bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of antigen-specific CD8 T cell responses in experimental cerebral malaria
Authors: Urban Stina Lisa , Pewe Lecia L , Harty John T
Published: January 2023

Malaria remains a major global health concern, infecting more than 200 million individuals and resulting in almost a half a million deaths per year. Most malaria-associated deaths are caused by cerebral malaria (CM). Due to ethical limitations of human experimentation, an experimental cerebral malaria (ECM) model is used to further our understanding of the pathogenesis of CM. C57BL/6 mice are susceptible to ECM upon Plasmodium berghei ANKA live sporozoite (spz) or infected RBC (iRBC) infection. CD8 T cells are required for ECM symptoms as depletion of CD8 T cells inhibits disease. Despite the known role of CD8 T cells in the pathogenesis of ECM, the kinetics, phenotype and function of antigen-specific response have not been described. Here, we characterize the antigen-specific CD8 T cell phenotype after spz or iRBC infection and correlate with parasitemia levels and development of ECM. Using surrogate activation markers to identify antigen-specific CD8 T cells, the frequency of CXCR3 expressing activated CD8 T cells decreased initially and subsequently increased during onset of ECM symptoms. Higher parasitemia levels and frequency of activated CD8 T cells correlated with ECM development in both spz and iRBC infection models. Based on these data, we hypothesize that ECM develops in a goldilocks-principle manner. If a specific threshold of parasitemia and activated CD8 T cells are reached at the same time, ECM develops, but should the CD8 T cell response occur too early or late, ECM does not develop and mice will eventually succumb to hyperparasitemia and severe anemia.
Targeting TIGIT to modulate immunity and disease during experimental malaria
Authors: Graham Amy C , Obeng-Adjei Nyamekye , Guthmiller Jenna J , Zander Ryan A , Wohlfert Elizabeth A , Crompton Peter D , Butler Noah S
Published: January 2023

Plasmodium infections and the disease malaria remain global health emergencies. CD4 T cells are essential for coordinating protective immunity against Plasmodium infection and promoting parasite clearance, yet long-lived immunity is rarely acquired. We have linked immune failures during human and experimental Plasmodium infection to the expression of immuno-regulatory networks that impair CD4 T cell activity. Here we show the immuno-inhibitory molecule, TIGIT, is highly expressed by parasite-specific CD4 T cells during human and experimental Plasmodium infection, but not experimental Toxoplasma gondii infection. Strikingly, the bulk (>90%) of TIGIT+ parasite-specific CD4 T cells are T-bet+ Th1 effectors, not Foxp3+ T regulatory cells. Moreover, sustained expression of TIGIT is associated with CD4 T cell-expression of IL-10 and is regulated by systemic inflammation, not persistent antigen or chronic T cell stimulation. Preliminary studies using TIGIT-specific agonistic and antagonistic reagents support the biological relevance of sustained TIGIT expression during Plasmodium infection. Current studies are focused on genetic manipulation of TIGIT to dissect the CD4 T cell subset-intrinsic roles of TIGIT during malaria. Collectively, our data support that TIGIT represents an additional host factor that can be targeted to improve immunity against Plasmodium infection and limit disease severity.
Carbonic anhydrase enzymes regulate mast cell-mediated immunity to Trichinella spiralis.
Authors: Siracusa Mark C , Henry Everett K , Sy Chandler B , Inclan-Rico Juan M.
Published: January 2023

Type 2 cytokine responses are necessary for the development of protective immunity to helminth parasites but also cause the detrimental inflammation associated with allergies and asthma. Recent studies have found that peripheral hematopoietic progenitor cell populations contribute to type 2 cytokine-mediated inflammation through their enhanced ability to develop into mast cells. Here we identify that carbonic anhydrase (Car) enzymes are upregulated in type 2-asscoaited progenitor cells and demonstrate that Car enzyme inhibition was sufficient to prevent murine mast cell development, type 2 cytokine-mediated inflammation and protective immunity to Trichinella spiralis. Moreover, we show that Car enzyme inhibition was also sufficient to prevent intestinal mast cell responses in a murine model of food allergy-like disease. Finally, we performed translational studies and demonstrated that Car enzymes can be targeted with an FDA-approved inhibitor to prevent human mast cell development. Collectively these studies identify a previously unrecognized role for Car enzymes in regulating mast cell lineage commitment and suggest that FDA-approved Car enzyme inhibitors may possess additional off-label therapeutic potential that can be employed to treat mast cell-mediated inflammation.
Defining the correlates of protective immunity mediated by Ly6C+CD44+CD62L−CD4+ T effector (TEFF) cells against Leishmania major infection: implications for vaccination against intra-phagosomal pathogens
Authors: Hohman Leah S , Carneiro Matheus BH , Peters Nathan C
Published: January 2023

The protozoan Leishmania parasite is a human obligate intra-cellular pathogen and the causative agent of leishmaniasis, a chronic neglected tropical disease of existing and growing global importance. Despite considerable effort a human vaccine does not exist. In the C57Bl/6 mouse model of naturally acquired protective immunity, chronic primary infection maintains short-lived Ly6C+ TEFF cells that mediate protective immunity against secondary challenge. These cells, not memory cells or cells derived from memory cells following challenge, emulate the rapid IFN-g response associated with protective immunity against natural infected sand fly challenge. Defining the mechanisms by which these cells mediate their protective effect will be essential for informing future vaccine design. To elucidate the importance of the rapidity of the Ly6C-mediated response as a protective correlate, the influence of these cells on innate immune cells, and ultimately their capacity to facilitate the priming and activation of Leishmania-specific naïve cells, we devised an adoptive transfer system in which Ly6C+ TEFF cells from chronic mice are transferred into recipient mice at the time of infection or 4 days following infection, just prior to the initiation of parasite replication. Disease progression was followed for 3 weeks and the activation of phagocytes, antigen-presenting cell function, and IFN-g production by both adoptively transferred and recipient populations was analyzed. Our observations are likely to enhance our understanding of the correlates of protective immunity beyond the simple generation of IFN-g producing memory cells, a strategy that has so far failed to generate an effective human vaccine.
Immunomodulation by Leishmania-derived surface oligosaccharides promotes pro-inflammatory cytokine secretion and reduces L. major parasite load
Authors: Grinnage-Pulley Tara , Roychoudhury Rajarshi , Schaut Robert , Osanya Alex , Martinez Pedro , Gibson-Corley Katherine , Toepp Angela , Lamb Ian , Pohl Nicola , Petersen Christine A
Published: January 2023

Lipophosphoglycans (LPG) of Leishmania spp. are known to alter innate immune responses and be critical for parasite binding to the vector gut wall. LPG displays various oligosaccharides capping the terminal and branch apexes. However, it is unknown how these capping oligosaccharides serve as immunomodulators in the absence of a conjugated protein. Several oligosaccharides including dimannose and trimannose, were synthesized and covalently linked to latex beads to study their effect in a Leishmania infection model. In vitro incubation of activated J774 or bone marrow derived macrophages with di- and tri-mannose resulted in differential modulation of IL-12p40, a key cytokine to control intracellular pathogens by driving a Th1 response. Co-inoculation of trimannose and L. major into the footpad of C57BL/6 mice increased IL-12p40 and IFN-γ production 48 hours post-infection, a significantly Th1 skewed response. Noticeably at 14 days post-infection, L. major infected, trimannose treated mice had significantly decreased lesion size and decreased parasite load compared to infected untreated, controls. Cytokines from draining lymph node supernatants measured via Luminex at 14 days post infection showed decreased IL-12p40 and IL-10 secretion and increased IFN-γ in the co-inoculated mice. In vitro, the effects of trimannose were mediated by macrophage TLR and mannose receptors as well as T cell proliferation. Leishmania-derived trimannose represents a novel immunomodulator that provides early Th1 skewed cytokine production to control parasite load and alter the course of infection.
Exogenous induction of type I IFN protects mice from Cryptococcus neoformans and Cryptococcus gattii infections by distinct CD4+ T cell and CD8+ T cell-mediated mechanisms.
Authors: Davis Michael J , Sionov Edward , Mayer-Barber Katrin D. , Barber Daniel L , Chang Yun C. , Kwon-Chung Kyung J.
Published: January 2023

Rational

Cryptococcus neoformans (Cn) causes deadly mycosis in AIDS patients whereas the Vancouver outbreak strain of Cryptococcus gattii (Cg) mostly infects otherwise healthy individuals but not AIDS patients even where the strains are equally prevalent in the environment. As HIV infection induces type I IFN (t1I) we hypothesized that exogenously induced t1I would differentially affect infections by Cn and Cg.

Results

Cn or Cg infected mice were dosed with poly(I:C)LC (PIC) to induce t1I. PIC-treated mice were protected from Cn and Cg infection-mediated death with reduced fungal burdens. Both Cn and Cg infection produced a non-protective immune response dominated by Th2 cell recruitment, M2/DC2 polarization and eosinophilia. PIC-treatment reversed the Th2 polarization toward increased Th1/17 cells, M1/DC1 polarization and reduced eosinophilia. PIC also induced activation of CD8 T cells in both Cg and Cn infected mice. Cg-infected CD8 cell-deficient mice were not protected by PIC treatment while PIC protection was intact in Cn-infected CD8 cell-deficient mice. In contrast, PIC protection was intact in Cg-infected CD4 cell-deficient mice while Cn-infected CD4 cell-deficient mice were not protected by PIC treatment.

Conclusions

These data show that t1I induction protects mice from Cn and Cg however protection was mediated by distinct immune mechanisms. PIC-mediated protection from Cn requires CD4 but not CD8 cells while PIC-mediated protection from Cg requires CD8 but not CD4 cells. Notably, Cn but not Cg was able to grow in CD4-deficient t1I-induced mice, which somewhat resemble the immunological condition of AIDS patients, suggesting a novel explanation for the observed AIDS patient tropism of these cryptococcal species.
Loss of efficacy of the antifungal caspofungin is mediated by increased chitin exposure and concomitant eosinophilia in invasive pulmonary aspergillosis
Authors: Amarsaikhan Nansalmaa , Tsoggerel Angar , Sands Ethan M , Templeton Steven P
Published: January 2023

Recently significant interest has focused on the composite recognition of fungal molecular patterns that program innate and adaptive immunity to the human fungal pathogen Aspergillus fumigatus. We observed that decreased relative levels of cell wall β-glucan to chitin in A. fumigatus strains correlated with increased airway eosinophil recruitment associated with decreased fungal clearance by type 2 immune responses in invasive pulmonary aspergillosis. Furthermore, an increase in surface chitin exposure driven by growth and germination in the presence of the anti-fungal, β-glucan targeting drug caspofungin resulted in increased airway eosinophilia upon aspiration of A. fumigatus conidia. More importantly, in a model of invasive aspergillosis, we observed a decrease in fungal clearance upon caspofungin treatment that was reversed in eosinophil-deficient mice. Eosinophil recruitment was partially dependent on γδ T cells, and our preliminary results show a similar increase in caspofungin mediated clearance in γδ-T cell-deficient mice. Our results provide a novel mechanism that may explain the widely reported paradoxical loss of efficacy in response to caspofungin, and we are currently working to define the relationship between drug treatment, eosinophil activation, and fungal burden in clinical samples.
Dual roles of scavenger receptor MARCO in innate versus adaptive immune responses during Cryptococcus neoformans infection
Authors: XU JINTAO , Flaczyk Adam , Eastman Alison J , Neal Lori , Zhao Guolei , Malachowski Antoni , Akolkar Jay , Osterholzer John J , Olszewski Michal A
Published: January 2023

Resistance to the opportunistic fungal pathogen Cryptococcus neoformans (Cn) is associated with development of Th1/Th17 immune bias while a Th2 immune response results in increased susceptibility. Macrophage Receptor with Collagenous Structure (MARCO) is important for recognition of some pathogens and subsequent activation and polarization of the immune responses by DC and macrophages, however, its role during Cn infection remains unknown.

Here we found that in spite of diminished early (innate) control of Cn in the infected lungs, Cn-infected MARCO−/− mice showed improved fungal clearance during the adaptive phase (wk 5) compared to MARCO+/+ mice. MARCO−/− mice showed: 1) improved accumulation of exudate macrophages and CD11b+ DC and their improved activation phenotype (CD80+, CD86+ and MHCIIhigh); 2) increased intracellular IFN-γ and IL-17 production by pulmonary CD4+ and CD8+ T cells; 3) decreased IL-4, IL-5, IL-10 and increased IFN-γ and IL-17 production by splenocytes treated with Cn antigen; 5) diminished systemic level of IgE. Interestingly, in spite of improved clearance, MARCO−/− mice showed decreased frequency of lung resident CD44highCD62LlowCD69+ CD103+CD11a+CCR7− effector memory T cells. We conclude that while important for the innate control of Cn in the lungs, MARCO can be exploited by Cn to interfere with immune polarization leading to an altered, non-protective efferent immune bias, which outweighs potentially beneficial effects of MARCO.
Th17 cells are more protective than Th1 cells against the intracellular parasite Trypanosoma cruzi.
Authors: Cai Catherine Wen , Blase Jennifer R. , Eickhoff Christopher S. , Hoft Daniel F.
Published: January 2023

Th1 cells are considered the major CD4+T helper subset responsible for the control of intracellular pathogens, including Trypanosoma cruzi, the protozoan parasite of Chagas disease. Th17 cells are known for their involvement in autoimmunity and the defense against some extracellular organisms, but are generally not believed to have a significant function during intracellular infections.

In contrast, we show that Th17 cells are highly protective against the intracellular pathogen T. cruzi. We reconstituted RAG KO mice lacking endogenous T cells with polyclonal CD8+ T cells and parasite-specific Th1 or Th17 cells, generated from novel T cell receptor transgenic mice recently developed by us. After adoptive cell transfer, recipients were infected with a normally lethal dose of T. cruzi. Th17 cells impressively protected 100% of mice from mortality long-term, while Th1 cells protected only 38%.

Transfer of Th17 cells alone failed to protect, implicating helper effects on CD8+ T cells as the basic mechanism of Th17-mediated intracellular immunity. We confirmed this using in vitro assays, and discovered that Th17 cells help activate CD8+ T cells through IL-21 secretion, indicating a protective mechanism surprisingly independent of the main Th17 cytokine IL-17A. In vivo, co-transfer of IL-21R knock-out CD8+ T cells with Th17 cells failed to protect mice, confirming the importance of IL-21 signaling.

In summary, our data define an important new role for Th17 cells in the defense against a major intracellular human pathogen and define novel mechanisms of Th17 cell effects. This work increases our understanding of the protective immune interactions in T. cruzi infection, and more broadly, illuminates the growing diversity of Th17 cell roles.
Oxygen metabolism regulates macrophage susceptibility to Trypanosoma cruzi.
Authors: Koo Sue , Garg Nisha Jain
Published: January 2023

Macrophage activation rely on the metabolism which supports the generation of compounds crucial for proper cell function. Aerobic metabolism is dependent on mitochondrial oxidative phosphorylation, and is essential for the activation of immunomodulatory macrophages (M2). Conversely, mitochondria-independent, anaerobic metabolism provides substrates to produce anti-microbial mediators by pro-inflammatory macrophages (M1). Reactive oxygen species (ROS) and nitric oxide (NO) are key M1 molecules for host defense against intracellular parasites, including the etiologic agent of Chagas disease, Trypanosoma cruzi. In this study, we report deficient production of ROS and NO by macrophages infected with T. cruzi (H2DCFDA, Amplex Red and Griess assays), and show that mitochondrial gene expression (qPCR) and function (Seahorse Extracellular Flux Analyzer) are in concordance with the M2 phenotype and macrophage susceptibility to T. cruzi infection. These findings indicate the possibility of manipulating oxygen metabolism to improve the pro-inflammatory function and pathogen clearance by macrophages.
Short-chain fatty acids induce effector T helper cells and cause inflammatory responses in the renal system.
Authors: Park Jeongho , Goergen Craig , HogenEsch Harm , Kim Chang H
Published: January 2023

Short-chain fatty acids (SCFAs) are major products of gut microbial fermentation and profoundly affect host health and disease. SCFAs generate IL-10+ regulatory T cells, which may promote immune tolerance. SCFAs can also induce Th1 and Th17 cells during active immune responses and, therefore, have the potential to induce inflammatory responses. However, the potential function of SCFAs in induction of tissue inflammation has not been documented to date. We investigated, in depth, the impact of elevated SCFA levels on T cells and tissue inflammation in mice. Orally administered SCFAs induced effector (Th1 and Th17) and regulatory T cells in ureter and kidney tissues, and induced T-cell mediated ureteritis leading to kidney hydronephrosis (hereafter called C2RD). Kidney hydronephrosis in C2RD was caused by ureteral obstruction, which was, in turn, induced by SCFA-induced inflammation in the ureteropelvic junction (UPJ) and proximal ureter. Oral administration of all major SCFAs, such as acetate, propionate, and butyrate, induced the disease. We found that C2RD development is dependent on mTOR activation, T cell-derived inflammatory cytokines such as IFNg and IL-17, and gut microbiota. In a manner similar to many types of chronic renal diseases in humans, young or male animals were more susceptible than old or female animals respectively. However, SCFA receptor (GPR41 or GPR43) deficiency did not affect C2RD development. Our study provide evidence for the first time that SCFAs can cause dysregulated T cell responses and tissue inflammation in a specific organ in the body. We believe that our results have significant ramifications in therapeutic applications of SCFAs.
Deep phenotypic profiling of small clinical samples through MultiSpectral Imaging Cytometry (MuSIC) in nanowell arrays
Authors: Gierahn Todd M , Yalcin Ayca , Loginov Denis , Love J. Christopher
Published: January 2023

Many disease relevant immune processes occur within tissues, making them challenging to monitor clinically using standard blood based assays. Comprehensive phenotyping of the scarce cells acquired directly from tissue samples is limited by inefficiencies in sample handling and data collection inherent in current flow-based profiling methods. To overcome this barrier, we developed Multi-Spectral Imaging Cytometry (MuSIC), a method that combines an array of sub-nanoliter wells (nanowells) for efficient cell capture with multi-spectral imaging to resolve up to 16 fluorescent markers on ~103–105 cells. Importantly, the cells remain viable for further functional characterization, enabling linkage of deep cell surface phenotypes with functional activity. Using MuSIC in combination with a single-cell protein secretion assay, we were able to identify unique T cell populations present in human cervical biopsies. MuSIC provides an effective and accessible means to acquire deep phenotypic profiles of leukocytes from small clinical samples.
Digital Vivarium™ Platform Enables Automated Drug Efficacy Assessment Correlated with Pathology in Rheumatoid Arthritis
Authors: Schaevitz Laura , Cha Paulyn , Ford Daniel , Heath Kyle , Jadzinsky Pablo , Lim Maria , Robertson Timothy
Published: January 2023

Current methods for evaluating new therapeutics in rodent disease models and improving understanding of disease mechanisms are frequently labor-intensive and require the same technician to perform key measurements for consistency. To address these limitations, we established a novel Digital Vivarium™ platform, which monitors subjects continuously for disease and health-related endpoints, without human interaction, to evaluate drug efficacy. As a proof of principal, we validated automated disease scoring in a standard model of arthritis with an algorithm that captures salient motion metrics during the animals’ active period to compute an arthritis score. This Digital Arthritis Index™ score reproducibly correlated to disease pathology and was better at detecting mild disease pathology as compared to joint swelling across standard-of-care compounds including Methotrexate, Etanercept, and Dexamethasone. Additional health endpoints, include breathing rate, overall activity, and circadian rhythms, are also monitored continuously and stored in auditable electronic and videographic records. The platform was then used to screen 9 re- purposed drugs at appropriate dose ranges for efficacy. Study-specific disease and health endpoints are available real-time remotely over the internet to enable rapid decisions. Our results demonstrate a low-touch digital platform that will pave new ways for more rapid, reproducible, drug discovery and evaluation, not only for RA, but also more broadly in other autoimmune diseases such as MS and Lupus, as well as outside of immunology such as cancer, CNS and toxicology.
Ubiquitin ligases and immune response: Quantitative analysis of their substrates and drug discovery
Authors: Lu Xiaolong , Rapp Matthew , Corson Joy , Strickler James
Published: January 2023

Protein ubiquitylation has emerged as a key regulatory mechanism of innate and adaptive immune systems. In particular, Cbl-b (Casitas B-lineage lymphoma proto-oncogene b), a RING finger E3-ubiquitin ligase primarily expressed in immune cells, acts as an intracellular checkpoint and a master negative regulator of both CTLs and NK cells. As such, Cbl-b is a novel target for developing small molecule cancer immunotherapy agents. Nedd4-family E3 ligases including Itch are known to negatively regulate inflammatory immune responses by suppressing both TH2 and TH17 differentiation and cytokine production. Modulators of Itch are expected to have anti-inflammatory properties. In addition, deubiquitylases such as USP7 and USP4 are implicated in modulating immune responses. Thus, several ubiquitin pathway enzymes have emerged as therapeutic targets for a variety of immune disorders. Therapeutic modulation of these ubiquitin pathway enzymes is expected to alter the ubiquitylation of their substrate proteins and documentation of such changes is critical to establish mechanism of action in the cell and develop physiological screens for target validation and drug discovery. Current methods to study polyubiquitin on substrates are cumbersome, inefficient, and low throughput. Here we describe a powerful technology that allows selective and quantitative analysis of polyubiquitylated proteins in high throughput format in the cell. Validation is provided using small molecule modulators of E3 ligase and deubiquitylase enzymes. This technology allows detection of changes in highly dynamic poly-ubiquitylated substrates, thus elucidating the role of these enzymes in the cell and the mechanism of action of drug molecules.
Estrogen induces multiple regulatory B cell subtypes and promotes M2 microglia and neuroprotection during experimental autoimmune encephalomyelitis
Authors: Zhang Jun , Benedek Gil , Bodhankar Sheetal , Vandenbark Arthur A , Offner Halina
Published: January 2023

Sex hormones promote potent immunoregulatory effects on autoimmune diseases, including multiple sclerosis (MS). Our previous studies demonstrated a critical role for regulatory B cells in mediating estrogen (17β-estradiol, E2)-mediated protection against experimental autoimmune encephalomyelitis (EAE), a mouse model for MS, leading to elevated numbers of IL-10 secreting CD1dhiCD5+B regulatory cells (Bregs) in wild type mice. The current study evaluated estrogen effects on regulatory B cells and resident CNS microglia during EAE and demonstrates for the first time an E2-dependent induction of multiple regulatory B cell markers indicative of IL-10 dependent as well as IFN-γ dependent pathways. Moreover, although E2 pretreatment of female EAE mice inhibited the infiltration of pro-inflammatory immune cells into the CNS compared with sham pretreated mice, it enhanced the frequency of regulatory B cells and M2 microglia in the CNS. Our study suggests that estrogen has a broad effect on the development of regulatory B cells during EAE, which in turn could promote neuroprotection in the CNS.
The role of donor memory-like NK cell infusion in allogeneic hematopoietic stem cell transplantation
Authors: Liu Haiyan , Hu Bo , Song Yuan
Published: January 2023

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the only curative therapy for patients with chemotherapy-resistant hematologic malignancies and some solid tumors. However, relapse after allo-HSCT is common in those with high-risk disease and typically portends a poor prognosis. Previous studies showed that donor NK cell infusion could prevent disease recurrence possibly through killing residual leukemia cells. However, NK cell-based immunotherapy is hampered by their transient effector function. Recently, IL-12/15/18-preactivated memory-like NK cells were shown to persist with sustained effector function, we investigated whether memory-like donor NK cells could improve donor NK cell’s GVL effect in murine allo-HSCT. NK cells preactivated with IL-12/18 or IL-12/15/18 produce high levels of IFN-γ upon restimulation by IL-12/15 after 4d of in vitro culture. The infusion of IL-12/15/18 or IL-12/18-preactivated NK cells significantly promoted the GVL effects compared to donor HSCs infusion alone. After adoptive transfer, IL-12/18 and IL-12/15/18 preactivated NK cells were found in higher numbers in spleen, liver and lung compared with the control NK cells. The proliferation of the transferred NK cells was greatly promoted by IL-12/18 and IL-12/15/18 preactivation. Despite of the strong GVL effect of the IL-12/15/18-preactivated NK cells, they aggravated acute GVHD whereas IL-12/18-preactivated NK cell infusion significantly prolonged the GVHD-related survival compared with the HSCs alone or control NK cells. Therefore, IL-12/18-preactivated memory-like donor NK cell infusion may be a more effective and safer strategy for preventing or treating relapse after allo-HSCT.
The effects of mesenchymal stem cells in a murine sclerodermatous model of graft-versus-host disease
Authors: Lim Ji-Young , Ryu Da-Bin , Lee Sung-Eun , Oh Il-Hoan , Min Chang-Ki
Published: January 2023

Human chronic graft-versus-host disease (CGVHD) shares clinical characteristics with a murine sclerodermatous GVHD (scl GVHD) model that is characterized by skin and lung fibrosis. We used B10.D2 → BALB/c model of scl GVHD, which differ at minor histocompatibility loci, to address the therapeutic effect of mesenchymal stem cells (MSCs) on the development of CGVHD. MSCs were intravenously treated on days 3, 5 and 7 post-transplantation. The clinical severity of cutaneous scl GVHD was significantly attenuated after the treatment of MSCs. Pathologically, animals treated with MSCs showed less fibrosis and inflammatory cell infiltrations in skin and ear but not in visceral organs including lung and liver. Accordingly, the mRNA expression of collagen type 1 α1, 1 α2 and 3 α1 as well as collagen production were significantly reduced after the MSC treatment only in skin compared to the allogeneic scl GVHD controls. MSCs down-regulated mRNA of proinflammatory cytokines mainly in skin tissues and among them TGF-β expression was consistently reduced 28 days after HSCT. The effects of MSCs on molecular markers implicated in persistent TGF-β signaling and fibrosis, such as phosphatase and tensin homolog (PTEN), p-Smad2/3 and matrix metaloprotease-1 (MMP-1) were observed at the skin tissues. MSCs inhibited infiltration of immune cells into skin through down-regulating CCR4, CCR8 and CCR10 on donor CD4 T cells and CCR1 on CD11b monocytes. Moreover, MSCs diminished the expression of mRNA for chemokines such as CCL1, CCL8, CCL17, CCL22 and CCL3, in skin. In conclusion, treatment of MSCs early after transplant attenuated cutaneous scl GVHD by selectively blocking of immune cell migration and down-regulating the chemokines and chemokine receptors.
Learning from cancer: Using D-lactate for therapeutic immunosuppression
Authors: Beier Ulf H , Jiao Jing , Xiao Haiyan , Angelin Alessia , Wallace Douglas C , Levine Matthew , Hancock Wayne W
Published: January 2023

Accumulation of lactic acid in the tumor microenvironment contributes to local immunosuppressive conditions that weaken anti-tumor immunity. In mammals including humans, lactate is present almost entirely as the L-lactate optical isomer. We hypothesized that D-lactate could have similar immune modulatory effects that maybe exploited for therapeutic immunosuppression. 5–40 mM of added sodium D- and L-lactate caused marked impairment of murine, and human, CD4 and CD8 T cell proliferation in vitro, with stronger effects of D-lactate than L-lactate. Neither D- nor L-lactate affected cell viability or apoptosis. Adding 20 mM D-lactate to CD4+ Foxp3− T cells under polarizing conditions increased Foxp3+ induced regulatory T cell (iTreg) formation by 46.2 ±31.5% (P<0.03, n=4). L-lactate also augmented Treg induction but to a lesser degree. Next, we adoptively transferred 1×106 conventional T cells, with or without 1.25×105 Tregs, into Rag1−/− mice, treated each group with 90 mmol/kg/d D-lactate or NaCl for 5 days. After 7 days, T cell proliferation was reduced by 62.1% using D-lactate treatment (P<0.03, n=8). While Treg suppressive function was unaffected, de-novo Foxp3+ Treg formation was increased in D-lactate (14.9 ±3.4%) vs. NaCl (10.6 ±2.3%) treated animals (P<0.01, n=8). Adding D-lactate to proliferating T cells increased oxygen consumption rates compared to NaCl controls, by 56.1 ±35.1% (p<0.05, n=4). In contrast, methyl-esters of L- and D-lactate resistant to either lactate dehydrogenase isoform did not reduce T cell homeostatic proliferation. Our data show that D-lactate has in vivo immunosuppressive effects and can induce de-novo iTregs. The process of lactate oxidation is likely required for its immune modulatory effects.
Induction of donor-specific immune tolerance using bioengineered thymus organoid
Authors: Pradhan Isha , Tajima Asako , Bertera Suzzane , Rudert William , Trucco Massimo , Fan Yong
Published: January 2023

Extended use of immunosuppression is a major setback in organ transplantation. Hence, establishment of donor specific immune unresponsiveness, without the need of immunosuppression, is critical and remains a major challenge. Immunological tolerance is known to occur through two main mechanisms: central (thymus) and peripheral. Our lab has recently reported that bioengineered thymus organoids, made of de-cellularized thymus scaffolds populated with thymic epithelial cells (TECs), can successfully support T-cell generation in athymic nude mice. Here, we aim to test the hypothesis that transplantation of bioengineered thymus, incorporated with donor TECs, can induce central tolerance to donor antigens. Thymus scaffold were reconstructed with either TECs harvested from F1 offspring of B6 (H-2b) and NOD (H-2b/g7) or TECs from both B6 and CBA/J (H-2k) mixed in 1:1 ratio. These reconstructed thymus organoids are transplanted to the B6. Nude recipient, that are subsequently challenged with syngeneic, allogeneic and third party skin graft. Results from recipients with transplanted bioengineered thymus consisting F1 TECs demonstrated successful engraftment of skin transplants from both syngeneic (B6) and allogeneic (B6.H-2b/g7) whereas, clear rejection of the third party (CBA/J). Similarly, we observed prolonged survival of allograft skin (CBA/J) than third party (Balb/C) in recipient mice with reconstructed thymus organoids containing TECS from both donor (B6) and recipient (Balb/c). Furthermore, immune unresponsiveness to allograft was shown in MLR assay. Overall, our findings suggest reconstructed thymus-containing donor TECs could potentially have clinical application to induce-donor specific immune tolerance.
Monitoring the Cytomegalovirus-specific cellular immunity in lung transplant recipients: A comparative analysis of two assay systems
Authors: Boeckl Katharina , Striebinger Hannah , Kneidinger Nikolaus , Barabas Sascha , Neurohr Claus , Nitschko Hans , Jäger Gundula
Published: January 2023

Objective

Monitoring the cellular immunity for Cytomegalovirus (CMV) in organ transplant recipients is a promising tool to support prevention strategies for posttransplant CMV infection or reactivation. Commercially available in vitro test systems differ substantially in their capacity to stimulate subpopulations of T-lymphocytes. We compared two assays for CMV immune monitoring in respect of their clinical practicability and significance.

Methods

Blood samples of 30 lung transplant recipients (LuTRs) were examined before transplantation and over a period of six months afterwards with T-Track® CMV (Lophius Biosciences GmbH, Regensburg) and QuantiFERON®-CMV (Cellestis GmbH, Darmstadt). The T-Track® CMV is based on ELISpot-technology, allowing quantification of interferon-gamma (IFNγ) secreting CD4+ and CD8+ T-cells after specific stimulation. Contrarily, the QuantiFERON®-CMV assay is restricted to detection of IFNγ secreted by CD8+ T-lymphocytes with ELISA. The data are evaluated in the context of transplant outcome and determination of viral load in plasma by qPCR.

Results and Conclusion

Both approaches provide similar results while exhibiting certain advantages and limitations. Early during immune suppressive therapy, QuantiFERON®-CMV generates often indeterminate results as depletion of T-cells is not taken into account. Although a comparatively large volume of blood is required, T-Track® CMV circumvents this drawback. In addition, T-Track® CMV reaches a higher sensitivity prior to LuTx and after onset of immunosuppression.
Donor γδT cells promote graft-versus-leukemia effect and ameliorate acute graft-versus-host disease in allogeneic hematopoietic stem cell transplantation
Authors: Yuan Song , Bo Hu , Ying Zhu , Haiyan Liu
Published: January 2023

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is one of the most effective way of treating leukemia and other malignant diseases. However, disease recurrence and GVHD are still the major causes of death after allo-HSCT. Previous studies showed that immune cells involved in graft versus leukemia (GVL) effect traditionally include CD8+ T, CD4+ T and NK cells. Unfortunately, some of these T-cell subsets also cause graft-versus-host disease (GVHD). The role of donor γδ T cells after transplantation is less well defined. γδ T cells display unique features in terms of MHC-unrestricted recognition of lymphoid malignancies, which makes them promising candidates as anti-leukemia effectors. We detected direct anti-leukemia cytotoxicity of γδ T cells in vitro and the cytotoxicity of Vγ1 was stronger than the Vγ4 subset. To evaluate the role of donor γδT cells in aGVHD and GVL, we established the murine allo-HSCT GVL/GVHD model. Our results showed that TCRδ−/− donor bone marrow cells caused enhanced disease progression and aggravated aGVHD compared with wildtype donors. Furthermore, flow cytometry analysis demonstrated γδ T cells mediated anti-leukemia effects through promoting IFN-γ production by CD8+ T cells while suppressed aGVHD by inhibiting the activation of CD4+ T cells in liver, lung and IEL. The experiment of adoptive transfer of in vitro expanded γδT/Vγ1/Vγ4 cells indicated that the subset of donor γδT cells to alleviate murine aGVHD and enhance GVL effect might be Vγ4 γδT cells. The GVL effect of γδ T cells in vivo could be mediated by both direct cytotoxicity and regulation of CD8+ T cell function. These findings suggest donor γδT cells infusion as a novel strategy to improve patients’ overall survival after allo-HSCT.
Natural Killer (NK) Cells’ Response to Epstein - Barr virus Infections and its Influence on the Risk of Developing Post-transplant Lymphoproliferative Disease (PTLD) after Allogeneic Hematopoietic Cell Transplantation: Effect of Donor Killer Ig-like Receptor Genes and Motifs Copy Numbers
Authors: Faridi Rehan M , Kemp Taylor J , Dharmani-Khan Poonam , Lewis Victor A , Berka Noureddine , Storek Jan , Khan Faisal M
Published: January 2023

BACKGROUND

Uncontrolled reactivation of Epstein-Barr virus (EBV) leading to post-transplant lymphoproliferative disorder (PTLD) is one of the major complications after T-cell depleted HCT. Recovering within weeks after HCT, natural killer (NK) cells are deemed important in the immunopathogenesis of EBV complications. Their role however remains elusive. NK cell responses are regulated by a series of activating and inhibitory cell surface receptors, central to which are the Killer Ig-like Receptors (KIR). Here we hypothesized and tested whether diverse NK cell receptor repertoires can titrate NK cell functional responses to EBV and can potentially modify the risk of developing PTLD.

METHODS

KIR genotypes, centromeric and telomeric motifs and their variants were determined for 356 allo-HCT donors through next generation sequencing of KIR locus. PBMNCs from KIR typed healthy volunteers were co-cultured with EBV-transformed cells and degranulation and IFNγ producing NK cells were enumerated using multi-parameter flow cytometry. Effect of donor KIR profile on PTLD was tested using competing risks regression statistics. Segregation of NK cell response to EBV across various KIR repertoires was tested by Mann-Whitney U statistics

RESULTS

At least one copy of Donor tA01 motifs was required for a strong protection against PTLD (p=0.0001, SHR=0.17). The number of EBV induced NK cells increased with increasing tA01 motifs. There was no influence of recipients’ KIR repertoire on the risk of developing PTLD.

CONCLUSIONS

KIR-regulated NK cells have a profound effect on the risk of PTLD. KIR gene profile based identification of HCT recipients at high risk of PTLD will enable closer monitoring of EBV DNAemia and facilitate prompt therapy.
Deletion of a Class IIb Histone/protein Deacetylase (HDAC), HDAC10, Enhances FoxP3+ T-regulatory (Treg) Cell Suppressive Function, Gene Expression and Metabolism, and Promotes Allograft Survival
Authors: Dahiya Satinder , Wang Liqing , Beier Ulf H , Angelin Alessia , Han Rongxiang , Wallace Douglas C , Hancock Wayne W
Published: January 2023

Treg cells control immune responses that cause unwarranted inflammation and promote allograft rejection, leading to interest in their therapeutic applications. Targeting HDACs to modulate the function of Tregs has practical advantages over injecting ex-vivo-expanded Tregs that may not survive long post-injection. Several of the 11 classical HDAC enzymes are necessary for optimal Treg function while others are dispensable. We evaluated the function of HDAC10 in murine Tregs. We showed that HDAC10 bound to Foxp3 in co-immunoprecipitation assays, and that germ-line knockout of this hitherto enigmatic HDAC had no effect on the health of targeted mice. We found that HDAC10 deletion: (i) enhanced Treg suppressive function both in vitro (Treg suppression assays) and in vivo (homeostatic proliferation assays), compared to wild-type (WT) Tregs; (ii) increased Treg expression of genes including Foxp3, Granzyme-B, IL-10 and others (microarray, qPCR); (iii) increased acetylation of NFkB/p65 at lysine 310 and thereby prolonged the nuclear retention of p65 and increased transcription from p65-dependent promoters; and (iv) increased oxidative phosphorylation in Tregs compared to WT Tregs (assays of mitochondrial function on the Seahorse platform). Lastly, heterotopic cardiac allograft studies (BALB/c->C57BL/6) showed that compared to WT recipients, HDAC10−/− allograft recipients had markedly prolonged allograft survival (p<0.01). While additional studies of the in vivo effects of HDAC10 deletion in transplant and autoimmune models, including colitis, are underway our data show promise for the therapeutic targeting of HDAC10 as a novel approach for transplant rejection and other immunologically mediated disorders.
The role of PI3K in modulating NKT cell responses to Mantle Cell Lymphoma
Authors: Shissler Susannah Claire , Webb Tonya J
Published: January 2023

Natural Killer T (NKT) cells are important in the initiation and regulation of immune responses to cancers. The observation that mantle cell lymphoma (MCL) patients have a significant reduction in both NKT cell number and function suggests that the antitumor effects mediated by these cells may be compromised. MCL is characterized by the chromosomal translocation and nuclear overexpression of Cyclin D1. Secondary chromosomal abnormalities and dysregulation of cell cycle machinery also contribute to MCL pathogenesis, such as aberrant or constitutive activation of phosphatidylinositol 3 kinase (PI3K). We hypothesize that constitutive activation of PI3K in MCL alters the repertoire of lipids in these cells, ultimately resulting in decreased CD1d-mediated NKT responses to MCL. In this study, we used small molecule pharmacologic inhibitors of PI3K (PI3Ki) to investigate the role of PI3K signaling on MCL survival and immunogenicity both in vitro and in vivo. Treatment with PI3Ki induced apoptosis in human MCL cell lines and enhanced their immunogenicity. In addition, we have found that treatment of peripheral blood mononuclear cells with isoform-specific PI3Ki does not suppress T cell activation; whereas, treatment with a pan PI3Ki, BEZ-235, completely abrogates NKT/T cell responses. These studies suggest inhibitors targeting individual isoforms may have higher therapeutic efficacy. Collectively, this work will advance our understanding of differences in the modes of PI3K signaling in MCL survival and in antigen processing and presentation.
Splenic B cells contribute to intraocular tumor progression
Authors: Whitehill Brooke M , Andersen Jessica , Kopy Stephen , McKenna Kyle C
Published: January 2023

Primary intraocular melanomas heavily infiltrated by CD8+ T cells and macrophages are generally larger, more heavily vascularized tumors with a genetic profile indicating greater risk of metastasis to the liver. To better understand how the tumoricidal activity of these immune cell populations is inhibited within the eye, a site of immune privilege, we developed a mouse model of intraocular tumor development in which immunogenic E.G7-OVA tumor cells transplanted in the anterior chamber form progressively growing intraocular tumors in C57Bl/6 mice despite infiltration by tumor-specific CD8+ T Cells and macrophages. Recently, we demonstrated that splenectomy prior to tumor challenge restored tumoricidal activity of CD8+ T cells and macrophages and promoted complete elimination of established intraocular tumors. Herein, we show that E.G7-OVA tumors transplanted in the eye of B cell deficient μMT mice are rejected by CD8+ T cells. The rejection of intraocular tumors in μMT mice was not due to abnormal splenic architecture but rather stemmed from the absence of tumor-specific B cells as mice transgenic for an immunoglobulin molecule to an irrelevant antigen (hen egg lysozyme) also rejected intraocular E.G7-OVA tumors. These data suggest that intraocular tumor development induces the generation of tumor-specific regulatory B cells (Breg) in the spleen. How these Breg contribute to intraocular tumor growth is not completely understood. However, intraocular E.G7-OVA tumors grew progressively in mice deficient in activation-induced cytidine deaminase that cannot undergo isotype class switching suggesting that secreted tumor specific IgG does not contribute to inhibiting tumoricidal activity within the eye.
Modeling altered germinal center responses due to oncogenic activating mutations in CARMA1
Authors: Dutra Michelle N , Seymour Brenda , Sommer Karen , James Richard , Rawlings David
Published: January 2023

BCR engagement drives phosphorylation of the multi-adapter, CARMA1, and assembly of signaling complexes required for NF-κB activation. B cell lymphomas are frequently associated with constitutive NF-κB activation that sustains their survival. At least 10% of germinal center (GC) derived, activated B-cell like diffuse large B-cell lymphomas (ABC-DLBCLs) exhibit gain-of-function mutations in CARMA1. In the current study, we directly assess the impact activated CARMA1 (aCARMA1) on GC biology. We developed a novel mouse model that permits inducible expression of aCARMA1. Following T-dependent immunization, mice with B cell intrinsic aCARMA1 exhibit an expanded plasma cell compartment and reduced GC B cell numbers. A similar phenotype was present with GC restricted aCARMA1 expression, leading to increased total and antigen-specific IgG. ABC-DLBCL tumors frequently exhibit somatic mutations predicted to block terminal B cell differentiation. To better define how aCARMA1 impacts GC biology and cooperates with these lesions, we crossed KI mice to Blimp1F/F mice. aCARMA1 expression, in the absence of Blimp1, lead to increased GC B cell numbers compared to controls. Current studies are focused on detailed analysis of this altered response. Our results demonstrate that aCARMA1 is sufficient to alter the GC response and B cell fate, favoring plasma cell development. Further, in the setting of inability to exit the GC, aCARMA1 may be sufficient to directly promote lymphomagenesis.
B-1 cells secrete antibodies with anti-tumor properties
Authors: Rodriguez-Zhurbenko Nely , Rabadek Maura , Quach Tam D , Hopkins Thomas J , Rothstein Thomas L , Hernandez Ana Maria
Published: January 2023

It is well known that B-1 cells are the main B cell subset responsible for the production of natural antibodies. Several studies suggest that natural IgM antibodies play an important role in immunosurveillance against transformed cells in humans, preferentially binding glycolipids and carbohydrate structures. NeuGcGM3 ganglioside is a sialic acid containing glycosphingolipid that has been considered an attractive target for cancer immunotherapy since is not naturally expressed in healthy human tissues but is overexpressed in several tumors. We previously described the existence of cytotoxic anti-tumor antibodies against NeuGcGM3 ganglioside in healthy young subjects. However, whether this anti-tumor antibody response has a “natural origin” is unknown. With the present work we aimed to elucidate whether B-1 cells have the potential to secrete anti-NeuGcGM3 antibodies. First we generated four mouse monoclonal antibodies from Balb/c mouse peritoneal B cells. All hibridomas secreted IgM which bound NeuGcGM3 but no other gangliosides and recognized and killed NeuGcGM3 positive tumor cells. All the anti-NeuGcGM3 IgM-producing hybridomas were CD5+ B-1 cells. Then naïve, memory, plasma and B-1 cells were purified from healthy human donors PBMC and stimulated in vitro to produce immunoglobulins. All the B-1 cell supernatants reacted with NeuGcGM3 and most of them also recognized tumor cells expressing this ganglioside. Only 40% of memory samples, and none from naïve or plasma cells, recognized this antigen. These data suggest a protective role for B-1 cells against malignant cells through the production of natural antibodies reactive to tumor associated antigens such as NeuGcGM3 ganglioside.