bluuebunny/crossref_metadata_2025 huggingface dataset:
Immunomodulation mediated by anti-angiogenic therapy improves viral vaccination against GL261 glioma tumors.
Authors: Malo Courtney Sarah , Renner Danielle N , Jin Fang , Hansen Michael J , Pavelko Kevin D , Johnson Aaron J
Published: January 2023

Glioblastoma multiforme (GBM) is a lethal cancer of the central nervous system with a median survival rate of 15 months with treatment. Thus, there is a critical need to develop novel therapies for GBM. Immunotherapy is emerging as a promising therapeutic strategy. However, current standard-of-care therapies for GBM, in particular anti-angiogenic therapies that block vascular endothelial growth factor (VEGF), may have undefined consequences on the efficacy of immunotherapy. While this treatment is primarily prescribed to reduce tumor vascularization, multiple immune cell types also express VEGF receptors, including the most potent antigen‑presenting cell, the dendritic cell (DC). Therefore, we assessed the role of anti-VEGF therapy in combination with a novel tumor antigen-specific picornaviral vaccination in the GL261 syngeneic murine model of glioma. We hypothesized that treatment of GL261 glioma-bearing mice with anti‑VEGF therapy will act synergistically with anti-tumor picornaviral vaccination by enhancing DC maturation. We found that VEGF blockade results in a more mature DC phenotype in the brain and tumor-draining lymph node, as demonstrated by an increase in the expression of the co‑stimulatory molecules B7-1 and B7-2. Furthermore, we observed an increase in brain infiltrating, tumor antigen-specific CD8 T cells. This change in immune response directly correlates with a significant reduction in tumor burden, as measured by bioluminescence imaging, and a significantly improved survival rate of mice receiving both anti-VEGF therapy and viral vaccination compared to either treatment alone. Thus, anti-angiogenic therapy can be used in conjunction with and enhance immunotherapy for GBM.
Chemotherapy-derived inflammatory responses promote the differentiation and immunosuppressive activity of monocytic-MDSC
Authors: Ding Chuanlin , Yan Jun
Published: January 2023

Emerging evidence has unveiled the contribution of tumor microenvironment to the regulation of chemotherapeutic outcomes. However, the impact of chemotherapy on anti-tumor immunity and immunosuppression still remains elusive. In this study, we investigated the role of chemotherapy-derived inflammatory responses in the differentiation of myeloid-derived suppressive cells (MDSC). The anti-cancer agents Gemcitabine and Carboplatin induced the activation of MAPK pathway in mouse mammary cancer cell line EO771 and also stimulated cytokine and chemokine production including GM-CSF and IL-6. The supernatants of Gemcitabine-treated tumor cells promoted more bone marrow-derived monocytic MDSC differentiation, and enhanced their immunosuppressive activity on effector T cells. The expression of MDSC associated molecules such as Arginase and iNOS significantly increased in M-MDSC induced by the supernatant of Gemcitabine-treated tumor cells. In vivo treatment of EO771 tumors with Gemcitabine increased the suppressive function of MDSC. Our findings also suggest that Dectin-1 activation by β-glucan could significantly reduce MDSC suppressive function. Together, these results describe a role of chemotherapy-derived inflammation in the modification of chemotherapy-treated tumor microenvironment and suggest the targeting of Dectin-1 signaling may benefit cancer patients by reprogramming MDSC in chemotherapy setting.
Peripheral Reprogrammed CD4+ CTL Efficiently Control Tumor Growth.
Authors: Thiault Nicolas , Husain Mushtaq , Iwaya Hitoshi , Cheroutre Hilde
Published: January 2023

The initial commitment to either the CTL- or Th-lineage is made during thymic development, where thymocytes expressing an MHC class II–reactive TCR commit to the CD4 helper T cell lineage, whereas thymocytes with specificity for MHC class I differentiate into the CD8 CTL lineage. The molecular regulation is controlled by the action and counteraction of transcription factors, such as t ThPOK, which promotes the Th fate and Runx3 which drives the differentiation of MHC class I restricted thymocytes into the CD8 CTL-lineage. This dichotomy persists in the periphery, where ThPOK continues to suppress the cytotoxic fate of MHC class II–restricted CD4+ Th cells. Recently, our team made a major breakthrough and showed that the thymic lineage commitment of CD4 Th cells is not fixed and that mature peripheral CD4+ T cells have the plasticity to lose ThPOK expression.

We demonstrated that the loss of Thpok expression and the Th fate coincides with the re-expression of CD8α and the induction of a typical CTL phenotype, including 2B4 and Granzyme B expression and IFN-γ and TNF-α cytokine production. Using an in vitro cytotoxic assay, our lab found that these reprogrammed ThPOK− CD4+ T cells display killer capacities.

In a new study we now investigate the role of CD4 CTL in anti-tumor immune surveillance. Using the well-described B16-melanoma tumor model, we demonstrated that in vitro differentiated OVA-specific CD4 CTL display anti-tumor capacity and efficiently killed OVA-expressing B16 tumor cells.

This studies on CD4 CTL in the cancer field will not only greatly expand our basic understanding of anti-tumor immunity but it will undoubtedly have major implications for translational research to design new cell therapies to treat devastating cancers.
Dissecting the use of recipient alloreactive responses to achieve anti-leukemic effect
Authors: Fast Loren D , Nevola Martha , Reagan John
Published: January 2023

Harnessing alloreactive responses to incur anti-leukemic effects has focused on the donor lymphocyte response directed toward recipient antigens. Limited studies have examined the anti-leukemic potential of recipient alloreactive responses to donor cell antigens. An example is the infusion of large numbers of haploidentical, G-CSF mobilized, donor T cells (1 – 2 × 108 CD3+ cells/kg) into patients with refractory hematological malignancies who had received100 cGy total body irradiation prior to infusion. Strong recipient immune responses were generated capable of eliminating the donor cells within two weeks (thereby preventing GVHD) with side effects of high fever (104°F), malaise, rash, and diarrhea. More than half of these patients also generated responses to the hematological malignancies(14/26, 9 major), including some complete remissions. The protocol was modified in the reopened clinical trial by elimination of both the G-CSF mobilization of donor cells and the recipient 100 cGy total body irradiation. These patients exhibited milder, self-resolving fevers and limited anti-leukemic responses (1/5 with transient response). Analysis of blood samples obtained at several different timepoints after infusion revealed diminished IL-6 levels. Increased levels of plasma IL-2 and granzyme B and increased expression of granzyme B and PD-1 by activated recipient T cells correlated with elimination of donor cells. Increased expression of PD-1 ligands on leukemic cells provides one explanation for the lack of anti-leukemic response. These results indicate that appropriate manipulation of both donor cells and patients in this protocol should improve the anti-leukemic responses of recipient lymphocytes.
Age and sex affect αCTLA-4 efficacy alone and combined with αB7-H1 or regulatory T cell depletion in a melanoma model.
Authors: Figueiredo Alvaro Souto Padron , Hurez Vincent , Liu Aijie , Curiel Tyler J
Published: January 2023

Background

Most cancer patients are aged but most preclinical studies are in young hosts. We tested age effects on immunotherapy to further our understanding and identify strategies.

Methods

Young (3 mos) and aged (~26 mos) BL6 mice were injected subcutaneously with syngeneic B16 melanoma cells. 7 days later, mice got 2 μg denileukin diftitox (DD), an FDA-approved IL-2/diphtheria toxin or 200 μg αCD25 (both to deplete Tregs) ± 50 μg αCTLA-4 or 100 μg αB7-H1. 5 days later mice got a 2nd treatment.

Results

αCTLA-4 depleted intra-tumor Tregs in young but not aged males, a major age effect. In young BL6 males or females αCTLA-4 + αB7-H1 rejected B16 tumors better than either single agent. In aged males, αCTLA4 + αB7-H1 treated B16, but surprisingly accelerated tumors in aged females. Individual agents were effective in young hosts but had negligible effects in the aged. αCTLA-4 + either DD or αCD25 to deplete Tregs significantly reduced tumor growth in young mice but DD was better than αCD25. Neither combo treated B16 in aged mice. This DD dose did not deplete Tregs in aged mice as well as young, but higher DD doses were equally effective. In support, in Foxp3DTR mice, in which we deplete ~100% Treg (versus 20–50% with DD or αCD25), aged mice potently rejected B16.

Conclusions

Cancer immunotherapy studies in young give an incomplete understanding of clinical effects and mechanisms in the aged most at risk for cancer. We identify major Treg effects and differential Treg depletion efficacy of αCTLA4 and DD contributing to treatment failures in the aged and means to mitigate effects. With appropriate treatments, even very old mice can mount effective anti-tumor immunity. Novel sex differences in aged are unlikely estrogen-related and require further study.
A topical chemo-immunotherapy for squamous cell carcinoma.
Authors: Friedman Blake , Donahue Cara , Erdos Geza , Falo Louis D
Published: January 2023

We have previously demonstrated that dissolvable microneedle arrays can co-deliver cargos directly to the skin while avoiding systemic distribution. As squamous cell carcinoma (SCC) has a lifetime incidence of 7–11% in the United States and is the second most common non-melanoma skin cancer in persons of white European descent, we sought to determine if MNA delivery of anti-tumor agents induce cell death and improve survival in a mouse model of SCC and in freshly excised human SCCs. To accomplish this, we evaluated the effectiveness of doxorubicin, a chemotherapeutic demonstrated to induce immunogenic cell death, as an MNA delivered therapeutic. Effectiveness was evaluated in mice inoculated with SCC cells and later treated with Blank MNAs (MNA-Blank) or MNAs integrating doxorubicin (MNA-Dox), and in human SCCs treated with MNA-Dox. In mice, MNA-Dox treatment resulted in increased levels of pro-inflammatory cytokine gene expression, increased levels of cell death as determined by TUNEL assays, attenuated tumor growth, and substantially improved survival over MNA-Blank treatment at 20 days post-inoculation (100% survival in MNA-Dox group versus 0% survival in MNA-Blank group). Survival advantage persisted with 40% of MNA-Dox-treated mice alive at 40 days post-inoculation. Similarly, freshly excised human SCCs treated with MNA-Dox demonstrated increased levels of cell death as determined by TUNEL assays. Taken together, these results demonstrate the potential of MNA administration of doxorubicin to effectively induce tumor cell death in a murine model of squamous cell carcinoma and in human SCCs supporting further clinical development.
CD26 expression on T helper cells correlates with cell persistence and antitumor activity in vivo
Authors: Bailey Stefanie R , Nelson Michelle , Butcher Aubrey , Bowers Jacob , Meek Megan , Neal Lillian , Paulos Chrystal
Published: January 2023

Adoptive T cell transfer has been an impressive therapy for treating cancer patients, but can be inconsequential if the transferred cells are unable to engraft and persist. Our lab has found that human CD4+ T cells that express intermediate or high levels of CD26—termed CD26intermediate and CD26high T cells, respectively—display striking antitumor activity in NSG mice bearing large, established mesothelioma. Importantly, these cells also exhibit increased engraftment and persistence compared to mice treated with bulk CD4+ or CD26negative T cells. While the multi-functional nature (IL-17, IFN-γ, IL-2, TNF-α, IL-22) and heightened cytotoxicity (CD107A, Granzyme B) of CD26high T cells can help explain their antitumor activity in vivo, their ability to persist despite their effector memory phenotype is intriguing. After further evaluation, we found that both CD26intermediate and CD26highT cells express elevated β-catenin and BCL-2, as well as decreased Caspase 3 cleavage, compared to CD4+ and CD26negative T cells. Excitingly, both CD26intermediate and CD26high T cells continued to display enhanced T cell persistence and significantly delayed tumor growth when transferred into mice bearing aggressive PANC1 pancreatic tumors. Furthermore, mice treated with bulk CD26+ T cells also displayed a better antitumor response than those treated with bulk CD4+ or CD26negative T cells. Strikingly, these effects were all seen in the absence of cytotoxic CD8+ T cells, which have often been thought to be crucial for effective antitumor responses. Overall, this data suggests that the presence of CD26 on transferred CD4+ T cells could be crucial for an effective antitumor response and can be utilized to enhance future immunotherapy strategies.
Hepatitis C virus-cross-reactive TCR gene-modified T cells: A model for immunotherapy against diseases with genomic instability
Authors: Spear Timothy T , Riley Timothy , Foley Kendra C , Murray David C , Simms Patricia E , Scurti Gina M , Wang Yuan , Hellman Lance M , McMahan Rachel H , Rosen Hugo R , Baker Brian M , Nishimura Michael I
Published: January 2023

A major obstacle hindering the development of effective immunity against viral infections, their associated disease, and certain cancers is their genomic instability. Accumulation of mutations can alter processing/presentation of antigens recognized by antibodies and T cells leading to immune escape variants. A therapeutic agent that can combat rapidly mutating viral or cancer-associated antigens would be advantageous in providing effective immunity. We propose T cells harboring cross-reactive TCRs may offer a solution in these instances. Using hepatitis C virus (HCV), known for its genomic instability, as a model for mutant antigen recognition, we demonstrate cross-reactivity of TCRs against immunogenic and mutagenic NS3:1406-1415 and NS3:1073-1081 epitopes in TCR-gene-modified T cells. Our approach includes (1) functional studies demonstrating crossreactive profiles; (2) epidemiological data supporting the relevance of mutant epitope prevalence in infected individuals; (3) TCR-pMHC structural modeling to rationalize how TCR structural properties accommodate recognition of certain mutated epitopes and how they impact the requirement of CD8; lastly, we (4) provide preliminary clinical evidence of cross-reactive TCR benefit in that T cell clones isolated from HCV-infected patients who spontaneously resolved infection exhibited broader cross-reactivity compared to those of individuals with chronic infection. Together this approach serves as a model to address diseases with genomic instability and highlights potential benefit of cross-reactive TCRs. A better understanding of such TCRs’ promiscuous behavior may allow for exploitation of these properties to develop novel adoptive T cell-based therapies.
Th17 cells retain antitumor efficacy after long-term ex vivo expansion
Authors: Bowers Jacob S , Nelson Michelle , Majchrzak Kinga , Bailey Stefanie R , Diven Marshall , Schwartz Kristina , Rohrer Baerbel , Gattinoni Luca , Paulos Chrystal
Published: January 2023

Immunotherapies have produced impressive responses in melanoma patients by re-training their immune system to fight their cancer. One of the most successful of these methods is adoptive T cell therapy (ACT), which enriches, expands, and educates a patient’s tumor-reactive T cells before returning them to the patient. This therapy depends on infusing large numbers of cells (upwards of 1010 cells/patient) to mediate tumor regression, while also requiring cells of durable quality capable of sustaining long-term remission. Unfortunately, CD8+ T cells which are most often used, lose their antitumor efficacy as they are expanded ex vivo to large numbers. To circumvent this dilemma, we propose the use of IL-17 producing CD4+ T cells (Th17 cells), for ACT. We found that similar to CD8+ T cells, treatment with Th17 cells was more effective with higher numbers. These numbers can be achieved as Th17 cells expanded up to 10,000 fold after three weeks of ex vivo culture. At the end of culture, Th17 cells were as effective at eradicating melanomas as Th17 cells cultured for shorter duration. Between early and late culture time points, Th17 cells retained an effector memory phenotype (CD44+CD62L−) without acquiring markers of full effector and exhausted status, converted to a Th1-like functional profile (from mainly secreting IL17 to co-secreting IFNγ and IL17), and maintained the ability to engraft and persist in the host. We also demonstrate the power of combining the high yield and durable potency of Th17 cells by using their total yield at late culture to control clinically significant end-stage disease. These results support the advancement of Th17 cells to clinical trials to increase the number, quality, and duration of patient responses to treatment.
CRISPR/Cas9-mediated PD-1 disruption enhances anti-tumor efficacy of human chimeric antigen receptor T cells
Authors: Rupp Levi , Schumann Kathrin , Roybal Kole T , Marson Alexander , Lim Wendell A
Published: January 2023

Immunotherapies with chimeric antigen receptor (CAR) T cells and checkpoint inhibitors (including antibodies that antagonize the programmed cell death protein 1 [PD-1] pathway) have both opened new avenues for cancer treatment, but the clinical potential of combination checkpoint blockade and CAR T cell therapy remains incompletely explored. Here we show that programmed death ligand 1 (PD-L1) expression on tumor cells can render human CAR T cells (anti-CD19 4-1BBζ) hypo-functional, resulting in impaired tumor clearance in a sub-cutaneous mouse xenograft model. To overcome this suppressed anti-tumor response, we developed a protocol for combined Cas9 ribonucleoprotein (Cas9 RNP)-mediated gene editing and lentiviral transduction to generate PD-1 deficient, anti-CD19 CAR T cells. PD-1 disruption augmented CAR T cell mediated killing of tumor cells in vitro and enhanced clearance of PD-L1+ tumor xenografts in vivo. This study demonstrates improved therapeutic efficacy of Cas9-edited human CAR T cells and highlights the potential of precision genome engineering to enhance next-generation cell therapies and uncover mechanisms regulating human immune cell function.
Targeted Gene Editing Restores Regulated CD40L Expression and Function in X-HIGM T cells.
Authors: Hubbard Nicholas Wesley , Hagin David , Sommer Karen , Song Yumei , Khan Iram , Clough Courtnee , Ochs Hans D , Rawlings David J , Scharenberg Andrew , Torgerson Troy R
Published: January 2023

Loss of CD40L expression or function results in X-Linked Hyper-IgM Syndrome (X-HIGM), characterized by recurrent infections due to impaired immunoglobulin class-switching and somatic hypermutation. Previous attempts using retroviral gene transfer to correct murine CD40L expression restored immune function; however, treated mice developed lymphoproliferative disease, likely due to viral-promoter dependent constitutive CD40L expression. These observations highlight the importance of preserving endogenous gene regulation in order to safely correct this disorder. Here we report efficient, on-target, homology directed repair (HDR) editing of the CD40LG locus in primary human T cells using a combination of a TALEN-induced double-strand break and a donor template delivered by recombinant Adeno-Associated Virus (rAAV). HDR mediated insertion of a coding sequence (GFP or CD40L) upstream of the translation start site within Exon 1 allowed transgene expression to be regulated by endogenous CD40LG promoter/enhancer elements. Additionally, inclusion of the CD40LG 3′-untranslated region (3′-UTR) in the transgene preserved post-transcriptional regulation. Expression kinetics of the transgene paralleled that of endogenous CD40L in unedited T cells, both at rest and in response to T cell stimulation. The use of this method to edit X-HIGM patient T cells restored normal expression of CD40L and CD40-muIg binding, and rescued IgG class switching of naïve B-cells in vitro. These results demonstrate the feasibility of engineered nuclease-directed gene repair to restore endogenously regulated CD40L, and the potential for its use in T cell therapy for X-HIGM syndrome.
Mesothelin Vaccination for the Prevention of Ovarian Cancer
Authors: Scholler Nathalie , Stein Paul , Repellin Claire , Kamat Kalika , Harrison M. Travis , Shoemaker Robert , Sambucetti Lidia
Published: January 2023

Survival of ovarian cancer patients with high-grade serous carcinoma (HGSC) remains low. We hypothesized that a tumor antigen-targeted vaccine capable of eliciting a robust cell-mediated immune response may substantially improve women’s health. Mesothelin is a glycosylphosphatidylinisotol (GPI)-anchored, conserved protein that is overexpressed by ovarian, lung and pancreatic cancers. We designed mesothelin vaccines containing cyclic dinucleotide (CDN)-based adjuvants or alum-based adjuvants to trigger CD8 T cell activation. Anti-mesothelin antibody titers were stable or slightly increased after 2 vaccine boosts, and the highest and most stable titers were obtained with CDN-based adjuvants (meso/CDN). In addition, splenocytes from all mice receiving CDN-containing vaccines secreted mesothelin-specific IFNγ. Next, immunized mice were injected orthotopically with luciferase-transduced mouse ovarian cancer cells (ID8). In vivo imaging performed 3 weeks after tumor injections revealed that most of the mice from the groups immunized with Alum-based vaccines (92%) presented a positive signal of bioluminescence, which corresponds to the routine take rate of the ID8 tumors. In contrast, only 1/3 of the mice in the meso/CDN group showed a positive signal compared with 2/3 of the mice immunized with the control CDN-only vaccine. These results suggest that the meso/CDN vaccine regimen delayed tumor establishment. Longer observation of the mice is ongoing to determine whether the vaccination regimen can prevent tumor development. We tentatively conclude that ovarian cancer prevention may be possible through vaccination against mesothelin tumor antigen in combination with adjuvants that trigger cellular immunity.
In vitro treatment of neonatal foal alveolar macrophages with CpG 2142 enhances their killing capacity of R. equi
Authors: Bordin Angela Ilha , Gressler Leticia Trevisan , Rocha Joana , Brake Courtney , Tanner Amelia , Juillard Veronique , Cohen Noah
Published: January 2023

Pneumonia caused by Rhodococcus equi, a facultative, intracellular bacterium that replicates within macrophages, is an important cause of disease and death in immunocompromised hosts. No vaccine is yet available, and control is based on treatment with antibiotics. The emergence of antibiotic resistance and the lack of effective alternative antimicrobials indicate novel approaches to control are needed, such as host-directed strategies. Cytosine-phosphate-guanine oligodeoxynucleotides (CpGs), Toll-like receptor (TLR) 9 agonists, have immunomodulatory function both in vitro and in vivo in foals. Our objective was to evaluate the impact of in vitro treatment of neonatal foal alveolar macrophages (AMs) with CpG 2142 during the first month after birth. Using broncho-alveolar lavage, AMs were collected from 6 foals at ages 2, 14, and 26 days. Effects of age and treatment on the intracellular killing of R. equi by foal AMs were determined using the ratio of colony-forming units at 48 hours (T48) to post-phagocytosis (T0) (i.e, T48:T0). Foal AMs had significantly (P < 0.05) greater killing capacity at 14 and 26 days than at 2 days of age. Relative to media control, CpG 2142 increased the killing capacity of R. equi by foal AMs at ages 2, 14, and 26 days by approximately 2, 10, and 8 fold (P < 0.05), respectively. Notably, CpG 2142 clearly enhanced killing of R. equi by foal AMs even at age 2 days, when neonatal foals are known to lack a fully functional immune system. CpG 2142 can stimulate innate immune cells including AMs to kill intracellular pathogens more efficiently, and might be effective when nebulized for host-directed therapy against pneumonia caused by R. equi and similar intracellular pathogens such as Mycobacterium tuberculosis.
Maternal vaccination with dPNAG protects foals against the intracellular pathogen Rhodococcus equi
Authors: Rocha Joana Nunes , Cywes-Bentley Colette , Bordin Angela I. , Brake Courtney N. , Pier Gerald B. , Cohen Noah D.
Published: January 2023

Rhodococcus equi is a facultative intracellular pathogenic bacterium that causes a form of pneumonia similar to tuberculosis in foals and immunocompromised humans. A vaccine against R. equi is lacking. We studied the efficacy of maternal vaccination with deacetylated poly-N-acetyl glucosamine (dPNAG) oligosaccharide conjugated to tetanus toxoid to protect foals against R. equi. Pregnant mares (n=9) were randomly assigned to intramuscular injection 6 and 3 weeks prior to foaling with either vaccine (n=5) or saline (n=4). Foals were infected intrabronchially at age 28 days with 1 × 106 of a single strain of live, virulent R. equi. Foals were examined twice daily for clinical signs of R. equi pneumonia (viz., coughing, tachypnea, dyspnea, temperature >103°F), and weekly by thoracic ultrasonography. Blood samples were collected at age 2, 14, 28, 42, 56, and 84 days from all foals to detect antibodies by ELISA against dPNAG and native PNAG in serum and IFNγ production from peripheral blood mononuclear cells (PBMCs). The proportion of foals with pneumonia was significantly (P<0.05) greater among the unvaccinated mares (3/4) than vaccinated mares (0/5). Titers of dPNAG and PNAG antibodies were significantly (P<0.05) higher at all ages for foals of vaccinated mares. IFNγ produced by PBMCs stimulated with R. equi antigen was significantly greater (P<0.05) at age 28 days (prior to infection) in foals of vaccinated mares. Results indicate the dPNAG vaccine protected foals against the intracellular pathogen R. equi and efficacy against R. equi was likely mediated by maternally-transferred antibodies and enhanced CMI responses, and contribute to a growing body of evidence for a key role of antibodies in protecting against intracellular pathogens.
Identification of helitron sequences in the coral genome and implications for host defense.
Authors: Garrison Keith E
Published: January 2023

Helitrons are mobile elements found in the genomes of numerous plant and animal species. Helitrons can mobilize flanking sequences when they transpose, and so have the potential to duplicate genes and remodel genomes. The stony coral Acropora has an expanded immune gene repertoire in comparison to the closely-related anemone species Nematostella, especially in the number of TLR and IL-1R genes. Mechanisms behind the expansion are not currently known. Although helitron sequences have been identified in Nematostella, no helitrons have been identified in Acropora. BLAST searches against the Acropora genome using Nematostella amino acid sequences as queries revealed seven candidate Acropora helitrons. Six of the seven Acropora candidates had large open reading frames in the forward direction. One candidate element was split between two adjacent reading frames. Conserved domain searches were performed within the open reading frames using the databases at NCBI. As expected, Helitron N-like and replicative protein A (RPA) domains were shared between Acropora and Nematostella elements. UPGMA trees were constructed using highly conserved regions, and one candidate Acropora element clustered more closely with the Nematostella reference helitron than with other Acropora sequences. Interestingly, P-loop domains were common to cnidarian helitrons and a subset of plant helitron reference sequences containing plant disease resistance gene motifs in regions overlapping with a nucleotide-binding domain critical for resistance gene function. Together, these studies will help to determine if helitrons played a role in the expansion of the Acropora immune gene repertoire.
Lutein Ameliorates Streptozotocin-Induced Cardiac Oxidative Stress in Rats
Authors: ALREJAIE SALIM , ALHUSAINI KHALID
Published: January 2023

Background and Aims

A number of clinical studies and animal models have established that hyperglycemia is the leading cause for the progression of diabetic cardiovascular complications. An elevated glucose levels induces structural and functional changes in the cardiomyocytes through the activation of several signal transduction pathways, one of them is lipid peroxidation. The present study was undertaken to evaluate the protective effects of lutein against cardiac oxidative stress in streptozotocin (STZ; 65 mg/kg)-induced diabetic rats.

Method

Lutein content (40, 80 and 160 mg/kg) diets were supplemented to diabetic rats for six weeks. Thiobarbituric acid reactive substances (TBARS) and glutathione (GSH) levels, and superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR) and glutathione-S-transferase (GST) activities were measured in cardiac tissue. Histopathological changes were observed in cardiac tissue.

Results

Lutein dietary supplementations significantly inhibited the diabetic induced increased in heart levels of TBARS and enhanced the decreased levels of GSH in dose dependent manner. Markedly inhibited the cardiac enzymatic activities of SOD, CAT, GPx, GR and GST in diabetic rats were significantly increased in lutein diet supplemented diabetic animals compared to diabetic vehicle group.

Conclusion

These findings suggest that lutein has the potential to ameliorate STZ-induced toxicity in cardiac tissue probably through its antioxidant properties.
Comparative efficacy of equine bone marrow derived stem cell culture protocols.
Authors: Pullen Nicholas A. , Ryan Joan M. , Schiltz Paul M.
Published: January 2023

Across species the differentiation and propagation of various stem cell subsets are essential processes for research into immune cell and regenerative biology, and the development of new clinical opportunities. Especially in the equine realm there is an ever-growing interest in the effective and replicable generation of multipotential mesenchymal stem cells (MSC) for clinical resolution of intra-articular pathologies. Bone marrow is a desirable equine MSC source due to ease of access and the inclusion/richness of hematopoietic stem cells (HSC) that can also be studied from the same patient; however, there are numerous competing protocols for the differentiation of MSC, mostly concerning the initial isolation and enrichment steps from patient samples. Using sternebral BM biopsies we compared the efficacy of MSC purification and culture protocols including: carbohydrate/density gradient cell centrifugation, erythrocyte lysis, and cell settling/immediate subculture methods. Furthermore, with substantially fewer standardized equine-specific reagents available for the differentiation of HSC, we performed preliminary culture assessments of cross-reactivity for cytokines from non-equine sources, specifically IL-3 and SCF. The data presented here contribute to clinical and research decision-making with respect to MSC under differing circumstances of sample size, availability, scale of return, and time in addition to shedding light on research opportunities for growth and differentiation of equine HSC lineages.
Reduced memory responses to vaccination in cows naturally infected with bovine leukemia virus.
Authors: Frie Meredith , Sordillo Lorraine , Bartlett Paul , Coussens Paul
Published: January 2023

Bovine leukemia virus (BLV) is an extremely prevalent retrovirus infecting US dairy herds. Approximately 83% of US dairy herds are BLV-positive, and the within-herd infection rate is typically between 25–50%, with the prevalence increasing as the herd size increases. Historically, BLV infection has been considered benign, leading to a lack of control for its spread. However, recent research suggests that BLV infection negatively impacts the health of the host immune system. If BLV impairs host immunity, it would have a large economic impact on the dairy industry. To assess the immune competence of dairy cattle naturally infected with BLV, both BLV- and BLV+ cows were boosted on their regular schedule with BoviShield Gold FP5 L5 HB. Samples were collected over 28 days to characterize the strength of the immune response to the vaccine stimulus. BLV+ cows produced significantly lower titers of antigen-specific antibodies in comparison to healthy, BLV- cows. In vitro assays suggest that the difference in antibody titers is related to lower interferon-γ production from T cells in response to antigenic stimulation. Our results support the hypothesis that BLV+ cows have a reduced immune response to a commonly used commercial vaccine, which suggests that BLV+ cows may be less protected by vaccines and, thus, at an increased risk of developing secondary infections.
Variegated expression of members of the WC1 pathogen recognition receptor and co-receptor multi-gene family on γδ T cells
Authors: Damani-Yokota Payal , Telfer Jancie C , Baldwin Cynthia L
Published: January 2023

Cells of the immune system recognize disease-causing pathogens and respond in a manner to stop the infection. While we know how conventional cells of the immune system do this, for some non-conventional cells such as γδ T cells this process is less clear. We have shown that bovine γδ T cells bear lineage-specific transmembrane glycoproteins, known as WC1, are coded by a multigenic family and that these molecules function both as pattern recognition receptors (PRR) and signaling co-receptors for cellular activation. For example, while a subpopulation known as WC1.1+ γδ T cells respond early and participate in a recall response to the spirochetes Leptospira interrogans and Leptospira borgpetersenii, other subpopulations that express a different set of the WC1 genes do not respond. Other experiments showed that WC1 molecules on γδ T cells that respond to Leptospira bind the bacteria and are involved in cell activation. Thus, we hypothesize that WC1 is essential for the recognition of bacterial pathogens by γδ T cells and that the WC1 family members expressed by a particular cell will determine its ability to respond to an infection. We are now evaluating the distribution of WC1 gene expression among individual T cells to understand how subpopulations respond to specific pathogens. WC1 expressing γδ T cell clones were generated by infection with Theileria parva or by generation of long-lived memory cells to Leptospira Ag. To date, our findings suggest that the number of WC1 family members per an individual γδ T cell is restricted and stably expressed.
Stress perception differentially regulates the inflammatory response in the CNS to a neurotropic viral pathogen
Authors: Steel Christina D , Machida Mayumi , Lundberg Patric S , Wellman Laurie , Sanford Larry D , Ciavarra Richard P
Published: January 2023

Intranasal application of neurotropic vesicular stomatitis virus (VSV) replicates in olfactory receptor neurons and then rapidly invades the olfactory bulb (OB). VSV has an established time course of progression through discrete brain regions and of onset and recovery from encephalitis. Coincident with viral progression is a bimodal brain cytokine response composed of multiple chemokines and cytokines responsible for the prominent neuroinflammatory response. Stress can have significant, but poorly understood, effects on immune function and even less is known on how stress perception may impact immune competency. Therefore, VSV infected mice were exposed to either controllable (modeled by escapable foot shock, ES) or uncontrollable (modeled by inescapable foot shock, IS) stress prior to infection and for 1–3 days post infection (PI). OB cytokine responses were initially blunted and then amplified and dysregulated at days 1 and 3 PI, respectively. With time, dysregulated responses in ES mice were associated with increased pathogenecity. Ingenuity Pathways Analysis (IPA) identified miRNA146a as an upstream regulator of this response and qPCR found elevated OB levels (~5 fold) in ES versus IS cohorts at day 1 PI. Surprisingly, brains from mice that had long ago cleared VSV still contained an infiltrate containing CD103+ cells, a marker associated with long-term brain T resident memory cells. To our knowledge, this is the first time that stress perception has been shown to differentially modulate neuroinflammation. Our data also suggest the microRNA likely responsible for the observed stress-induced changes in the antiviral immune response to a neurotropic viral pathogen.
Development of interferon mimetic peptides having antiviral activity
Authors: Mujtaba Mustafa G
Published: January 2023

Current treatment options are limited for ameliorating viral infections. Interferons are involved in immune responses to viral infections; however, these interferons are not induced by some viral infections and, furthermore, can have toxic side effect when given at high doses as a therapeutic. The objective of this research is to design peptide mimetics of interferons that are not toxic to cells but have antiviral activities similar to interferons. Eleven peptides were designed based on interferon sequences and their receptor binding sites. Their activity on cell proliferation, toxicity, and antiviral effects against vesicular stomatitis virus (VSV) challenge of HeLa cells were measured. Results show that none of the peptides had significant antiviral activity when peptides were preincubated or incubated at the same time with the cells before VSV challenge. However, one peptide (Bact2) did have antiviral activity at 60ug/mL when this peptide was preincubated with the VSV virus for 24 hours before the HeLa cells were added to the mixture, suggesting that the Bact2 peptide directly bound to the VSV virus for its antiviral effect. Further analysis of the toxicity of the Bact2 peptide incubated alone with HeLa cells showed no cellular toxicity at concentrations of 60ug/mL or lower. Thus, the results from this study show that short peptides could be developed to directly neutralize viruses and interfere with their infectivity of cells.
RIG-I-like Receptor-mediated Signaling Regulated by NSs through Its Interaction with MAVS in a Novel Bunyavirus Infection
Authors: Xing Zheng , Zhu Lili , Qi Xian , Wu Xiaodong , Cardona Carol J
Published: January 2023

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease, which is caused by SFTS virus (SFTSV), a novel bunyavirus transmitted by ticks from animals. The disease is characterized by high fever and drastic reduction of leukocytes and platelets with high mortality rates in severe cases. We have reported that SFTSV suppresses host antiviral immunity and the viral nonstructural protein NSs plays a role in the immune evasion by interacting with downstream interferon signaling effectors, which is probably significant in virus pathogenesis. We furthered our study and demonstrated in this report that NSs interacted with mitochondrial antiviral signaling protein MAVS, the essential linker for host innate responses. NSs formed unique inclusion bodies (IBs) in the cytoplasm of infected cells and, through NSs and MAVS interaction, MAVS was translocated into the IBs in both transfected and infected cells. The interaction was specific and NSs did not interact with RIG-I or MDA5. Co-localization of NSs and MAVS trapped a significant portion of MAVS in the cytoplasmic IBs and weakened the functions of MAVS in activating downstream NFκB and IRF3 signaling. We examined host responses in NSs-overexpressing or infected cells and observed significant reduction of pro-inflammatory cytokine (IL-6, IL-8, Fas) and IFNs (IFN-α, IFN-β). Our data suggest that MAVS is essential to anti-SFTSV response since the knockdown of MAVS promoted viral replication via decreased IFN-β production, which could be reversed by overexpression of MAVS in the cells. This report demonstrates another player key to host responses, targeted by NSs of SFTSV through sequestration into viral IBs, resulting in inhibited IFN and NF-κB responses.
A central role for Type I IFN in the induction of Th2 responses by dendritic cells
Authors: Webb Lauren M , Lundie Rachel J , Borger Jessica G , Cartwright Adam N , Cook Peter C , Brown Sheila L , Jackson-Jones Lucy , Phythian-Adams Alex T , Davis Daniel M , MacDonald Andrew S
Published: January 2023

Although dendritic cells (DCs) are critical for induction of Th2 immunity against helminths or allergens, relatively little is known about how they become activated and function in response to Th2-polarizing antigens. We have discovered a previously unrecognized role for Type I IFN (IFN-I) in the optimal activation and function of DCs following exposure to strongly Th2-polarizing antigens. To date, IFN-I has primarily been associated with anti-viral immunity, and its role in Th2 settings is currently unclear. DCs exposed to total egg antigens from the parasitic helminth Schistosoma mansoni, or the primary immunostimulatory component of S. mansoni eggs, omega-1, produced IFN-I in vitro. IFN-I was also detected in response to the common allergen house dust mite (HDM). DCs lacking the IFN-I receptor displayed a dramatically impaired ability to induce Th2 cytokines in vivo, but unimpaired ability to support CD4 T cell polarization in vitro. Further, Th2-promoting DCs depended on IFN-I signaling for optimal activation, efficient migration to the draining LN, and effective localization within the T cell zone. In vivo challenge of naïve mice with S. mansoni eggs or HDM in the absence of the IFN-I receptor induced significantly reduced levels of Th2 cytokines compared to controls. Together, our data suggest a key role for IFN-I to enable Th2 induction by DCs against Th2 Ag in vivo. Future work will address the wider role of IFN-I in Th2 inflammation, including during patent helminth infection in vivo.
TLR1/2 ligand Neisseria meningitidis PorB accelerates, antigen presenting cell activation
Authors: Lisk Christina , Reiser Michael , Mosaheb Munir , Wetzler Lee
Published: January 2023

Background

Vaccines are a powerful tool for preventing the transmission of diseases, however very few take advantage of specific mechanistic knowledge. In order for newer and better vaccines to be developed, a greater understanding of adjuvant mechanism and immune system priming needs to be achieved. Our laboratory has focused on the natural adjuvant, PorB, a pore forming protein isolated from Neisseria meningitidis. This protein has been shown to be a TLR2 agonist with potent adjuvant activity. PorB also increases antigen-specific antibody titers and T cell proliferation when co-administered with an antigen.

Hypothesis

In this study, we hypothesized that PorB increases intracellular trafficking of antigen, ovalbumin (OVA), faster than OVA alone in bone marrow derived dendritic cells (BMDCs).

Methods

Isolated BMDC from BL6 mice were divided into 3 groups – PBS control, fluorescently labeled OVA (used antigen control), and OVA+PorB. Stimulation lasted 0, 0.5, 1, 2, or 4 hours. Cells were then stained with early endosome antigen 1 (EEA1) and lysosome-associated membrane protein 1 (LAMP1) to track the labeled OVA.

Results

This experiment showed that OVA uptake was increased within BMDCs stimulated with PorB. Specifically, OVA co-localized with EEA1 and LAMP1 more frequently than OVA alone. OVA co-localization was also present earlier in stimulation when PorB was present compared to OVA alone.

Conclusion

These insights of how BMDCs traffic antigen with adjuvants can allow for more efficient antigen presentation, lymphocyte responses, and, overall, better vaccines.
Immuno-proteomic interrogation of antigen presentation during Dengue infection reveals novel and HLA haplotype-specific MHC-I antigens
Authors: Swaminathan Kavya , Lund Peder J , Olsson Niclas , Marceau Caleb , Carette Jan , Davis Mark M , Elias Josh E
Published: January 2023

Dengue (DEN) virus infection is a leading cause of hospitalization and death in the developing world and broadly effective vaccines and therapies remain elusive. Identifying infection-specific peptide antigens would open new avenues for developing T cell based interventions. Past efforts towards mapping viral antigens used computational predictions that only partially reflect actual antigens presented by the major histocompatibility complex (MHC). To identify DEN-specific antigens without relying on error prone predictions, we developed an immuno-proteomics approach for interrogating antigen presentation in DEN infected B-lymphocytes. This approach enabled three fundamental findings; First, we identified 86 viral MHC-I antigens (59 novel), and mapped them to presentation hotspots in the DEN genome. Second, we discovered post-translationally modified viral and host antigens and antigens derived from alternate reading frames. Predicting these antigens using computational methods alone would be infeasible. Third, we found antigens responsible for HLA-haplotype dependent immune responses against DEN infection: we genetically engineered B-lymphocytes to express HLA alleles associated with either strong or weak DEN-immune responses and identified corresponding allele specific antigens. Together these discoveries let us assay the immunogenic potentials of an unprecedented range of DEN-specific antigens. Ex-vivo assays including ELISPOT and HLA tetramer staining supported our identification of immunogenic antigens in DEN-specific CD8+ T cells. These antigens have potential as both diagnostic tools to characterize DEN-specific T cell immunity, and serve as candidates for designing effective DEN vaccines.
The modulation of cathepsin activities by HIV protease inhibitors alters the processing and cross-presentation of pathogens
Authors: Le Gall Sylvie , Kourjian Georgio , Rucevic Marijana , Berberich Matthew J , Boucau Julie
Published: January 2023

Immune recognition by T cells relies on the presentation of pathogen-derived peptides by infected cells but the persistence of chronic infections calls for new approaches to modulate immune recognition. Pathogen uptake in phagosomes initiates infection of macrophages and leads to the priming of T cell responses by dendritic cells. The original degradation of antigens is performed by pH-dependent endolysosomal cathepsins. We show that HIV protease inhibitors (PIs) prescribed to HIV-infected persons variably modulate cathepsin hydrolytic activities in human antigen presenting cells (APCs), dendritic cells and macrophages, and CD4 T cells, three cell subsets infected by HIV. Two HIV PIs acted in two complementary ways on cathepsin activities: They directly enhanced procathepsin maturation and increased cathepsin hydrolytic activities. They indirectly altered regulators of cathepsin functions by inhibiting Akt kinase activities, reducing NADPH oxidase 2 (NOX2) activation, lowering phagolysosomal ROS production and pH, which altogether led to enhanced cathepsin activities. HIV PIs modified endolysosomal degradation and epitope production of proteins from HIV and other pathogens in a sequence-dependent manner. They altered cross-presentation of antigens by dendritic cells and epitope-specific T cell-mediated killing by up to 2.5-fold. HIV PI-induced modulation of antigen processing partly changed the self MHC-peptidome displayed by primary human cells. This first identification of prescription drugs modifying the regulation of cathepsin activities and the MHC-peptidome may provide an alternate therapeutic approach to modulate immune recognition in immune disease beyond HIV.