+, CD11cint, MHCII+, Ly6Cint, CX3CR1int cells accumulate in the small intestine and express ARG1 and NOS2 during infection. Intracellular cytokine staining revealed an increase in IL-10 with a concurrent decrease in TNF-α. Macrophage accumulation was the same in wild-type and CCR2-deficient mice, and EdU labeling suggested in situ proliferation of resident macrophages rather than recruitment of inflammatory monocytes. Mice treated with anti-CSF1R eliminated the parasites as well as control mice.

This study demonstrates that macrophages with a regulatory phenotype accumulate in the small intestine during Giardia infections and may explain why intestinal inflammation is not present during Giardia infections.
A malaria protein factor induces IL-4 expression by dendritic cells via TLR/MyD88-independent signaling, promoting Th2 development
Authors: Wu Xianzhu , Gowda Nagaraj M. , Gowda Channe D.
Published: January 2023

Dendritic cells (DCs) and cytokines produced by DCs play crucial roles in inducing and regulating pro/anti-inflammatory and Th1/Th2 responses. While DCs are known to produce Th1-promoting IL-12 in response to malaria and other pathogenic infections, it is widely thought that DCs do not produce Th2-promoting IL-4. Here, we show that a protein factor of malaria parasites induces IL-4 responses by CD11chiMHCIIhiCD3ɛ−CD49b−CD19− FcɛRI− DCs via MyD88-independent signaling. Malaria parasite-activated wild type DCs induced IL-4 responses by T cells. Interestingly, lethal parasites, Plasmodium falciparum and P. berghei ANKA, induced IL-4 response primarily by CD8α− DCs, whereas nonlethal P. yoelii induced IL-4 by both CD8α+ and CD8α− DCs. In both P. Berghei ANKA- and P. yoelii-infected mice, IL-4-expressing CD8α− DCs did not express IL-12 and a distinct population expressed IL-12. Further, in P berghei ANKA-infection, CD8α+ DCs expressed IL-12 but not IL-4, whereas in P. yoelii infection CD8α+ DCs expressed IL-4 but not IL-12. This differential IL-4 and IL-12 responses by DC subpopulations may contribute to differential Th1 and Th2 development and different clinical outcomes in lethal and nonlethal malaria. Our results for the first time demonstrated that a malaria protein factor induces IL-4 production by DCs, revealing the molecular mechanism that initiate the promotes Th2 development.
The transcription factor Bhlhe40 is a novel regulator of large peritoneal macrophages and type 2 immunity
Authors: Jarjour Nicholas N. , Bradstreet Tara R. , Schwarzkopf Elizabeth A. , Lin Chih-Chung , Cook Melissa E. , Huang Stanley Ching-Cheng , Taneja Reshma , Randolph Gwendalyn J. , Urban Joseph F. , Edelson Brian T.
Published: January 2023

Tissue-resident macrophages occupy key roles in immunity and physiology within organ microenvironments. Many tissue macrophages derive from embryonic progenitors and self-maintain locally as unique tissue-specific populations. Resident macrophages can also expand in response to type 2 stimuli including parasites and contribute to control of infection via the alternative activation program. However, the transcriptional basis for this capacity to proliferate in situ is poorly understood. We have observed that the transcription factor basic helix-loop-helix, member e40 (Bhlhe40) is highly expressed in a subset of hematopoietic cell types, including large peritoneal macrophages (LPMs). Based on this data, we hypothesized that Bhlhe40 is a key part of the LPM transcriptional network. We have found that peritoneal macrophages are selectively reduced in Bhlhe40−/− mice, in contrast to other resident macrophages. Mixed bone marrow chimeras, conditional knockout mice, and other approaches demonstrated a specific, intrinsic defect in mature, Bhlhe40-deficient LPMs. Bhlhe40−/− LPMs exhibited an alternative activation-like profile and impaired self-renewal. These functional perturbations were correlated with altered expression of gene sets pertaining to the endoplasmic reticulum (ER) and protein homeostasis, as well as ER morphology changes in Bhlhe40−/− LPMs. Using models of peritoneal type 2 immunity, we observed near-total loss of expansion of Bhlhe40-deficient LPMs, correlated with impaired control of an intestinal helminth. Our findings demonstrate critical roles for Bhlhe40 as a tissue-specific regulator of resident macrophage self-renewal and expansion during type 2 immunity.
Malnutrition Decreases both Effector and Memory CD4 T cells in Malaria Infection
Authors: Pilotos Jennifer , Smith Margaret Rose , Johnson Corey M. , Campbell Logan , Ibitokou Samad , Stephens Robin , Opata Michael M.
Published: January 2023

Malaria and other chronic infections generate effector and effector memory T cells, but the infection is not completely controlled. While malnutrition has long been suggested to be protective against malaria, recent studies have shown that malnourished children are more susceptible to malaria infections, with increased mortality rates. Current vaccination protocols result in immunity that decays, but the influence of malnutrition in the target population has not been explored. To better understand how malnutrition affects the development of protective T cells in this infection, we used a diet of moderate malnutrition to determine the activation of effector (Teff), central memory (Tcm) and effector memory (Tem) T cells. C57Bl/6 mice were fed on either protein sufficient (16%) or moderately malnourished diets (3% protein, and deficient in both Iron and Zinc) for 5 weeks. Upon infection, we observed a significant decrease in the number of effector cells at day 7 post-infection. This was accompanied by a significant drop in weight among the malnourished mice. Surprisingly, there was no significant deference in parasitemia by day 7p.i. At the memory timepoints (d60 p.i), we observed that the spleens of the malnourished mice were much smaller than the well-nourished control mice. There was a significant drop in the number of CD4 T cells and Tcm but the proportion of Tem increased in the malnourished mice and a lower cell number due to the few total CD4 T cells in the malnourished group. Our data indicate that the nutritional status of the host affect the pool of memory CD4 T cells generated in malaria infection. This could be one mechanism as to why T cell immunity decays in malaria patients as the disease is prevalent in resource-limited nations.
Epithelial TSC1-mTOR controls RIPK3-dependent necroptosis and susceptibility to IBD by sensing dietary and microbial metabolites
Authors: Xiao Hui
Published: January 2023

Intestinal epithelial cells (IECs) are responsible for the sensing and uptake of dietary and microbial metabolites, yet how dietary and microbial components exert on intestinal homeostasis and Inflammatory Bowel Diseases (IBDs) has yet to be better understood. Here we firstly identified epithelial necroptosis as a key process influenced by nutrients protein and fatty acid, as feeding high-protein or high-fat diet to mice led to mTOR activation and aberrant necrosis in IECs. Notably, genetic ablation of TSC1 in the IECs mimicked the effect of high-protein/fat feeding, resulting in mTOR dysregulation, epithelial necrosis and hyper-susceptibility to colitis. Mechanistically, our data demonstrated that aberrant activation of mTOR could result in the overexpression of RIPK3 in enterocytes, whereby rendering epithelial necrosis and ensuing colitis, a process could be reversed by microbiota-depletion or rapamycin treatment. Remarkably, this mTOR-RIP3-necrosis axis seemed to underlie the pathogenesis of human IBDs as well. Furthermore, we found that mTOR-regulated selective autophagy might have contributed to the degradation of RIPK3 in IECs, revealing an intricate association between autophagy and necroptosis. Together, our data unravel an unsuspected link between metabolism and necroptosis, and highlight a crucial role for TSC1-mTOR in the maintenance of intestinal homeostasis and prevention of colonic inflammation through coordinating epithelial metabolism, autophagy and necroptosis.
Protective effect of IFN-γ during experimental autoimmune encephalomyelitis is associated with the induction of inducible nitric oxide synthase.
Authors: Dietz Alina Kay , Robinson Rachel R. , Forsthuber Thomas
Published: January 2023

Over 2.3 million people are affected by multiple sclerosis (MS), the most common demyelinating autoimmune disease of the CNS. The relapsing-remitting form of MS is the most common initial diagnosis; however, the vast majority of patients will eventually develop secondary-progressive MS. The mechanisms that contribute to the progression of MS are largely unknown. IFN-γ, a pro-inflammatory cytokine, has long been implicated in the pathogenesis of MS and its murine model, experimental autoimmune encephalomyelitis (EAE); however, protective functions have also been demonstrated. Previous studies in our lab revealed mice lacking IFN-γ signaling developed severe and progressive EAE due to an increased presence of myelin debris and lipid peroxidation in the CNS. Recently, we identified a significant increase of inducible nitric oxide synthase (iNOS) production in the brains of WT as compared with IFN-γR−/− mice at peak of disease by qRT-PCR. iNOS is an inducible enzyme that produces nitric oxide (NO) upon activation by immunological stimuli such as IFN-γ and LPS. There are conflicting data on the role of iNOS in the pathogenesis of EAE. Our preliminary findings indicate macrophages are the primary contributors to iNOS expression during disease progression. To further explore the role of iNOS in disease progression, we are studying the underlying mechanisms, key molecules involved in this process, and the effects of treatment with iNOS inhibitors on disease course, general pathology and biochemical composition of the CNS. Our results will contribute to a better understanding of the underlying mechanisms and may lead to new treatments to prevent progression of MS.
The Effect of Helminth-Induced Immunomodulatory Therapy (HINT) in Relapsing Remitting Multiple Sclerosis
Authors: Hernandez Gianna L. , Ganansky Caleb , Sandor Matyas , Fleming John , Fabry Zsuzsanna
Published: January 2023

This study examines the immune response stimulated by Trichuris suis ova when used as a treatment for Multiple Sclerosis. The hygiene hypothesis posits that insufficient exposure to microorganisms due to modern sanitation can predispose susceptible individuals to immune dysregulation. A pilot HINT 1 study indicated that TSO could effectively regulate the immune system of relapsing-remitting MS patients. Here, we investigate this hypothesis further with a longer treatment period. The HINT phase 2 clinical trial consisted of 16 treatment-naïve patients with newly diagnosed RRMS. Patients were given TSO buffered with 15mL of Phosphate buffered saline every two weeks. Anti-TSO immune response was measured by IgG1, IgA and IgE ELISAs of patient serum and eosinophilia count. Changes in immune cell populations were monitored by flow cytometry of peripheral blood mononuclear cells. CD4+ T cells were further analyzed as Th1, Th2, Th17, TREG, and TFH cells. During TSO treatment, the average number of new Gd+ lesions in the HINT patients was reduced. In addition, there was a significant increase in the anti-TSO immune responses as measured by IgG1 and IgE ELISAs, and increased eosinophilia during the second month of treatment. Also, TSO administration led to a decrease in the percentage of activated CD4+ and CD8+ T cells during therapy accompanied by an increase in the presence of TREG cells during treatment, a reduction in the presence on TFH cells in circulation, and an increase in IL-4-expressing T cells. This data implies a shift from a Th1 inflammatory state to a Th2 anti-inflammatory state during HINT II TSO treatment and suggests that immunoregulatory therapy through probiotic helminth treatment could be a viable option as a treatment for MS.
Oral Therapy with Colonization Factor Antigen I (CFA/I) Activates Stable Regulatory T Cells (Tregs) in the Pancreatic Lymph Nodes (PaLNs) of Non-Obese Diabetic (NOD) Mice
Authors: Nelson Andrew Scott , Maddaloni Massimo , Hoffman Carol , Abbott Jeffrey , Pascual David
Published: January 2023

Type 1 diabetes (T1D) is an autoimmune disease in which insulin-producing pancreatic β cells are destroyed by auto-aggressive T cells. Tregs in T1D have been shown to lose their suppressive function and/or become inflammatory, complicating Treg immunotherapy. Oral treatment with the adhesin from enterotoxigenic E. coli, CFA/I fimbriae, protects against autoimmunity. Although CFA/I fimbriae act in a bystander or in an antigen (Ag)-independent fashion, protection is ultimately dependent upon the induction and/or activation of auto-Ag-specific Tregs. Our initial findings showed that oral dosing with a Lactococcus lactis vector expressing CFA/I fimbriae (LL-CFA/I) reduces incidence of T1D in NOD mice by 45%. We hypothesized that dosing NOD mice more frequently would induce Foxp3+ Tregs in the pancreas and PaLNs to provide more robust protection. To test this, 4 wk-old NOD mice were orally dosed with LL-CFA/I varying the dosage and frequency of treatment. When dosed every 2 wks, the frequency of insulitis was reduced by more than half at 11 wks of age, and tetramer staining revealed a 2-fold reduction of insulin-specific CD4+ and CD8+ T cells within the PaLNs. Additional groups were followed until 16 wks of age, and examined for Treg and inflammatory T cell phenotypes. Treatment with LL-CFA/I reduced 2-fold IFN-γ-producing CD8+ T cells in the spleen and PaLNs. PaLN Foxp3+ T cells from the PBS and vector control groups, but not the LL-CFA/I-treated mice, co-expressed Tbet and IFN-γ, suggesting that Tregs from LL-CFA/I treated mice maintained their regulatory function at 16 wks. These data show that orally dosing with LL-CFA/I diminishes or halts T1D progression via activation of stably functional Foxp3+ Tregs. Work supported by AI121745.
Human alloreactive-regulatory T cell profile after ex vivo expansion using CD40L-stimulated B cells or monocyte-derived dendritic cells
Authors: Lee Linda M. , Zhang Hong , Liang Horace , Lee Karim , Thomson Angus W. , Tang Qizhi
Published: January 2023

Alloreactive regulatory T cells (arTregs) expanded with CD40L-stimulated B cells (sBcs) are being tested clinically to induce tolerance and control rejection in liver and kidney transplantation. In research settings, allogeneic monocyte-derived DCs (moDCs) have been shown to expand arTregs. We directly compared the efficacy of sBcs and cytokine-matured moDCs in expanding arTregs, and the phenotype of sBc-stimulated arTregs (sBc-arTregs) and moDC-stimulated arTregs (moDC-arTregs). arTregs expanded twice as much after stimulation with moDCs than with sBcs. Neither sBcs nor moDCs produced much TNFα, IL-1α, IL-1β, IL-6, IL-12P70, or IL-23 that can destabilize Tregs. sBc- and moDC-arTregs expressed similar percentages of Treg-defining markers - FOXP3, HELIOS, CD25, CD27, and CD62L. moDC-arTregs had slightly elevated FOXP3 and CD62L MFI. Compared to primary Tregs, sBc- and moDC-arTregs expressed similar or decreased levels of TBX21, GATA3, and RORC mRNA. However, greater percentages of moDC-arTregs produced IFNγ, IL-4, and IL-17A, and a greater percentage of sBc-arTregs produced IL-17A, but far less than those percentages produced by similarly stimulated conventional CD4+ T cells (Tconvs). The majority of cytokine-expressing arTregs were FOXP3+. Percentages of CXCR3 and CCR4, but not CCR6, increased after sBc and moDC stimulation. Using a 770-gene Nanostring panel and unsupervised clustering, sBc- and moDCs-arTregs were found to be mostly similar, but distinct from primary Tregs and further separated from Tconvs. Thus, arTregs expanded by sBcs or moDCs are phenotypically similar, likely stable, and had features that may enable them to traffic to inflammatory sites, e.g. transplanted organs undergoing alloimmune attack.
Prolonged cold ischemia storage of renal allografts shifts DSA specificity and enhances glomerular injury.
Authors: Gorbacheva Victoria Y. , Fan Ran , Baldwin William , Valujskikh Anna
Published: January 2023

Prolonged cold ischemia storage (CIS) of donor organs is a major risk factor for allograft injury. We tested how increased CIS time influences humoral alloimmunity in a mouse model of renal transplantation.

B6 (H-2b) mice received BALB/c (H-2d) renal allografts subjected to 0.5 h or 6 h CIS. At 14 d posttransplant, 6 h CIS recipients had increased titers of donor-MHC class II but not class I reactive IgG DSA, elevated frequencies of both class I and class II-reactive antibody secreting cells, higher frequencies of donor-reactive IFNγ-secreting T cells and higher proportion of graft glomeruli infiltrated with macrophages compared to 0.5 h CIS controls. B cell depletion inhibited DSA generation, intragraft C4d deposition and macrophage glomerular infiltration on d14.

Regardless of CIS duration, serum creatinine levels at d 60 posttransplant were comparable to non-transplanted mice. By d 60, 0.5 h CIS grafts had moderate segmental sclerosis in a limited number of glomeruli. In contrast, 6 h CIS grafts had extensive glomerular injury including thickened capillary loops, sclerosis, thrombotic microangiopathy, intracapillary fibrin thrombi, and mesangiolysis, and elevated intragraft levels of acute kidney injury markers NGAL and osteopontin. In addition, 6 h CIS recipients had increased serum levels of anti-class II but not anti-class I IgG DSA whereas the frequencies of IFNγ-producing donor-reactive T cells were low and comparable in both groups.

Our findings suggest that the augmented humoral rather than cellular immune responses account for the transplant glomerulopathy after prolonged CIS. Posttransplant inflammation spreads the specificity of DSA responses from class I to class II and may thus influence renal allograft pathology.
Vasoactive intestinal peptide synthesis in transplant recipients regulate donor T cell alloreactivity and graft-versus-host disease
Authors: Li Yiwen , Waller Edmund K.
Published: January 2023

Vasoactive intestinal polypeptide (VIP) is an immunosuppressive neuropeptide with pleiotropic effects. Activation of donor T cells leading to graft-versus-host disease (GvHD) in allogeneic bone marrow transplant (allo-BMT) causes secondary upregulation of immune co-inhibitory pathways, including VIP expression in T cells and dendritic cells. VIP signaling favorably regulates the activation status and the graft-versus-leukemia (GvL) activities of donor T cells (Li et al., Cancer Research 2016). We tested the hypothesis that absence of VIP expression by recipient cells would enhance donor T cell activation and increase GvHD in allo-BMT. VIP-KO mice transplanted with TCD BM alone had only 43.3% survival at day 75 compared with 73.3% survival among the WT recipients with no different in levels of donor chimerism. The addition of donor splenocytes to the graft resulted in significantly more GvHD-related mortality in VIP-KO recipients compared with WT recipients, with 80% vs 100% day 75 survival among recipients of 1×106 splenocytes (p=NS) and 0% vs 40% survival (p<0.01) among recipients of 3×106 donor splenocytes. VIP-KO recipients transplanted with wildtype cells had similar co-inhibitory pathway expression (PD1, Lag3, Tim3) but elevated cytokine production of IL2, IL4, IL10, IFNγ and TNFa by donor CD4+ and CD8+T cells post allo-BMT compared to WT recipients. VIP is expressed by residual host leukocytes up to 10 days post-transplant confirmed by confocal microscopic imaging. Host cells express GFP transgene under the control of VIP promoter, consistent with the hypothesis that VIP production by host antigen presenting cells represents a critical pathway in regulatory immune activation of donor T cells.
Recipient mucosal-associated invariant T cells control graft-versus-host-disease within the colon.
Authors: Varelias Antiopi , Bunting Mark , Ormerod Kate , Koyama Motoko , Olver Stuart , Straube Jasmin , Kuns Rachel , Robb Renee , Henden Andrea , Cooper Leanne , Lachner Nancy , Gartlan Kate , Lantz Olivier J. , Kjer-Nielsen Lars , Mak Jeffrey , Fairlie David , Clouston Andrew , McCluskey James , Rossjohn Jamie , Lane Steven , Hugenholtz Phil , Hill Geoff
Published: January 2023

Mucosal-associated invariant T (MAIT) cells are a unique innate-like T-cell subset that responds to a wide array of bacteria and yeast through recognition of riboflavin metabolites presented by the MHC I-like molecule, MR1. Here we demonstrate using MR1 tetramers that recipient MAIT cells are present in small but definable numbers in graft-versus-host disease (GVHD) target organs and protect from acute GVHD in the colon following bone marrow transplantation (BMT). Consistent with their preferential juxtaposition to microbial signals in the colon, recipient MAIT cells generate large amounts of IL-17A, promote gastrointestinal tract integrity, and limit the donor alloantigen presentation that in turn drives donor Th1 and Th17 expansion specifically in the colon after BMT. Allogeneic BMT recipients deficient in IL-17A also develop accelerated GVHD, suggesting MAIT cells regulate GVHD, at least in part, by the generation of this cytokine. Indeed, analysis of stool microbiota and colon tissue from IL-17A−/− and MR1−/− mice identified analogous shifts in microbiome operational taxonomic units (OTU) and mediators of barrier integrity which represent pathways controlled by similar, IL-17A-dependent mechanisms. Thus, MAIT cells act to control intestinal microbiota and barrier function to attenuate pathogenic T-cell responses in the colon, and given their very high frequency in humans, likely represent an important population in clinical BMT.
DISTINCT IL-6 SIGNALING PATHWAYS DRIVE ALTERNATE PATHOGENIC T CELL DIFFERENTIATION AND GVHD AFTER TRANSPLANT IN VIVO
Authors: Wilkinson Andrew N. , Kuns Rachel D. , Varelias Antiopi , Vuckovic Slavica , Rose-John Stefan , Gartlan Kate H. , Hill Geoffrey R.
Published: January 2023

Introduction

Dysregulation of interleukin 6 (IL-6) is pivotal for the development of graft-versus-host-disease (GVHD) after allogeneic stem cell transplantation. IL-6 drives many protective as well as inflammatory effects, the latter involving differentiation of T cells into pathogenic Th22, Th17, and Tc17 subsets. IL-6 signals through three principal pathways; classical, trans, and cluster, but their contribution to T cell differentiation and GVHD remains unclear. Importantly, these pathways are differentially targeted by clinical IL-6 inhibitors.

Aims

To dissect the contribution of the IL-6 signaling pathways to T cell differentiation and GVHD in order to guide therapy.

Methods

Donor bone marrow and T cells were transplanted from lineage-specific Cre × IL-6fl/flor IL-6Rfl/fl mice into irradiated, MHC disparate, wild-type or sgp130:Fc transgenic recipient mice with or without IL-6R mAb. Donor T cell differentiation and GVHD was assessed after transplant.

Results

Surprisingly, IL-6 trans-signaling was not required for T cell differentiation and GVHD, but instead regulated Th22 cell development. Indeed, inhibition of IL-6 trans-signaling in sgp130:Fc recipients promoted the expansion of Th22 cells and generated severe cutaneous GVHD. In contrast, IL-6 classical-signaling was critical for the development of Th22 and Th17 cells and its inhibition significantly attenuated GVHD broadly. Interestingly, unlike Th17 differentiation, Tc17 differentiation was highly IL-6-dependent but independent of classical and trans-signaling pathways, consistent with induction by cluster-signaling.

Conclusion

These data highlight cluster, in addition to classical IL-6-signalling as key therapeutic targets.
Depletion of macrophages from Cytomegalovirus (CMV) latently infected donor allografts decreases CMV mediated immunopathology and prevents CMV-accelerated chronic rejection
Authors: Haese Nicole , Burg Jennifer , Ando Takeshi , Jones Iris , Kreklywich Craig , Orloff Susan , Streblow Daniel
Published: January 2023

Cytomegalovirus (CMV), a ubiquitous β-herpesvirus, is linked to acceleration of solid organ transplant vascular sclerosis (TVS) and chronic rejection (CR). Latent CMV infection increases cardiac-resident macrophages and T cells that may increase inflammation and promote allograft rejection. To investigate the role of cardiac macrophages in CMV mediated TVS/CR, macrophages were depleted from CMV+ donor allografts prior to transplantation using clodronate liposomes (clod) in our rat heart transplant model. Latently rat CMV infected donor F344 rats were treated with clod or PBS or control liposomes 3 days prior to heterotopic cardiac transplant into CMV-naïve Lewis rats and macrophage depletion was validated. Donor hearts were transplanted and monitored daily for rejection. Clod treatment significantly increased graft survival from 61 days post-transplant to 84 days. The severity of graft vessel disease was decreased compared to controls. Clod treatment significant decreased macrophage and T cell infiltration into allograft tissue versus controls. Clod treatment also altered allograft tissue protein and gene expression cytokine/chemokine profiles toward a phenotype that promotes graft survival by reducing the allograft inflammation. Clod treatment did not prevent CMV reactivation since all recipients seroconverted and CMV DNA was detectable in recipient salivary glands. Depletion of donor macrophages prior to transplant represents a novel method to modulate allograft rejection immunity and prevent the negative impact of CMV on allograft recipient survival.
PD-L1 expression of monocytes may be used a new marker of the classification for the cancer progression in hepatocellular carcinoma.
Authors: Asai Akira , Yasuoka Hidetaka , Tsuchimoto Yusuke , Ohama Hideko , Fukunishi Shinya , Tsuda Yasuhiro , Kobayashi Makiko , Higuchi Kazuhide , Suzuki Fujio
Published: January 2023

Hepatocellular carcinoma (HCC) is the fifth common cancer in the world. In previous study, we reported that M2b monocytes which predominately generated in advanced-stage HCC patients were important target for HCC immunotherapy. Recently, an anti-PD-1 antibody has been approved for the treatment against HCC. In this study, we tried to analyzed for immunological characters of monocytes isolated from patients with several stages of HCC.

From 2017 August to 2017 December, nine patients diagnosed as advanced-stage HCC patients and five patients diagnosed as early-stage HCC patients were enrolled in this study. Monocytes from peripheral blood of these patients were stained for anti-PD-1 antibody, anti-PD-L1 antibody and anti-PD-L2 antibody. These cells were analyzed for flow cytometry. Furthermore, five healthy donors were used for the control groups.

In the results, a majority of monocytes from advanced-stage HCC patients were expressed PD-L1 (81.5 ± 14.7%) and PD-L2 (71.6 ± 18.5%), but not PD-1(20.2 ± 18.6%). And, monocytes from early-stage HCC patients were expressed PD-L1 (9.9 ± 4.1%), PD-L2 (38.74 ± 9.9%) and PD-1 (23.5 ± 9.8%). As the contorls, monocytes from healthy donors were not expressed PD-L1 (21.7 ± 5.5%), PD-L2 (31.7 ± 23.8%) and PD-1 (11.6 ± 6.4%). The PD-L1 expression in monocytes was increased as HCC progressed.

These results indicate the PD-L1 expression may be used as a new marker for the classification of the cancer progression. And, PD-1−PD-L1+PD-L2+ monocytes which were predominantly generated in advanced-stage HCC patients, may target for the treatment of anti-PD-L1 antibody.