bluuebunny/crossref_metadata_2025 huggingface dataset:
Inhibition of Cdk2 promotes the generation of inducible CD8+ T regulatory cells by abrogating binding of the EZH2 methyltransferase on the Foxp3 promoter.
Authors: Li Lequn , Barchan Kankana , Weaver Jessica D , Patsoukis Nick , Boussiotis Vassiliki A
Published: January 2023

Cdk2 has a central role in cell cycle re-entry and Cdk2 inhibition is mandatory for induction of T cell tolerance. To determine the effect of Cdk2 inhibition on T cell alloresponses we used the B6D2F1 model of bone marrow transplantation (BMT) and two Cdk2 inhibitors (Cdk2i), CYC202 and CYC205. B6D2F1(Kd) recipients infused with BM and T cells from C57BL/6(Kb) donors were treated with each Cdk2i for 3 weeks and graft versus host disease (GvHD) was assessed over a 70-day period. Cdk2i treatment decreased GvHD mortality (p=0.0054). To examine if Cdk2i affected Treg differentiation we used T cells from Foxp3.GFP-KI mice during BMT. Cdk2i-treated recipients had no difference in CD4+GFP+ Treg compared to control groups in peripheral blood, splenocytes, lymph nodes and intestinal lymphoid cells (ILC) but had increased CD8+GFP+ Treg, predominantly in ILCs (p=0.05). In Treg polarizing culture Cdk2i had only minimal effect on CD4+GFP+ cells but induced significant increase of CD8+GFP+ cells. However, TGF-b-mediated signaling, as determined by Smad3 phosphorylation was unaffected. FOX family members are regulated by transcriptional and epigenetic mechanisms. The Polycomb group (PcG) protein, enhancer of zeste homologue 2 (EZH2) induces histone H3 lysine 27 trimethylation (H3K27me3) and epigenetic gene silencing. Cdk2 phosphorylates EZH2 Thr350, which is important for maintenance of H3K27me3 at EZH2 target loci. We determined that Cdk2i inhibited EZH2 Thr350 phosphorylation in CD8+ T cells, which coincided with increased Foxp3 expression. ChIP-PCR identified EZH2 binding on three sites of Foxp3 promoter, which was abrogated by Cdk2i. Thus, Cdk2 inhibition induces CD8+ Treg via EZH2-mediated epigenetic mechanisms.
Using patients’ stated preferences for immunoglobulin therapies to evaluate administration features in current treatments
Authors: Runken Michael Chris , Gonzalez Juan Marcos
Published: January 2023

In 2012, Mohamed et al.(J Med Econ) estimated preference weights for changes in the administration features of immunoglobulin (IG) therapies among patients with self-reported primary immunodeficiency disorder (PIDD). The preference weights were estimated using a discrete-choice experiment survey that asked respondents to choose between hypothetical treatments with varying administration features. However, the authors did not discuss the implications of their results on patients’ preferences for currently available IG therapies with specific combinations of the elicited features.

Transferring preference results from Mohamed et al. allowed us to evaluate current treatment options not considered during the original study. This transfer of preference information is commonplace in economics, but not in health applications where it is done almost exclusively with treatment outcomes and not treatment administration features.

The aim of this study was to assess patients’ relative preferences for currently available IG treatments in terms of their administration features as elicited by Mohamed et al. To achieve this objective, we approximated a preference-weight function through visual examination of the weights plotted in the publication. This function provides a calculation of preference scores for any IG treatment described with the attributes studied in Mohamed et al.

This research suggests a general tendency in PIDD patients to prefer the administration features associated with intravenous therapies over subcutaneous therapies. The evaluation of IG therapies with the approximated preference function highlights how attribute preferences can be considered jointly in clinically meaningful treatment profiles.
Hypersplenism, EPO resistance and chronic Salmonella infection
Authors: Loomis Wendy P , Delaney Martha A. , Johnson Matthew L. , Cookson Brad T
Published: January 2023

Chronic diseases, such as AIDS and malaria, are associated with increased susceptibility to non-typhoidal Salmonella (NTS) infection. Among the key clinical manifestations shared by these immunocompromised individuals are anemia and splenomegaly (i.e. hypersplenism), conditions also associated with NTS susceptibility. To better understand the immunological basis of hypersplenism, we developed a model of chronic NTS infection in resistant 129X1/SvJ mice transiently depleted of CD4+ T cells. NTS infection induced chronic peripheral anemia that became more severe in the absence of CD4+ T cells. While serum erythropoietin (EPO) peaked on day 21 post infection in mock-depleted mice, EPO levels continued to rise in CD4-depleted animals. The failure to resolve anemia despite high levels of EPO parallels the development of EPO resistance in patients receiving recombinant human EPO therapy. Increased accumulation of mature erythrocytes and immature erythroid precursor cells within the enlarged spleens of CD4-depleted mice suggests that chronic inflammation leads to splenic sequestration of damaged erythrocytes, thus contributing to persistent anemia. We demonstrate that sequestration of erythroid cells drives the expansion of splenic macrophages, a known reservoir for NTS survival. Our findings provide a potential mechanism to link anemia of chronic disease, hypersplenism and increased susceptibility to NTS infections.
CD169+ marginal zone macrophages orchestrate innate immune responses to bacterial infection.
Authors: Perez Oriana A , Qiu Zhijuan , Romagnoli Pablo A , Benechet Alexandre P , Maher Leigh , Tanaka Masato , Khanna Kamal Mohan
Published: January 2023

The spleen is an important site for generating protective immune responses against pathogens. Immune cells in the spleen undergo rapid reorganization to initiate and maintain local inflammatory responses against pathogens. How the spatial dynamics of cellular communication are regulated in the spleen after infection remains unclear. Respectively, CD169+ macrophages are a population of tissue resident macrophages that are positioned in the marginal zone to rapidly encounter invading pathogens. However, the role of splenic CD169+ macrophages during infections is not well understood. Here we show that splenic CD169+ macrophages serve as a primary cellular host to blood-borne bacteria Listeria monocytogens (Lm) and are also essential for the clearance of other bacterial and viral pathogens. In addition to controlling initial bacterial growth, CD169+ macrophages orchestrated a second phase of innate protection by mediating the transport of Lm to the splenic T cell zones and driving the subsequent reorganization of neutrophils, monocytes, and NK cells into hierarchical clusters. Specifically our study revealed that bacterial transport to the T cell zones was mediated by trans-infection of closely interacting CD8α+ DCs with Lm-infected CD169+ macrophages. Although, CD8α+ DCs are required for a productive Lm infection in the spleen, to our surprise mice that lacked both CD8α+ DCs and CD169+ macrophages were unable to control Lm infection. These results demonstrated that CD169+ macrophages regulated bacterial access to the pathogen favorable CD8α+ DC niche, ultimately providing a rationale for reassessing the current paradigm regarding the temporal role of CD8α+ DCs during Lm infection.
Identification of two T cell subsets that protect and survive long-term in malaria infection
Authors: Opata Michael , Norwood Karis , Ibitokou Samad A , Carpio Victor Hugo , Wilson Kyle , Dillon Brian , Stephens Robin
Published: January 2023

Malaria and other chronic infections generate effector and effector memory T cells, but the infection is not completely controlled. While the parasite is present, some level of protection is provided, but this decays as specific cytokine production disappears. This indicates that protection in chronic malaria is partly provided by continuously activated CD4 T cells. Current vaccination protocols also result in immunity that decays. In order to invent new vaccination strategies to combat this problem, we need to understand what T cell types are potentially both protective and long-lived. Therefore, we tested effector (Teff), central (Tcm), and effector memory (Tem) T cell subsets for their ability to both protect and survive in vivo. Using transgenic T cells specific for the Merozoite Surface Protein-1 of Plasmodium chabaudi, and our previously defined effector and memory subsets, we sort-purified individual cell populations, and tested their ability to protect in combination with immune B cells. Strikingly, Teff reduce parasitemia by half, at all stages of effector maturation. When Teff subsets were rested in naïve animals to allow generation of memory, the least mature Teff subset, TeffEarly, which we have shown generate the most memory cells, decreased parasitemia the most on infection of adoptive hosts. Interestingly, these earliest activated T cells survive as well as the memory subsets, of which only Tem Late (CD62LloCD27−) reduced parasite by 10%. Therefore, we have identified two long-lived T cell subsets that may represent a correlate of protection or a target for longer-lived vaccine-induced protection against malaria.
Lactic acid suppresses cytokine production and expression of miR-155 and 146a following LPS activation in mast cells.
Authors: Caslin Heather , Abebayehu Daniel , Qayum Amina Abdul , Spence Andrew , Ryan John J
Published: January 2023

A considerable health burden, sepsis is a clinical inflammatory condition with mortality rates from 30–50% and no effective treatments. Septic patients and animal models show increased blood lactate concentrations and microRNA(miR)-155 and miR-146a expression, but how these factors are related is not clear. Lactic acid has been shown to have a suppressive effect on LPS activation and subsequent NFkB induction in macrophages and dendritic cells, but mast cells have not been examined. Our data suggests that lactic acid suppresses LPS-induced cytokine and chemokine production in bone marrow derived mast cells (BMMCs) and peritoneal mast cells. This effect is dependent upon the concentration of lactic acid, pH, and transport through MCT-1. Additionally, it is known that miR-155 and 146a affect cytokine production downstream of NFkB and TLR4, however the effects of lactic acid on these miRs is unknown. Our results show that LPS enhances miR-155 and miR-146a expression, an effect that is antagonized by lactic acid. Interestingly, miR-155 is historically considered pro-inflammatory, while miR-146a is considered anti-inflammatory. Current studies seek to determine the contribution of these miRs to septic inflammation and its modulation by lactic acid.
RNF144b is a negative regulator in TLR2-mediated NF-kB activation
Authors: Baek Suk-Hwan , Huang Bin , Chang Hyeun Wook
Published: January 2023

NF-κB regulates the expression of a various genes involved in diverse cellular processes including inflammation and immunity. Activation of NF-κB requires ubiquitination, a highly conserved and versatile modification that can regulate cell signaling through both proteasome dependent and independent mechanisms. Ubiquitination process is a representative of post-translational modification involved in NF-κB activation of TNFR and TLR signaling. We demonstrated that RNF144b, an E3 ubiquitin ligase, is important factor for the NF-κB regulation in macrophages upon stimulation with TNFa or TLR2 ligand. Treatment of TNFa or TLR2 ligand (Pam3CSK4) decreased the expression of RNF144b mRNA on macrophages. TLR2 ligand has a stronger effect than TNFa in the expression of RNF144b mRNA. Knock-down of RNF144b potentiated activation of NF-kB. And according to the increase of NF-κB activity, the formation of foam cells and the production of cytokines (IL-1β, IL-6, CCL2 and CCL3) following TLR2 stimulation increased. On the contrary, overexpression of RNF144b decreased NF-κB activity and cytokines production in the TLR2-stimuated macrophages. RNF144b binds and ubiquitinates TRAF6 and these effects are dependent of its E3 ligase activity. Mutation of Cys201 to Ala abolishes activity of RNF144b to activate TRAF6 ubiquitination and also partially attenuates NF-κB activity. Taken together, these results suggest that RNF144b stimulates NF-κB activity via TRAF6 ubiquitination and then produces inflammatory cytokines and chemokines, and eventually controls foam cell formation in the TLR2-mediated macrophages.
Autocrine and paracrine IL-2 signals collaborate to regulate distinct phases of CD8 T cell memory
Authors: Yuzefpolskiy Yevgeniy , Penny Laura Anne , Baumann Florian Martin , Smith Kendall A , Sarkar Surojit , Kalia Vandana
Published: January 2023

Proliferation is a defining feature of clonal cytotoxic T lymphocyte (CTL) responses upon activation. However, how proliferative events during primary expansion relate to divergent CD8 T cell fate outcomes remains poorly defined. Emerging evidence from our laboratory and others in the field suggest that death-fated effectors undergo more pronounced proliferation compared to memory-fated cells during antigen-driven T cell expansion. Adoptive transfer of activated CD8 T cell-subsets, purified based on their extent of proliferation, showed that in vivo differentiation of CD8 T cells and memory fate are linked to proliferation. We found that the less-divided effector CTL subset preferentially contributed to the central memory lineage. Interestingly, these less-divided memory precursor cells were also programmed to produce more IL-2 compared to their more-divided terminal effector counterparts. How do memory-fated cells that make their own IL-2 (a pro-proliferative cytokine) proliferate less and how does this make them better fit for the long-lived memory lineage? To answer this question, we generated CD8 T cells that could be conditionally ablated for il2. We ablated IL-2 specifically in antigen-specific CD8 T cells, during distinct phases of effector and memory differentiation. Our data showed that while paracrine IL-2 is critical for driving the early in vivo proliferative burst, the programmed life-long autochthonous IL-2 in memory-fated cells was necessary for antigen-independent maintenance, and robust secondary expansion of memory cells. Thus, autocrine and paracrine IL-2 signals collaborate during distinct stages of T cell differentiation to direct diverse CTL lineages through effects on proliferation.
Asymmetric integrin segregation regulates CD8+ T cell fate during cell division
Authors: Capece Tara , Walling Brandon Lee , Hyun Young-Min , Kim Tae-Hyoun , Lim Kihong , Kim Minsoo
Published: January 2023

Recent evidence suggests lineage commitment of effector and memory T cells occurs as early as the first T cell division, using the evolutionarily conserved mechanism of asymmetric cell division. In addition, successful activation of the adaptive immune response requires precise cellular localization of divided T cells in critical lymphoid niches. Although protein inheritance of asymmetrically divided T cells has been characterized and leads to two differentially fated daughter T cells, little is known about how asymmetric cell division regulates T cell migration in the lymph node and how it controls T cell differentiation. We demonstrated distinct migration and T-APC conjugation patterns and localization after the first T cell division both in vitro and in vivo. Expression of the integrin LFA-1 was the only relevant surface change during division. Using our newly generated CD11a-mYFP knock-in mice, we discovered naïve T cells reserve an intracellular store of LFA-1 located in the uropod during T cell migration. Intracellular LFA-1 quickly translocated in a Rab27-dependent manner to the cell surface within minutes of contact with antigenic stimulus. Importantly, the redistribution of both surface and intracellular LFA-1 led to a molecular asymmetry that was maintained during cell division, giving rise to daughter cells with distinct fates. We hypothesize the redistribution and asymmetric segregation of intracellular LFA-1 controls subsequent T cell encounters, localization, and cell fate.
T cell restricted Notch signaling contributes to pulmonary Th1 and Th2 polarization during Cryptococcus neoformans infection
Authors: Neal Lori M , Qiu Yafeng , Chung Jooho , Xing Enze , Cho Woosung , Malachowski Antoni , Osterholzer John J , Maillard Ivan , Olszewski Michal A
Published: January 2023

Notch is a key signaling pathway regulating T cell development, activation and function. Notch signaling has been shown to affect the differentiation and functional responses of Th cells in several models of infectious disease and therapeutic targeting of Notch signaling has been proposed as treatments for autoimmune diseases. However, the contribution of Notch signaling to Th cell differentiation and responses during fungal infections is unknown. To elucidate the role of Notch signaling during fungal infections we infected mice expressing a dominant negative Notch inhibitor specifically within mature T cells with the fungal pathogen Cryptococcus neoformans. Inhibition of Notch resulted in impaired leukocyte recruitment, failure to control fungal burden in the lungs and failure to prevent dissemination to the CNS. Furthermore, we show that inhibition of Notch signaling impaired both protective Th1 and non-protective Th2 anti-cryptococcal immune responses. Leukocyte cultures and T cells from Notch deficient mice produced less IFN-γ, IL-5 and IL-13 than WT cells and had reduced expression of Th1 and Th2 associated transcription factors, Tbet and Gata3. Treatment with the Notch inhibitor DAPT did not impair cytokine production in WT cells indicating that Notch impacted the differentiation of these cells rather than biasing the magnitude of responses. Th17 responses were largely unaffected by Notch; although we also measured an increase in the frequency of Tregs in Notch deprived C. neoformans infected mice. Overall, these data suggest that Notch signaling controls the differentiation of Th1 and Th2 cells during C. neoformans infection and that targeting Notch may inadvertently render hosts more susceptible to fungal infections.
IFN-gamma mediated inflammatory monocyte recruitment neutralizes iNOS-dependent parasite killing by expanding the permissive host cell reservoir during early Leishmania amazonensis infection
Authors: Carneiro Matheus Batista , de Moura Lopes Mateus Eustaquio , Romano Audrey , Campos Alexandre Camargo , Sacks David , Vieira Leda Q , Peters Nathan Colin
Published: January 2023

IFN-γ is a key factor in the elimination of intracellular parasites. However, during early L. amazonensis infection IFN-γ production does not correlate with a reduction in parasite growth and IFN-γ−/− mice do not have enhanced parasite loads. The reason why IFN-γ production does not reduce parasite loads during early L.a. infection is controversial. In order to investigate the role of IFN-γ during early L.a. infection we infected C57BL/6 Wt and IFN-γ−/− mice with 104 RFP+ parasites in the ear and followed disease for 6 wks. IFN-γ−/− mice showed equivalent lesion sizes and parasite numbers compared to Wt mice for the first 4 wks p.i., despite higher expression of the putative disease promoting factors IL-4 and arginase I. IFN-γ−/− mice had no CD11b+iNOS+ phagocytes while iNOS production was detected as early as 2 wks p.i. in Wt mice. Interestingly, Wt mice had higher numbers of inflammatory monocytes per ear during early infection. Inflammatory monocytes represented the majority of total infected cells and parasites within inflammatory monocytes cells were viable. Inflammatory monocytes were a small proportion of CD11b+iNOS+ cells in Wt mice and had significantly reduced levels of IFN-γR versus dendritic cells and macrophages. The enhanced host cell infiltrate observed in Wt mice may counteract the protective role of IFN-γ mediated iNOS expression. This could explain, in part, why IFN-γ production does not lead to a reduction in parasite load during early L. amazonesis infection. We propose IFN-γ plays a dual role, inducing both the expression of iNOS and parasite killing and the recruitment of inflammatory monocytes that support parasite replication during early infection.
Local antigen in non-lymphoid tissue promotes resident memory CD8+ T cell formation during viral infection.
Authors: Nolz Jeffrey C , Khan Tahsin N , Mooster Jana L , Kilgore Augustus M , Osborn Jossef F
Published: January 2023

Tissue-resident memory (TRM) CD8+ T cells are functionally distinct from their circulating counterparts and are potent mediators of host protection against re-infection. One largely accepted paradigm is that activated CD8+ T cells are able to freely migrate to non-lymphoid tissues and differentiate into TRM independent of local antigen recognition. In contrast, using skin infections with Vaccinia virus (VacV) expressing model antigens, we found that local antigen recognition had a profound impact on TRM formation. Activated CD8+ T cells trafficked to VacV-infected skin in an inflammation-dependent, but antigen-independent manner. However, following viral clearance, there was a subsequent ~50-fold increase in CD103+/CD69+ TRM formation when antigen was present in the tissue microenvironment. Secondary antigen stimulation in non-lymphoid tissue caused CD8+ T cells to rapidly express CD69 and be retained at the site of infection. Finally, TRM CD8+ T cells that formed during VacV infection in an antigen-dependent manner became potent stimulators of localized antigen-specific inflammatory responses in the skin. Thus, our studies indicate that the presence of antigen in the non-lymphoid tissue microenvironment plays a critical role in the formation of functional TRM CD8+ T cell populations, a finding with relevance for both vaccine design and prevention of inflammatory disorders.
Route of immunization governs the development of distinct resident memory CD8 T cells and protective immunity in the intestinal mucosa
Authors: Qiu Zhijuan , Sheridan Brian S
Published: January 2023

We previously demonstrated that oral immunization with a murinized Listeria monocytogenes induced rapid formation of intestinal resident memory CD8 T (TRM) cells that were capable of providing secondary protection. It is well appreciated that systemic vaccination also induces a population of protective TRM cells in the intestine. We sought to understand the impact of immunization route on the development of intestinal TRM cells and protective immunity. Oral immunization resulted in significantly more antigen-specific effector CD8 T cells in the intestinal compartments than intravenous (i.v.) immunization, likely due to differential imprinting and migration of T cells. While both oral and i.v. immunization induced a4b7 expression on CD8 T cells, only oral immunization resulted in CCR9 expression. Moreover, compared to i.v.-induced CD8 T cells, oral-induced cells rapidly adopted a CD127+ KLRG-1− memory precursor phenotype and a CD103+ CD69+ residency phenotype. Furthermore, oral immunization led to a substantially larger TRM population in the intestine compared to i.v. immunization. While the majority of oral-induced TRM cells were CD103+, a substantial proportion of i.v.-induced TRM cells did not express CD103. Collectively, these data suggest that oral immunization would provide an optimal sentinel population of memory cells that are more efficient in providing protection against secondary intestinal exposure. Indeed, oral-immunized mice had reduced bacterial burden after challenge infection compared to i.v.-immunized mice. In conclusion, our studies demonstrate that route of immunization greatly affects the induction and maintenance of TRM cells and thus the outcome of immunization.
Failure of gut-associated pDCs to express membrane bound APRIL and BAFF prevents their ability to promote low-affinity IgA expression in ME/CFS
Authors: Lombardi Vincent C , Khaiboullina Svetlana F , De Meirleir Kenny L , Mijatovic Tanja , Hulstaert Jan
Published: January 2023

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a heterogeneous illness characterized by a number of comorbid conditions; gastrointestinal (GI) dysregulation make up one subgroup of this disease. IgA is the most abundant antibody isotype found in mucosal secretions including the gut. In a process of class switch recombination (CSR), that relies on the interaction of plasmacytoid dendritic cells (pDCs) with B cells, in a T cell independent (TI) manner, low-affinity IgA are produced that limit the adhesion of commensal bacteria to intestinal epithelia without neutralizing them. These low-affinity antibodies also limit bacterial overgrowth and potential bacterial translocation thus maintaining gut homeostasis. This process is known as “immune exclusion”. Two ligands on the surface of pDCs that are obligatory for the process; the membrane bound form of APRIL and BAFF. The upregulation of APRIL and BAFF on the surface of pDCs is dependent on low-level expression of type I interferon (IFN) which is produced by intestinal stromal cells in response to Toll-like receptor (TLR) engagement. Previous studies suggest that peripheral pDCs are significantly lower in subjects with ME/CFS when compared to controls and studies conducted by us further suggest these cells likely redistribute from the periphery to the gut. We have observed that, in contrast to controls, gut-associated pDCs in subjects with ME/CFS lack APRIL and BAFF expression. These data support a model of gut pathology in ME/CFS whereby dysregulated pDCs fail to promote the production of low-affinity IgA through the process of TI activation of B cells, thereby leading to bacterial overgrowth, dysbiosis, bacterial translocation and systemic immune activation.
Investigating distinct immunization routes in zebrafish on mucosal immunity
Authors: Young Matt , Li Ellen S. , Branham Erica , Browne Zachary , Koufteros Christiana A , Pham Sydney E L , Kim Joshua C , Stebbins Elyssa A , Wenck Fallon E , Zhang Annie L , Kommein Swathi , Russo Maximillian A X , Criscitiello Michael F
Published: January 2023

Model organisms such as Danio rerio are used to explore and model physiological reactions to stress, illness, and disease. Immune response is measured by quantifying lymphocyte presence and the production of mRNA encoding different immunoglobulin proteins. Because the method through which D. rerio is exposed to an antigen affects its immune response and the degree to which immunity is developed, we will expand on experiments comparing the immune response after visceral or mucosal introduction of DNP-KLH in D. rerio. In our visceral treatment, we will combine DNP-KLH with Freund’s incomplete adjuvant and inject the mixture into the fish (100 uL at 1 mg/mL). For our mucosal exposure, we will combine DNP-KLH with cholera toxin as an adjuvant and introduce the mixture to their water (35 mg/L). Two control groups will account for intravenous immune response by injections solely with sterile saline solution as well as a control for the anesthetic used, MS-222. Viscera tissue samples will be collected from the immunized fish and two control groups of fish at weekly points throughout the experiment and will be analyzed for lymphocyte versus granulocyte ratios with flow cytometry and expression of IgM, IgZ-1, and IgZ-2 levels via qPCR. We expect to see an increase in lymphocyte percentages for both immunized groups compared to the control groups, with a higher lymphocyte ratio in the mucosal group than the visceral group. Furthermore, we expect more mucosal IgZ isotypes in the mucosal group and higher levels of the IgM, IgZ-1, and IgZ-2 antibodies in the visceral group than in the mucosal group. For future experiments, we would like to determine specific tissues of interest that provide data for analysis of mucosal and visceral immunological responses.
3H-1,2-Dithiole-3-thione as a novel therapeutic agent for the treatment of experimental autoimmune encephalomyelitis
Authors: Kuo Ping-Chang , Brown Dennis A. , Scofield Barbara A. , Yu I-Chen , Chang Fen-Lei , Yen Jui-Hung
Published: January 2023

3H-1,2-Dithiole-3-thione (D3T), the simplest member of the sulfur-containing dithiolethiones, is found in cruciferous vegetables, and has been previously reported to be a potent inducer of antioxidant genes and glutathione biosynthesis by activation of the transcription factor Nrf2. D3T is a cancer chemopreventive agent and possesses anti-inflammatory properties. Although D3T has been shown to protect against neoplasia, the effect of D3T in the autoimmune inflammatory disease Multiple Sclerosis/Experimental Autoimmune Encephalomyelitis (EAE) is unknown. The present study is the first report of the therapeutic effect of D3T in EAE. Our results show D3T, administered post immunization, not only delays disease onset but also dramatically reduces disease severity in EAE. Strikingly, D3T, administered post disease onset of EAE, effectively prevents disease progression and exacerbation. Mechanistic studies revealed that D3T suppresses the expression of co-stimulatory molecules and production of pro-inflammatory cytokines by dendritic cells, inhibits pathogenic Th1 and Th17 differentiation, reduces the production of inflammatory mediators by microglia, and induces the expression of antioxidant phase II enzymes in microglia. In summary, these results indicate that D3T affects both innate and adaptive immune cells, and the protective effect of D3T in EAE might be attributed to its effects on modulating dendritic cell and microglia activation and pathogenic T cell differentiation.
Inhibition of MIF as a novel treatment for autoimmune myocarditis and dilated cardiomyopathy.
Authors: Nalawade Saisha Abhay , Forsthuber Thomas Gunter
Published: January 2023

Myocarditis is an inflammatory disease of the myocardium and a major cause of sudden death in young adults. It is characterized by the presence of immune infiltrates and necrotic myocytes in the myocardium. Importantly, patients often progress to a more severe form of the disease termed dilated cardiomyopathy (DCM), which leads to ventricular dilation and impaired cardiac function. Despite the inflammatory and autoimmune nature of this disease condition, immunosuppressive treatments such as corticosteroids (CSs) have not been very effective in preventing myocarditis and its progression to DCM. We hypothesized that macrophage migration inhibitory factor (MIF) may play a role in resistance to CSs, as it is the only known pro-inflammatory cytokine to be induced by CSs. Importantly, MIF counter-regulates CS-mediated immunosuppression. Using the experimental autoimmune myocarditis (EAM) animal model, we observed that MIF−/− mice treated with Dexamethasone (Dex) were highly resistant to EAM and progression to DCM. Furthermore, using small molecule inhibitors of MIF combined with Dex treatment recapitulated this phenotype in wild type mice. Investigating the mechanism we found that treatment with MIF inhibitors and Dex decreased the expression of key chemokines and adhesion molecules and implicated these molecules in the progression of EAM to DCM. Our results suggest that MIF promotes the recruitment of inflammatory cells to the myocardium to promote DCM. Moreover, the results suggest therapeutic inhibition of MIF in combination with CSs as a novel treatment approach to prevent DCM. Last, our studies may provide new insights into the mechanisms driving DCM.
Th17 and Th1 play distinct roles in obliterative bronchiolitis pathogenesis after transplantation.
Authors: Xu Jiangnan , Zhang Yamei , Wang Lifeng , Chen Qirui , Li Hui , Bromberg Jonathan S , Ding Yaozhong
Published: January 2023

Development of obliterative bronchiolitis (OB) is the biggest obstacle that limits long-term allograft survival and clinical application of lung transplantation. Mouse orthotopic trachea or lung transplantations were utilized to examine roles of Th17 and Th1 during OB pathogenesis after transplantation. Allografts of wild type recipients displayed elevated levels of Th1 and Th17 cytokines IFNγ, IL-6 and IL-17 expression. Recipients deficient in Th1 key transcriptional factor T-bet resulted in decreased cell infiltration and improved airway integrity in orthotopic transplanted trachea grafts; Lack of Th2 transcriptional factor Stat6 had little effect; While absence of Th17 master transcriptional factor RORγt had striking effects in abolishing airway epithelial injury, a leading factor of OB pathogenesis. Administering anti-IL-6 or anti-IL-17 but not anti-IFNγ mAbs restored normal airway epithelial morphology. Furthermore, we found that anti-CD40L treatment resulted in increased FoxP3 and regulatory T cells, and decreased IFNγ expression; However it had very limited effect on IL-17 suppression and airway epithelial protection. While CTLA4-Ig treatment significantly inhibit IL-17 expression and reduced epithelial injury, and the combination of CTLA4-Ig and anti-CD40L markedly restored airway morphology of grafts. Together, our findings provide new insights for understanding Th17 and Th1 responses during OB pathogenesis and demonstrate the critical importance of suppressing Th17 for OB development after transplantation.
LRBA is essential for allogeneic responses in bone marrow transplantation
Authors: Sudan Raki , Park Mi Young , Srivastava Neetu , Neelam Sudha , Youngs Christie , Wang Jia-Wang , Engelman Robert W , Kerr William
Published: January 2023

Members of the PH-BEACH-WD40 repeat gene family (PBW) play a role in coordinating receptor signaling and intracellular vesicle trafficking. LPS-Responsive Beige-like Anchor (LRBA) is a PBW protein whose immune function has not been defined. Here we show that LRBA−/− mice are viable with normal and healthful life span, but exhibit compromised rejection of allogeneic, xenogeneic and missing self bone marrow grafts. Further we demonstrate that LRBA−/− Natural Killer (NK) cells exhibit impaired signaling by the key NK activating receptors, NKp46 and NKG2D. However, induction of γ-IFN by co-culture with IL12 and IL18 remains intact, indicating LRBA selectively facilitates signals by receptors for ligands expressed on the surface of NK targets. Surprisingly, LRBA limits immunoregulatory cell numbers in tissues where Graft versus Host Disease (GvHD) is primed or initiated, and consistent with this LRBA−/− mice demonstrate resistance to lethal GvHD. These findings demonstrate that LRBA is redundant for host longevity while being essential for both host and donor-mediated immune responses in allogeneic transplantation and thus represent a unique and novel molecular target in transplant immunology.
Thymus is required for T cell reconstitution following lymphoablation
Authors: Ayasoufi Katayoun , Fan Ran , Valujskikh Anna
Published: January 2023

Lymphoablation is used as induction therapy in transplantation. Following lymphoablation, peripheral lymphopenia triggers homeostatic T cell proliferation and thymopoiesis. However, the relative contribution of each pathway to T cell recovery is unknown. The goal of this study was to test the role of thymus during T cell reconstitution following lymphoablation. We use a mouse model of heterotopic heart transplantation (BALB/c to B6) and a murine analog of Thymoglobulin (mATG). We previously reported that the recovery of CD8 T cells after mATG depletion requires residual memory CD4 T cells and CD40 expressing B cells. CD4 T cell depletion or CD154 blockade significantly reduced the numbers of single positive CD8 thymocytes generated in mATG treated mice. To test if thymus is required for T cell reconstitution, thymectomized and euthymic heart allograft recipients were treated with mATG on days 0 and 4. Thymectomy impaired recovery of CD4 and CD8 T cells compared to euthymic mice (10% vs. 1.7% CD4 and 3% vs 0.06% CD8 T cells on d 12). The transplantation of thymi from B6 WT or RAG1−/− mice under the kidney capsule of thymectomized mice partially restored T cell reconstitution following depletion. Adoptive transfer of CD4 T cells (10×106 i.v. d −1) or treatment with agonistic anti-CD40 mAb (0.1mg i.v. d 0, 2) also restored T cell recovery in thymectomized mice. These results indicate that depletion-resistant memory CD4 T cells and thymus are both required for effective CD8 T cell recovery. Our findings may lead to therapies that specifically target reconstitution of naïve vs memory T cells following lymphoablation.
Generation of alloantigen-specific T regulatory cells via a chimeric antigen receptor targeting HLA-A2
Authors: MacDonald Katherine Grace , Hoeppli Romy , Huang Qing , Gillies Jana , Luciani Dan , Orban Paul , Broady Raewyn , Levings Megan K
Published: January 2023

CD4+ T regulatory cells (Tregs) are of clinical interest due to their potent immunosuppressive activity in both transplantation and autoimmunity models. Clinical efficacy in humans has been limited by aspects of Treg biology, including the difficulty of isolating sufficient numbers for potency without sacrificing purity and thus function. Enriching for antigen-specific Tregs greatly increases potency, however, antigen-specific Tregs are extremely rare. The success of chimeric antigen receptor (CAR) technology in generating antigen-specific effector T cells suggested that a similar approach could be used to generate alloantigen-specific Tregs for use in transplantation. We generated a new CAR specific for HLA-A2 (A2-CAR) and tested its application in the generation of alloantigen-specific human Tregs. Naïve Tregs were sorted from human peripheral blood, activated, and transduced with A2-CAR lentivirus. Stimulation through the A2-CAR upregulated Treg activation markers and functional molecules, and resulted in proliferation. Importantly, in vitro studies showed that A2-CAR-expressing Tregs maintained their expected phenotype and suppressive function, before, during and after HLA-A2-mediated stimulation. In vivo, A2-CAR Tregs were superior to Tregs expressing an irrelevant CAR at preventing xenogeneic graft-versus-host disease caused by HLA-A2+ T cells. Together, these data suggest that alloantigen-specific Tregs generated by the introduction of an A2-CAR may be a potent and specific immunosuppressive treatment in transplantation.
MicroRNA let 7i regulates DCs maturation targeting IL-10 via JAK1-STAT3 signal pathway subsequently induces prolonged cardiac allograft survival in rats
Authors: Jin Xiangyuan , Sun Yong , Liu Xianglan , Wang Junfeng , Zhang Maomao , Yu Bo , Wu Jian
Published: January 2023

Objective

In this study, we investigated whether microRNA (MiR) let 7i regulates DCs maturation targeting IL-10 via JAK1-STAT3 signal pathway subsequently prolongs rat cardiac allograft survival.

Methods

QRT-PCR, ELISA, dual luciferase assay were performed to verify whether IL-10 was the target of let 7i. Tregs were assessed by flow cytometry and immunohistochemical study, and JAK1, STAT3 and pSTAT3 expressions were detected by western bolt. Lewis recipients of DA heart were transfused with PBS, LPS-mDCs, or let 7i inhibitor-mDCs. Allograft survival times were recorded, and histologic study were performed.

Results

Expression of IL-10 mRNA level and production of IL-10 were increased in let 7i inhibitor-mDCs as compared to LPS-mDCs. Luciferase activity showed that the translational level of the IL-10 luciferase reporter was decreased by let 7i mimic, but increased by let 7i inhibitor. MiR let 7i inhibitor suppressed DC maturation by depressing CD80 and CD86, however， pretreatment of IL-10 SiRNA attenuated the suppression. Expressions of JAK1, STAT3, and pSTAT3 in mDCs were suppressed by let 7i mimic and pretreatment of IL-10 SiRNA, however up regulated by let 7i inhibitor. Lewis recipients transfused with let 7i inhibitor-mDCs significantly prolonged DA cardiac allograft survival. The allografts transfused with let 7i inhibitor-mDCs showed slight cell infiltration and significantly preserved graft structure. Inhibition of let 7i increased Tregs and modulated cytokine profiles both in vivo and in vitro.

Conclusion

MiR let 7i regulated DCs maturation and function targeting the IL-10 through JAK1-STAT3 pathway. Moreover, transfusion of let 7i inhibitor-mDCs induced prolonged cardiac allograft survival and generated Tregs.
The NK killer receptor Ncr1 shapes tumor architecture to prevent metastasis
Authors: Glasner Ariella , Mandelboim Ofer
Published: January 2023

Natural killer (NK) cells were so-named because of their excellent ability to kill tumors. NKp46 (Ncr1 in mice) is a prominent NK killer receptor, critically involved in controlling various pathogens as well as tumors and tumor metastases. We and others and have demonstrated the ability of the NK killer receptor Ncr1 to contain tumor growth, however, the mechanisms underlying the ability of Ncr1 to control tumor growth and metastases in vivo are yet undetermined

Metastatic melanoma is an extremely aggressive disease and the leading cause of death from skin cancers. Here, we used the spontaneous metastases model B16F10.9 (B16), the Ncr1 knockout mice (Ncr1gfp/gfp), Ifnγ−/− and Tnfα−/− knockout mice to visualize the tumors’ architecture in-vivo, using Reflectance Confocal Microscopy (RCM).

We demonstrate that Ncr1, through Ifnγ prevents tumor metastasis by a novel mechanism, modulating the structural properties of the developing tumors, at the level of the tissue organization.

Using RNAseq we have analyzed the transcriptome of Ifnγ treated B16 cells and reveled a 24 fold increase in the integrin binding ECM protein Fibronictin1 (FN1), the absence of which was reported to correlate with increased metastases.

The finding that Ncr1, through Ifnγ, shapes the tumors’ structures and thus controls metastasis may lead to generation of new drugs aimed at targeting specifically the tumors’ architecture and new treatment options for melanoma patients.
Interleukin (IL)-15 superagonist mediates immunotoxicity by inducing hyperproliferation of activated NK cells and production of IFNγ
Authors: Guo Yin , Luan Liming , Rabacal Whitney , Bohannon Julia , Fensterheim Benjamin , Hernandez Antonio , Sherwood Edward
Published: January 2023

Interleukin (IL)-15 is currently undergoing clinical trials to assess its anti-tumor efficacy. IL-15 is essential for the generation, function and survival of natural killer (NK), natural killer T (NKT) and memory phenotype (MP) CD8+T cells. The combination of IL-15 with its soluble IL-15 receptor α (IL-15Rα) generates a complex termed IL-15 superagonist (IL-15 SA) that exhibits more profound biological activities on NK, NKT and MP CD8+T cells than IL-15 alone. Thus, IL-15 SA is a potentially more attractive therapeutic agent for use against advanced tumors. However, the safety of IL-15 SA administration in vivo is not clearly elucidated. This study was designed to evaluate toxic consequences of IL-15 SA in mice. Administration of 2 μg IL-15 SA for 4 days caused a time- and dose-dependent immune-toxicity, characterized by hypothermia, weight loss, liver damage and death. At 2ug, IL-15 SA expanded NK cells in spleens and livers compared to vehicle and significantly increased NK cell expression of CD69, NKG2D and NKp46 as well as production of IFNγ, granzyme B and perforin. IL-15 SA also prompted NK cell maturity with a preferential expansion of the pro-inflammatory (CD27+ CD11b+) NK subset. Mice depleted of NK cells by anti-asialoGM1or anti-NK1.1 were resistant to IL-15 SA-induced toxicity, whereas adoptive transfer of wild type NK cells to Rag2−/−γc−/− mice re-established IL-15 SA toxicity. While IL-15 SA expanded NKT and mCD8+ T cells, mice depleted of NKT or CD8+ T cells remained sensitive to IL-15 SA-mediated toxicity. In addition, IFNγ KO, but not TNF-α KO or perforin KO, mice were resistant to IL-15 SA-induced toxicity. In conclusion, high-dose IL-15 SA causes systemic immunotoxicity via induction of NK cell proliferation and activation.
FIP200 targets Ago2 and mitochondria in naive T cells to control tumor immunity
Authors: Xia Houjun , Wang Wei , Kryczek Ilona , Wei Shuang , Crespo Joel L , Guan Junlin , Zou Weiping
Published: January 2023

Naive T cells are poorly studied in cancer patients. Here, we report that naïve T cells are susceptible to apoptosis and lost in human and mouse cancer. Accompanied with this feature is a selective loss of autophagy component, FIP200 in naïve T cells in tumor bearing hosts. FIP200-deficient T cells functionally resemble their counterparts in tumor bearing hosts and mediate poor anti-tumor immunity in vivo. Mechanistically, loss of FIP200 disables the balance between pro- and anti-apoptotic Bcl-2 family members via elevating argonaute (Ago) 2 ubiquitination and subsequently eliminating microRNA1198-5p expression and consequently lifting microRNA1198-5p-mediated repression on pro-apoptotic gene Bak1 expression. In vivo rebalance of the Bcl-2 family genes via enforcing Bcl-2 expression partially rescues FIP200-deficient T cell apoptosis. In parallel, specific FIP200 deficiency causes potent mitochondria activation and reactive oxygen species (ROS) release in T cells. In vivo ROS blockade prevents FIP200-deficient T cell apoptosis. Thus, tumor may trigger naïve T cell apoptosis via targeting its FIP200 to escape anti-tumor immunity.
First in-depth investigation of multiple inhibitory molecule expression by bona fide tumor-reactive T cells in ovarian cancer.
Authors: Chacon Jessica A , Yamamoto Tori , Garrabrant Thomas , Kari Gabor , Best Andrew , Ye Qunrui , Powell Daniel J
Published: January 2023

Ovarian cancer causes more deaths than any other cancer of the female reproductive system. However, novel therapies, including immunotherapy are now being utilized to treat this disease. In addition, efforts have been made to identify and evaluate the subsets of cells within the tumor environment that correspond with patient overall survival. The presence of intraepithelial CD3+ T cells and CD8+ T cells have been correlated with ovarian cancer patient survival and our lab has recently identified CD137 as a biomarker for naturally-occurring tumor-reactive T cells in melanoma and ovarian cancers. However, a thorough elucidation of the immune landscape of endogenous bulk and tumor-reactive T-cell has not been conducted. In our study, we used an in-depth multi-parameter flow cytometry panel to determine the expression of multiple inhibitory molecules, Th1 cytokines, and the effector phenotype in the bulk and tumor-reactive subsets in human ovarian cancer. We observed that the CD4+ and CD8+ T cells found within the tumor environment express inhibitory receptors, very limited Th1 cytokine producing cells and a decreased expression of Granzyme B within the CD8+ subset. The tumor-reactive subset expressed inhibitory molecules, but was still able to elicit an effector phenotype and comprise Th1 cytokine producing cells.

In conclusion, using multi-parameter flow cytometry enabled us to investigate the expression of inhibitory molecules and determine the effector phenotype in the bulk TIL and tumor-reactive CD137+ subsets.