bluuebunny/crossref_metadata_2025 huggingface dataset:
Changes in MHC-II/peptide binding affinity following protein glycation, as observed in Diabetes
Authors: Santambrogio Laura , Clement Cristina , Negroni Pia , Stern Lawrence
Published: January 2023

Increased oxidized ribose, glucose, glyoxal and methylglyoxal concentrations are observed in several metabolic diseases including diabetes. These highly reactive moieties bind to the cellular proteome to form advanced glycation end-products (AGEs), which can alter the structure and function of targeted proteins. To address whether the AGE-mediated modification of immunologically relevant proteins could have functional consequences we incubated soluble recombinant HLA- DR1 (HLA-DRA*01:01/DRB1*01:01) in presence of glucose at concentration commonly observed in diabetic subjects (15–20 mM). Analysis of the AGE adducts was achieved by using bottom-up proteomics and tandem mass spectrometry analysis. Major observed modifications were glyoxal-derived hydroimiadazolone, argpyrimidine, dihydroxy and methylglyoxal adducts on arginine residues, pyralline and glyceraldehyde-derived pyridinium compound adducts of lysine and carboxy ethylation and methylation of both arginine and lysine residues. These AGE-related modifications were comparable to what observed on MHCII, immunoprecipitated from the PBMC of Diabetic patients. AGE PTMs mapped to both the binding groove and HLA-DM binding domains. AGE-modified and unmodified HLA-DR1 were then assessed for their binding affinity of several relevant peptides in a fluorescence polarization assay in the presence or absence of HLA-DM. Changes in binding affinity for the selected peptides was observed in AGE-HLA-DR1 as compared to the unmodified HLA-DR1. Altogether our data indicate that protein PTM, as commonly observed in conditions of metabolic and oxidative stress can change HLA-DR1/peptide binding affinity thus compromising MHC-II restricted immune responses.
PB1 as a potential target for increasing the breadth of T-cell mediated immunity to Influenza A
Authors: Uddback Ida Elin Maria , Steffensen Maria A. , Pedersen Sara Ram , Nazerai Loulieta , Thomsen Allan Randrup , Christensen Jan Pravsgaard
Published: January 2023

Current influenza vaccines are inducing antibody-mediated immunity toward the surface proteins hemagglutinin and neuraminidase. High variability of these proteins results in costly annual evaluation and production of new vaccines, not providing protection against heterosubtypic or emerging pandemic strains. Consequently, there is a need for a new universal influenza vaccine strategy. Unlike the surface proteins, the internal proteins of influenza are highly conserved. By vaccinating systemically and locally with nucleoprotein encoded in an adenovirus (AdNP) we could induce a protective CD8 T-cell response, that lasted up to at least 8 month post vaccination. To increase the breadth of our vaccine by combining construct, we studied the vaccine potential of another influenza protein. We showed that an adenovirus encoding PB1 elicits high numbers of antigen-specific CD8 T cells in mice. However, in vivo killing of peptide loaded cells was lower in PB1-vaccinated mice than in NP-vaccinated mice. This correlated with a poorer protection against challenge. To explain this, T cells from vaccinated mice were co-incubated with naïve splenocytes loaded with decreasing concentrations of peptide. If T cells were immediately added to the co-cultures, similar dose/response curves regarding IFNγ production was observed. However, if the T cells were added 6 hours after loading, cells from PB1-vaccinated mice required a much higher initial concentration of peptide to be activated compared with NP specific cells, indicating a lower stability of the MHC-PB1peptide complex. These results underscore that not only are T-cell quality and quantity crucial to consider in future vaccine design, but also MHC-peptide interactions.
Live attenuated Leishmania donovani Centrin knock out parasites generate non-inferior protective immune response in aged mice against Visceral Leishmaniasis
Authors: Bhattacharya Parna , Dey Ranadhir , Dagur Pradeep K , Joshi Amritanshu B , Ismail Nevien , Gannavaram Sreenivas , Debrabant Alain , Akue Adovi D , KuKuruga Mark , Selvapandiyan Angamuthu , McCoy John P , Nakhasi Hira L
Published: January 2023

Visceral Leishmaniasis (VL) causes significant mortality. Age appears to be critical in determining the clinical outcome of VL and there is no effective vaccine available against VL for any age group. Previously, we showed that gene deleted live attenuated L. donovani parasites (LdCen−/−) induced a strong protective immune response in young mice. In this study we analyzed LdCen−/− mediated modulation of immune response in aged mice (18 months) and compared to young (2 months) mice. Analysis of innate response in dendritic cells (DCs) from both young and aged mice upon infection with LdCen−/− parasites, showed significant enhancement of innate effector responses, which consequently augmented CD4+ Th1 cell effector function compared to LdWT infected DCs in vitro. Similarly, parasitized splenic DCs from LdCen−/− infected young and aged mice revealed induction of proinflammatory cytokines and down regulation of anti-inflammatory cytokine genes compared to LdWT infected mice. Furthermore, immunization with LdCen−/− induced higher IgG2a antibodies, lymphoproliferative response and stimulated splenocytes for heightened leishmanicidal activity in young and aged mice. Additionally, upon virulent L. donovani challenge, LdCen−/− immunized mice from both age groups displayed multifunctional Th1-type CD4 and cytotoxic CD8 T cells leading to significantly reduced parasite burden in the spleen and liver compared to naïve mice. Although, there was no difference in the LdCen−/− induced innate response in DCs between aged and young, the adaptive response in terms of T cell and B cell activation in aged was reduced, which correlated with LdCen−/− induced significant but diminished protective response in aged compared to young mice against VL.
Development of a Multivalent Subunit Vaccine against Respiratory Tularemia
Authors: Mansour Ahd A , Banik Sukalyani , Malik Meenakshi , McCormick Alison A , Bakshi Chandra Shekhar
Published: January 2023

Francisella tularensis (Ft); the causative agent of a fatal human disease tularemia is classified as a Category A Select Agent. No licensed vaccine is available for prevention of tularemia in the U.S.A. In this study, we used a novel Tobacco Mosaic Virus (TMV) based delivery platform for development of a fully protective multi-antigen subunit tularemia vaccine. Previously we have published that a trivalent TMV-conjugate vaccine confers 50% protection in immunized mice against respiratory Ft LVS challenge. In this study, we refined TMV-conjugate vaccine formulation to improve the level of protection in immunized C57BL/6 mice against respiratory tularemia.

We developed a tetravalent vaccine by conjugating OmpA, DnaK, Tul4 and SucB proteins of Francisella to TMV. CpG adjuvant was also included in the vaccine formulation. C57BL/6 mice were immunized intranasally (i.n.) on days 0, 14 and 28 and challenged with 10LD100 of Ft LVS on day 49 post-primary immunization (PPI). Mice were monitored for survival, weight loss, antibody and recall responses, and duration of immune protection.

100% of immunized mice were protected against a 10LD100 i.n. challenge dose of Ft LVS. Mice vaccinated with TMV-tetravalent vaccine showed high levels of TH1 antibodies than those with trivalent vaccine formulation. The challenged mice exhibited significantly reduced bacterial burden in lungs, liver and spleen. Strong ex-vivo recall responses were observed in immunized mice as late as day 84 PPI and 80% of mice survived when challenged 163 days PPI. Collectively, this study demonstrates that tetravalent vaccine formulation provides complete protection, induces strong protective and memory immune responses against respiratory challenge with F. tularensis.
Vaccine self-assembling immune matrix is a non-viral delivery platform that improves overall vaccine performance while maintaining safety and stability
Authors: Shollenberger Lisa M , Harn Donald A
Published: January 2023

Vaccination remains the most effective public health tool to prevent infectious disease. Many vaccines are marginally effective, and are less so when administered to immune-compromised populations. To enhance vaccine immunogenicity, we exploited the biphasic property of the (RADA)4 synthetic oligopeptide to create a new vaccine delivery method, VacSIM® (vaccine self-assembling immune matrix). Vaccine components are mixed with the VacSIM® solution for injection, after which the (RADA)4 peptides self-assemble into hydrated nanofiber gel-matrices, forming a vaccine depot with the vaccine antigens in the aqueous phase, allowing for slow-release of vaccine components over time. Thus, we have a non-viral, inert, biodegradable delivery system, not requiring formulation that can deliver various types of vaccines. We hypothesize that slow-release of vaccine components provides antigen persistence, driving enhanced vaccine-specific responses that are improved in both quantity and quality. We have tested VacSIM® with live bacterial vectors, inactivated virus and multiple recombinant protein vaccines. VacSIM® delivery of different protein immunogens, including Hepatitis B and HIV Envelope, is superior to delivery in licensed and unlicensed adjuvants and requires only a single injection. VacSIM® improves existing vaccines by reducing the number of injections needed, improving vaccine performance, especially in difficult-to-protect populations like the elderly and immune compromised, and reducing unwelcome side effects. VacSIM® can be stockpiled and stored without a cold chain, which is especially beneficial in resource-limited settings where vaccine access and compliance are issues.
Maternal Pertussis Vaccination with a Mono-component Pertussis Toxoid Vaccine is Sufficient to Protect Newborns
Authors: Kapil Parul , Zimmerman Lindsey I , Wagner Leslie D , Papin James F , Merkel Tod J
Published: January 2023

Bordetella pertussis is a highly infectious human pathogen causing serious illness in infants, children and adults. The first pertussis vaccination is administered at two months of age, therefore, infants under two months of age are most vulnerable to infection. Rising incidence of pertussis resulted in the 2012 recommendation by the Advisory Committee on Immunization Practices (ACIP) to immunize all pregnant women to protect infants from birth until their first vaccination. Using a baboon model, we demonstrated that maternal vaccination with the three or four component acellular pertussis vaccines protected newborn baboons from the disease but not from the colonization. We hypothesized this protection was due to the transfer of maternal anti-PT antibodies to the infant. Therefore we evaluated the protection conferred by maternal vaccination with a pertussis toxoid (PTx) only vaccine. Infant baboons born to mothers vaccinated with the PTx-only vaccine were free from disease and had low WBC counts following exposure to B. pertussis at five weeks of age. In contrast, infants born to unvaccinated mothers experienced severe disease with high WBC counts. Our results demonstrate that vaccination of baboons during pregnancy with a PTx-only vaccine is sufficient to protect newborn baboons from disease following exposure to pertussis. Because pertussis toxin is a secreted protein that can be purified from bacterial supernatants, a single-component vaccine comprised of pertussis toxoid is very likely the most cost-effective acellular pertussis vaccine possible. The development and use of mono-component PTx vaccines may enable the extension of maternal vaccine programs to low and middle income countries.
Combination CpG and MPL agonists heighten vaccine-induced protection against Influenza A virus challenge
Authors: Lampe Anna T , Hain Jacey L , Brown Deborah M
Published: January 2023

Current strategies for Influenza A Virus (IAV) vaccination result in antibodies against strain specific viral coat proteins. These antibodies do not provide protection against heterologous IAV strains and leave the population vulnerable to pandemics of highly pathogenic IAV. Vaccines may be improved by targeting T cells, which unlike B cells, are able to recognize inner, conserved IAV antigens, such as nucleoprotein (NP), increasing the potential for protection against different IAV stains. During live IAV infection multiple pattern recognition receptors (PRRs) on innate immune cells are activated; therefore vaccines triggering multiple PRRs may elicit better protection. As dendritic cells are the most potent antigen presenting cells for T cells, bone marrow derived dendritic cells (BMDCs) were activated by combined agonists that trigger separate, yet complementary PRR signaling pathways. Here we show that combined PRR agonists MPL and CpG, which activate TLR4 and TLR9 respectively, increased in vitro cytokine and chemokine expression by BMDCs. Further, intramuscular vaccination of mice with IAV pdm09 H1N1 hemagglutinin (HA) protein plus combined CpG and MPL, as adjuvants, induced the formation of pdm09 HA specific IgG2c antibodies. Combined CpG and MPL recipient mice experienced less weight loss and lower mortality after lethal challenge with pdm09 H1N1 compared to mice receiving lone agonists, suggesting that combined agonists improve protection. T cell activation in response to combined CpG and MPL is currently being investigated, as well as responses generated via mucosal, intranasal vaccination. Thus combined agonists that promote B and T cell mediated immunity could be incorporated into next generation IAV vaccines.
Biodegradable nanoparticle delivery of inactivated swine influenza virus vaccine provides heterologous cell-mediated immune response in pigs
Authors: Dhakal Santosh , Hiremath Jagadish , Bondra Kathryn , Lakshmanappa Yashavanth Shaan , Shyu Duan-Liang , Ouyang Kang , Kang Kyung-il , Binjawadagi Basavaraj , Goodman Jonathan , Tabynov Kairat , Krakowka Steven , Narasimhan Balaji , Lee Chang Won , Renukaradhya Gourapura J
Published: January 2023

Swine influenza virus (SwIV) causes considerable economic loss to pig industry, and some SwIV are zoonotic. This study was conducted to evaluate the cross-protective efficacy of PLGA (poly lactic-co-glycolic acid) nanoparticles (NPs) encapsulated SwIV vaccine in pigs. Killed SwIV H1N2 (δ lineage) antigens (KAg) were encapsulated in PLGA NPs of 200–300 nm (PLGA-KAg NPs), and influenza antibody-free pigs were prime-boost vaccinated intranasally as mist and challenged using a heterologous, virulent and zoonotic SwIV H1N1 (γ lineage). PLGA-KAg NPs induced maturation of pig macrophages and dendritic cells in vitro. In vaccinated pigs, PLGA-KAg NPs induced antigen specific lymphocyte proliferation and enhanced the frequency of T-helper/memory cells and cytotoxic T cells in peripheral blood mononuclear cells (PBMCs). In virus challenged pigs, the PLGA-KAg NPs vaccine rescued virus induced clinical fever, reduced the gross lung pathology, reduced the virus load in the lung sections with complete clearance of the virus from the lungs of most of the pigs; but the nasal virus shedding was not reduced. Immunologically, at post-challenge day 6 in a recall response in PBMCs of PLGA KAg NPs vaccinated pigs, a significant increase in IFN-γ secreting T cells against both vaccine and challenge viruses were detected. However, humoral immune response in those pigs was not augmented. In conclusion, intranasal delivery of PLGA NPs based SwIV induced cross-protective response through specific cell-mediated response. Future studies are aimed at boosting the mucosal antibody response.
Protein kinase D1 is essential for Saccharopolyspora rectivirgula-induced pulmonary inflammation and alveolitis.
Authors: Yoon Taewon , Kim Young-In , Fitzpatrick Elizabeth , Yi Ae-Kyung
Published: January 2023

Saccharopolyspora rectivirgula (SR) causes farmers’ lung disease, a form of hypersensitivity pneumonitis (HP). The initial proinflammatory responses to SR is dependent on Toll-like receptors (TLR) 2 and 9, and their signaling adaptor MyD88. We found previously that protein kinase D1 (PKD1), a serine/threonine kinase, can be activated through a MyD88-dependent TLR signaling pathway and plays a pivotal role in TLR/MyD88-dependent production of proinflammatory mediators. SR activates PKD1 via a MyD88-dependent manner in murine cells and SR-induced alveolitis is substantially suppressed by pretreatment with a PKC/PKD inhibitor Gö6976. However, it is currently unknown whether PKD1 plays a regulatory role in SR-mediated proinflammatory responses and alveolitis in vivo. In the present study, to further understand contribution of PKD1 in the SR-induced pulmonary inflammation, tamoxifen-inducible PKD1-deficient mice were generated. Wild-type mice and PKD1-deficient mice were challenged intranasally with SR. We found that the levels of proinflammatory mediators (including TNFα, IL-6, IL-12, and IFNγ), alveolitis, and neutrophil influx in the bronchoalveolar lavage fluid and interstitial lung tissue after single intranasal expose to SR were significantly lower in PKD1-deficient mice compared to those in wild-type mice. Our results demonstrate that PKD1 is essential for SR-mediated proinflammatory responses and neutrophil influx in the lung. Our findings also suggest that PKD1 might be one of the critical factors that contributes to the development of HP, and could be a therapeutic target for certain forms of HP, such as farmers’ lung disease.
Differential nutrient uptake by lung resident and spleen effector memory T cells following influenza virus infection
Authors: Cartwright Emily Kathleen , Kohlmeier Jacob E
Published: January 2023

Pulmonary virus infection generates a distinct population of CD8 T cells that reside within the lungs following antigen clearance. Resident memory T cells (TRM) are critical mediators of host defense, however, their ability to provide protection wanes over time. The protective immunity provided directly corresponds to the number of antigen-specific CD8+ TRM within the lungs at the time of secondary infection. Mechanisms underlying the differential loss of CD8 lung TRM remain poorly understood. Recent work has highlighted the importance of cellular metabolism in relation to cellular function and survival, but little is known about the metabolic phenotype of lung TRM following influenza infection. We investigated whether metabolic differences could explain the differential loss of lung TRM compared to circulating TEM. Using fluorescently labeled glucose and palmitate, we compared the nutrient uptake capacity of nucleoprotein (NP)-specific CD8 T cells in bronchealveolar lavage (BAL), mediastinal lymph node (mLN), spleen and lung TRM in vivo following influenza infection. We find lung TRM have differential capacity for nutrient uptake compared to NP-specific cells in other tissues. In addition, we utilized the Seahorse XF analyzer to measure oxygen consumption rate (OCR) and determine the spare respiratory capacity (SRC) of lung TRM compared to circulating TCM and TEM. We find that lung TRM tend to have lower OCR at baseline, as well as decreased SRC. Taken together, these data show that lung TRM have a different metabolic profile than circulating memory T cells. This work highlights the need to understand the differential metabolic requirements of TRM, which could facilitate future vaccine design strategies aimed at these pathways.
Glucagon-like peptide-1 receptor signaling attenuates RSV-induced type 2 responses and immunopathology
Authors: Bloodworth Melissa Harintho , Zhang Jian , Hotard Anne L. , Moore Martin L. , Hartert Tina V. , Niswender Kevin D. , Peebles R. Stokes
Published: January 2023

Glucagon-like peptide-1 receptor (GLP-1R) agonists are a well-accepted and safe treatment for Type II diabetes. Although GLP-1R agonists mainly act to potentiate insulin and suppress glucagon secretion, recent evidence suggests that GLP-1R signaling also has anti-inflammatory effects. Severe RSV-associated illness is partially caused by type 2-associated immunopathology. We hypothesized that GLP-1R signaling inhibits type 2-mediated immunopathology during infection with RSV 12/12-6, a strain of RSV that was isolated from a hospitalized infant with severe lower respiratory tract infection and bronchiolitis. We show here that GLP-1R agonist treatment decreased airway inflammation, airway reactivity, and airway mucus production in RSV 12/12-6-infected mice. GLP-1R agonist treatment decreased total lung IL-13 and IL-33 levels, with concurrent decreases in lung IL-13-producing group 2 innate lymphoid cell (ILC2), CD4+ Th2 cell, and basophil numbers as well as IL-33-producing epithelial cells. The GLP-1R agonist prevented airway inflammation, and did not impact viral load, anti-viral interferon and antibody production during secondary RSV infection. Relative to the respective mock-infected groups, RSV-infected GLP-1R agonist treated mice did not have increased weight loss compared to vehicle treated mice. A phenome-wide association study (PheWAS) identified a link between GLP-1R signaling and acute bronchitis and bronchiolitis in humans. These studies demonstrate that GLP-1R signaling protects against type 2-mediated immunopathology during RSV infection. GLP-1R agonists are the first known FDA-approved agents to inhibit IL-33 and may represent a novel treatment strategy for RSV bronchiolitis.
Induction of arginase-1 in MDSC requires exposure to CD3/CD28 activated T cells
Authors: Abdelaal Ahmed Mansour , Bian Zhen , Culpepper Courtney , Liu Yuan
Published: January 2023

Tumor-induced myeloid derived suppressive cells (MDSC) have been reported to inhibit T cell responses. In our study MDSC inhibit T cell proliferation through arginase-1 dependent manner. However, surprisingly we observe that freshly isolated MDSC from the bone marrow of tumor bearing mice do not constitutively express arginase-1 until after exposure to T cell proliferation system. The aim of this study is to determine the mechanism by which arginase-1 is induced in MDSC following exposure to a proliferative T cell environment. We show that treatment of MDSC with culture supernatant isolated from T cells activated with CD3/CD28 antibodies successfully induced arginase-1 expression and this process is independent of IL-10 and IFNγ. This suggest that arginase-1 induction in MDSC can occur independently of cell-cell contact. Interestingly IL-2, Con A or PMA activated T-cell supernatant as well as supernatant from multiple cancer cell lines failed to induce arginase-1 in MDSC. We also show that M-MDSC express higher levels of arginase-1 than G-MDSC after co-culture with CD3/CD28 activated T cells as well as its supernatant. In addition, other bone marrow cells have shown potential to express arginase-1 following exposure to the same conditions. For example, we observe that healthy Ly6C+ monocytes but not mature granulocytes successfully express arginase-1. These data demonstrate that T cells activated through stimulation of TCR elicit the immunosuppressive function of MDSC through induction of arginase-1.
Immune tolerance to recombinant immunotoxin LMB-100 using synthetic vaccine particles encapsulating rapamycin
Authors: Mazor Ronit , King Emily , Kishimoto Takashi Kei , Pastan Ira
Published: January 2023

Protein and cell based therapeutics have great potential to treat many human diseases. However, they often elicit an immune response that blocks their efficacy. Synthetic Vaccine Particles containing Rapamycin (SVP-R) is a novel approach to mitigate immunogenicity and induce specific immune tolerance.

We evaluated the efficacy of SVP-R to reduce anti-drug antibody (ADA) formation to a recombinant immunotoxin, LMB-100, a genetically engineered protein designed for cancer therapy. Due to the bacterial origin of the toxin, LMB-100 is highly foreign to the human immune system. Almost all patients treated with LMB-100 made ADAs after two cycles (6 doses) of treatment that neutralized the drug and greatly lowered efficacy.

Using an immune-competent mouse model we found that combining SVP-R with LMB-100 dramatically decreased ADA titers by 98%. The decrease persisted for more than six weeks despite additional challenges with LMB-100, indicating development of immunological tolerance to LMB-100. Transfer of splenocytes from tolerized mice to naïve mice, and challenge with LMB-100 resulted in a 66% decrease in titer, indicating that the immune tolerance is transferable.

SVP-R are being evaluated in humans to prevent ADA to Pegsiticase, an enzyme for refractory gout treatment. Our data indicates that combining SVP-R with LMB-100 should be useful in treating cancer by allowing more treatment cycles and better efficacy. This approach can be used to increase the efficacy of other immunogenic agents such as CAR-T cells, antibody drug conjugates and viral gene therapy vectors.
Phosphorylation of Y248 in the ITSM of PD-1 is indicative of PD-1-mediated inhibitory function
Authors: Bardhan Kankana , Weaver Jessica D , strauss laura , Patsoukis Nikolaos , Boussiotis Vassiliki A
Published: January 2023

PD-1 is an inhibitory receptor expressed on activated T-cells. SHP-2 tyrosine phosphatase interacts with PD-1 and is critical for PD-1-mediated inhibition. The cytoplasmic tail of PD-1 contains an immunoreceptor tyrosine based inhibitory motif (ITIM) and an immunoreceptor tyrosine-based switch motif (ITSM). It has been reported that SHP-2 may interact with either ITIM or ITSM of PD-1. We sought to identify the precise motif of PD-1 that is mandatory for SHP-2 interaction and PD-1 inhibitory function. Using GST-SHP-2 fusion protein we determined that PD-1 interacted with SHP-2 after PD-1 ligation with simultaneous TCR/CD3-mediated activation. This interaction required phosphorylation of the ITSM and was abrogated when the ITSM tyrosine Y248 was mutated to phenylalanine. In contrast, when the ITIM tyrosine Y223 was mutated, interaction of PD-1 with SHP-2 remained unaffected. Based on these findings, we hypothesized that phosphorylation of Y248 might be indicative of PD-1-mediated inhibitory signaling. We generated an antibody specific for Y248 and examined expression and function of PD-1pY248+ T cells in peripheral blood from healthy humans. PD-1pY248+ cells were detected within the CD4 but mostly CD8T cell populations, mainly in central memory and effector memory subsets and in much lower extent in terminally differentiated effectors. Although PD-1high did not correlate with altered ability of CD8 T cells to produce effector cytokines, PD-1pY248+ expression correlated with impaired production of IFN-g and TNFa in response to TCR/CD3+CD28-mediated stimulation. Thus, PD-1pY248 might serve as a biomarker indicative of PD-1 mediated inhibitory signaling in patients with chronic infections and cancer.
Multimeric Soluble 4-1BBL as a T Cell Stimulator for Adoptive Immunotherapy.
Authors: Kornbluth Richard S , Hamilton Victoria Snarsky , Stone Geoffrey W
Published: January 2023

Members of the TNF SuperFamily of ligands (TNFSFs) have significant potential as immuno-oncology agents. The TNFSFs are trimeric membrane proteins that can be cleaved into soluble single trimers. While the soluble single trimers can be easily prepared and studied, they have little or no activity in vivo. This deficiency is caused by the need to cluster their cognate receptors in the plane of the membrane in order to induce a supramolecular signaling complex on the cytoplasmic side of the plasma membrane. For the TNFSF ligands, this requires that they be used as many-trimer multimers that mimic the natural expression of many trimers on the surface of stimulating cells. To meet this need, we prepared fusion proteins comprised of the extracellular domains of TNFSF ligands joined to a natural protein that provides a multimerization scaffold. When surfactant protein D (SP-D) is used as a scaffold, the result is a 4-trimer TNFSF ligand product (UltraLigand™). Our published studies have described such multimeric forms of CD40L, OX40L, GITRL, CD27L/CD70, BAFF, RANKL, and TRAIL and shown that they are highly active in vitro and in vivo. As an extension of this work, 4-trimer forms of murine and human 4-1BBL (CD137L, TNFSF9) were constructed and expressed in CHO cells. As a co-stimulatory molecule, SP-D-4-1BBL (Ultra4-1BBL) activated both CD4+ and CD8+ T cells and promoted the growth of NK cells in vitro. Given the interest in 4-1BB (CD137) as a marker of therapeutically effective tumor-infiltrating lymphocytes (TILs), SP-D-4-1BBL should be a useful should be a useful growth factor for TIL manufacturing and T cell culturing in general.
Inhibitory cytokines mark distinct subpopulations of intratumoral regulatory T cells
Authors: Yano Hiroshi , Sawant Deepali V , Liao Mengting , Bruno Tullia , Chikina Maria , Workman Creg J , Vignali Dario AA
Published: January 2023

Regulatory T cells (Tregs) play a crucial role in the maintenance of self-tolerance and the resolution of inflammation; however, they also negatively regulate anti-tumor immunity by contributing to the immunosuppressive tumor microenvironment. Our previous studies have shown that the inhibitory cytokine interleukin-35 (IL35), a member of the IL12 cytokine family, is preferentially expressed by activated Tregs and the IL35 expression is required for the maximum suppressive activity of Tregs.

To investigate the role of Treg-derived IL35 in the tumor microenvironment, we generated a Treg-specific IL35 reporter knockin mouse as well as a Treg-specific IL35 deletion mouse. We demonstrated that IL-35+ Tregs are preferentially enriched in the B16 melanoma tumor microenvironment and actively involved in tumorigenesis by promoting the expression of multiple inhibitory receptors in CD8+ tumor-infiltrating T lymphocytes (TILs). However, the impact of IL35 relative to IL10 and the phenotype of IL35+ Tregs relative to other Treg subpopulations in the tumor microenvironment have not been fully elucidated.

We have assessed the expression of IL35 and IL10 in intratumoral Tregs and the phenotype and function of cytokine producing subpopulations using a triple Foxp3/Ebi3/Il10 reporter knockin mouse. We have also assessed IL35/IL10 expression in Tregs infiltrating several human tumors. Interestingly, there are distinct IL35-/IL10-expressing Treg subpopulations in mouse and human tumors. We continue to characterize the phenotype and function of intratumoral Treg subpopulations in mouse and human tumors.
Cancer testis autoantigens as germ cell autoantigens continuously egress from normal testes and maintain Treg-dependent tolerance
Authors: Tung Kenneth S K , Harakal Jessica , Qiao Hui , Rival Claudia , Paul Liesbeth , Hardy Daniel M. , Cheng C. Yan , Goldberg Erwin
Published: January 2023

We investigated tolerance for: 1) testis antigens (mitotic germ cells and somatic cells) located external to the blood-testis barrier (BTB) and 2) testis antigens (sperm and meiotic germ cells) located internal to the BTB. Transiently Treg-depleted B6AF1-DEREG mice produced autoantibodies to mitotic germ cells known to express NY-ESO-1 and MAGE-A (target cancer testis antigens (CTA) in cancer vaccine trials) and to Leydig cells. Unexpectedly, Treg-depleted mice also produced autoantibodies to sperm antigens. These plasma membrane and cytoplasmic sperm antigens continuously egressed from normal seminiferous tubules via the residual bodies. They were recognized by infused circulating antibodies to form local immune complexes outside the BTB and were sensed by antigen-specific T cells in regional lymph nodes. In contrast, sequestered sperm antigens, excluded from residual bodies, were not tolerogenic. Therefore, we provided direct evidence against the “complete testis antigen sequestration” dogma and established Tregs as a critical physiological tolerance mechanism for non-sequestered testis autoantigens. These findings are germane to both autoimmunity and tumor immunity. We found that vasectomized mice responded to sequestered sperm antigens, whereas Treg-depleted mice with autoimmune orchitis responded to non-sequestered sperm antigens. We also detected both tolerogenic and non-tolerogenic sperm antigens as CTA in many types of human cancer. We posit that the existence of two sets of CTA as normal germ cells antigens with distinct tolerogenic properties will impact the outcome of anti-tumor immunity in male patients and will influence the selection of optimal immunogens in cancer vaccine development.
Nonredundant role for GM-CSF in autoimmune pathology of the neureretina in the absence of IFN-γ and IL-17A
Authors: Bing So Jin , Silver Phyllis , Jittayasothorn Yingyos , Horai Reiko , Caspi Rachel R
Published: January 2023

Autoimmune uveitis is a complex group of sight-threatening CNS diseases caused by activated retina-specific Th1 or Th17 cells that acquired the ability to cross the blood-retinal barrier, but their respective contribution to disease is not fully understood. In this study, we used mice deficient in IL-17A, IFN-γ (GKO), or both (DKO) to dissect the role of Th1 and Th17 lineage-specific cytokines in pathogenesis of uveitis. Experimental autoimmune uveitis (EAU) was induced by active immunization with the retinal autoantigen IRBP in CFA. IL-17A−/− mice were protected from EAU, whereas GKO mice had exacerbated disease. Surprisingly, DKO mice were fully susceptible to EAU with scores similar to WT controls. The frequency of macrophages and production of proinflammatory cytokines (GM-CSF, IL-6, and TNF-α) was increased in the spleen and eyes of DKO mice. To test whether other T cell lineage-related cytokines contribute to pathology in the absence of IL-17A and IFN-γ, EAU-challenged DKO mice were treated with blocking Abs to IL-17F, IL-22, TNF-α, or GM-CSF. Blockade of IL-17F, IL-22, or TNF-α in DKO mice did not affect the severity of EAU. By contrast, treatment of DKO mice with an anti-GM-CSF Ab during the expression stage of disease significantly suppressed disease severity in DKO, but not in WT mice. These results suggest that IL-17F, IL-22 or TNF-α individually are dispensable, but GM-CSF appears to play a major and nonredundant role as a pathogenic cytokine in EAU when IFN-γ and IL-17A are both absent. Our results highlight that ‘classical’ Th1 and Th17 cells are not the only pathogenic effectors, and that other inflammatory cytokine(s), such as GM-CSF, can also drive uveitis.
The impact of MHCII-peptide editing by H2-DM on the generation of neonatal regulatory T cells
Authors: Tuncel Jonatan , Yang Siyoung , Fujikado Noriyuki , Benoist Christophe , Mathis Diane J
Published: January 2023

The transcription factor Aire drives the expression of peripheral-tissue antigens (PTAs) in a subset of thymic medullary epithelial cells (MECs). Presentation of PTAs by MHCII on MECs and thymic DCs induces negative selection of self-reactive thymocytes and positive selection of Tregs. Individuals with mutations in Aire develop a combination of autoimmune diseases. In mice, Aire expression during the first weeks of life is sufficient to protect against ‘Aire-less’ disease. During this ‘neonatal window’, a population of long-lived Aire-dependent Tregs is selected that is important for the suppression of self-reactive T-cells. The mechanism involved in the selection of these Tregs is still unknown, as is their function and property to colonize lymphoid and non-lymphoid organs in the adult mouse. We have previously shown that the expression of Aire-induced genes is not dependent on age; however, age affects the expression of H2-DM, a protein that catalyzes the removal of CLIP from the MHCII peptide-groove and which is more abundant in MECs from young mice. This finding suggested that MECs from young and old mice might present different repertoires of PTAs. To probe how the expression of H2-DM by MECs influences the selection of neonatal Tregs, we have now generated mice that are either H2-DM deficient or express reduced levels of this protein. Transfer of neonatal Tregs from these mice to Aire-KO neonatal recipients may provide important insights into the relevance of MHCII-peptide-editing for the selection of specific Treg populations.
The role of MHC class II polymorphisms for DM-mediated peptide editing and autoimmunity
Authors: Haag Sabrina , Pos Wouter , Pyrdol Jason , Dougan Stephanie K , Wucherpfennig Kai W
Published: January 2023

Polymorphisms in MHC class II (MHCII) genes are the strongest risk factors for the development of autoimmune diseases, such as type I diabetes (T1D). T1D is associated with allelic variants of the MHCII molecule HLA-DQ. In the nonobese diabetic (NOD) mouse, the orthologous molecule, I-Ag7, also predisposes mice to spontaneous diabetes development. To investigate the functional impact of natural MHCII polymorphisms on DM binding and T1D we introduced selected mutations in the genome of the NOD mouse by CRISPR/Cas9 genomic editing.

Our approach was to first identify unique polymorphisms in the putative interface of I-Ag7, and then assess the impact of these polymorphisms on DM-mediated peptide editing in vitro. Several polymorphisms increased the interaction between I-Ag7 and DM and augmented peptide binding. The substitution of lysine (K) 40 in I-Aα with glutamic acid (E) had the strongest effect on DM editing and accelerated peptide binding more than 9-fold. Lysine at position α40 is unique to the diabetes associated I-Ag7 molecule and was therefore selected for in vivo evaluation. Homozygous and heterozygous K40E mutant mice showed increased H2-DM protein expression compared to WT littermate controls. Increased H2-DM levels were accompanied by an increase in intra- and extracellular I-Ag7 expression. This suggests that the K40E mutation promotes the interaction between H2-DM and I-Ag7, and likely, represents increased intracellular dimerization and more stable I-Ag7/peptide complexes on the cell surface. Currently, we are evaluating whether increased editing of the MHCII peptide repertoire mediated by the K40E mutation influences spontaneous diabetes development.
The role of IL-10 and FasL in regulating Th1 and Th17 cell responses toward recoverin in autoimmune retinopathy
Authors: Nikoopour Enayat , Ohara Ray , Lin Cheng-mao , Lundy Steven K , Heckenlively John R
Published: January 2023

Autoimmune retinopathy (AIR) is a condition that manifests in acute and progressive vision loss in humans. Antibodies against the retinal protein recoverin are frequently detected in AIR patients. We have established a murine model to better understand the immunopathology of AIR. Mice were immunized subcutaneously with recombinant mouse recoverin (100–200 μg) emulsified in complete Freund’s adjuvant (CFA) and intraperitoneally with pertussis toxin (400 ng). After three weeks, the immunized mice were scanned with optical coherence tomography (OCT) for detection of pathological changes in the eyes. Cells from spleen and draining lymph nodes were restimulated with recoverin (5 μg/ml) for 4 days. Immunization of C57BL/6 and BALB/c mice with mouse recoverin resulted in recoverin-specific T cell responses. Production of IFN-γ and IL-17 were observed in cells harvested from draining lymph nodes and spleen of the immunized mice upon restimulation in vitro with recoverin. Immunization also led to production of high titers of recoverin-specific antibodies (1/400) in the immunized mice. The recoverin-specific Th1 and Th17 cell response was augmented in IL-10 KO mice, whereas humoral response was unaffected. More importantly, the number of lymphocytes infiltrated into the eyes dramatically increased in IL-10 KO mice upon immunization with recoverin. Immunization of Fas ligand deficient GLD mice with recoverin antigen led to a predominant Th17 cell response toward recoverin. These new insights regarding the anti-recoverin immune response have led us to hypothesize that skewing of recoverin-specific T cell response toward inflammatory Th1 and/or Th17 responses can drive the pathogenesis of AIR.
MOG35-55, not NFM15-35, is the critical autoantigen for inducing demyelinating autoimmune disease.
Authors: Blanchfield Jennifer Lori , Evavold Brian D
Published: January 2023

Here we examine functional significance of CD4+ T cell cross-recognition between MOG35-55 (myelin oligodendrocyte glycoprotein) and NFM15-35 (neurofilament medium polypeptide) in demyelinating autoimmune disease. These epitopes are interesting for synergist potential in promoting autoimmunity due to identical amino acids at proposed TCR contacts while displaying variances at MHC. Surprisingly, NFM15-35 peptide is not encephalitogenic in polyclonal models and we proposed to understand why by studying how NFM15-35 effects the known encephalitogenic MOG CD4+ T cell population using peptide:MHC (pMHC) technologies. We found a NFM15-35 challenge was less potent than MOG35-55 in expanding tetramer positive, MOG38-49:I-Ab T cells. Diminished tetramer staining indicated the expanded polyclonal T cells from NFM challenge were of low affinity for MOG38-49, because tetramer enriches for T cells with high affinity for pMHC. We also found that NFM18-30 specific cells in the CNS of mice with MOG35-55 induced EAE predominantly cross-recognized MOG38-49:I-Ab using the 2-dimensional micropipette adhesion frequency assay. These data suggested MOG35-55, not NFM15-35, was the critical encephalitogenic antigen and we confirmed this by showing that demyelinating disease in NFM deficient mice exhibited identical disease course to wild type mice. Interestingly, alterations of NFM15-35 N-terminal amino acids expanded T cells that cross-recognized MOG38-49 tetramer, a phenotype concomitant with restoring encephalitogenic potential in altered NFM15-35 peptides. Overall, we support MOG35-55 as the critical autoantigen for induced demyelinating disease with the contribution of NFM15-35 being dictated by cross-recognition of MOG.
Adoptive transfer of a novel MIF receptor (CD74+) expressing memory T cell subpopulation is sufficient to transfer inflammatory arthritis
Authors: Doherty Edward H , Piecychna Marta , Leng Lin , Bucala Richard
Published: January 2023

Rheumatoid arthritis (RA) is characterized by relapsing disease and progressive autoimmune joint destruction. The mechanism(s) for disease recurrence in previously affected joints is unknown. High expression alleles of the cytokine macrophage migration inhibitory factor (MIF) are associated with severe erosive RA. We report a novel subpopulation of T cells expressing the MIF receptor CD74, which previously has not been considered to be present in T lineage cells. CD74+ T cells comprise 1% of lymph node T cell and exhibit an effector memory phenotype (CD3+ CD74+ CD44+, CD62Llow). Notably, whole genome expression profiling (RNAseq) of CD74+ T cells revealed reduced expression of the immune checkpoint Ctla4 (3.5-fold, p=0.009), and increased expression of Stat4 and Ptpn22 (1.4-fold, p=0.001 and 0.86-fold, p=0.045), three genes that regulate T cell activation and also are linked genetically to RA pathogenesis. In the collagen-induced arthritis (CIA) mouse model of RA, we observed a 2-fold (p=0.023) expansion of CD74+ T cells after disease induction when compared to Mif−/− mice, which show no expansion of CD74+ T cells and reduced disease severity (disease score: WT: 7.8; Mif −/−: 1.4, p>0.001). Notably, adoptive transfer of CD74+ T cells from mice with established CIA into naive mice recapitulated joint inflammation (disease score: 4.3, p=0.001). Disease was not detectable in naïve mice that received CD74-T cells from CIA hosts. This study identifies a novel population of effector memory T cells that are capable of transferring autoimmune joint inflammation. We hypothesize that MIF responsive CD74+ effector memory T cells mediate recurrent joint inflammation and may be amenable to specific targeting by emerging MIF-directed therapies.
The role of programmed death-1 in regulating islet-specific CD4 T cells
Authors: Martinov Tijana , Spanier Justin , Fife Brian T
Published: January 2023

The inhibitory receptor programmed death (PD)-1 and its ligand PD-L1 negatively regulate T cell function. PD-1 blockade is used in the clinic to reinvigorate anti-tumor immunity. However, patients often experience autoimmune-like symptoms, or develop autoimmunity, including type 1 diabetes. We used peptide:MHC Class II tetramers to track islet-specific CD4 T cells in diabetes-prone (NOD) mice and ask how PD-1 regulated this population. We found that insulin-specific cells with the highest affinity for cognate antigen expressed the most PD-1. In turn, PD-1 blockade promoted the accumulation of high-affinity cells in the pancreas, and exacerbated diabetes. Given that 70% of insulin-specific cells are anergic, we investigated whether PD-1 was required for the maintenance of anergic state, using an adoptive cell transfer model. Anergic cells remained functionally blunted in comparison to effector cells after anti-PD-L1 treatment, suggesting that anergy maintenance depends on other signaling pathways. This work highlights how the differentiation status of a T cell may predetermine its susceptibility to PD-1 blockade, and has implications for cancer immunotherapy and autoimmunity.
Membrane-bound Dickkopf-1 in Foxp3+ regulatory T cells suppresses T cell-mediated autoimmune colitis
Authors: Chae WookJin , Hao Liming , Henegariu Octavian , Yilmaz Saliha , Bothwell Alfred L M
Published: January 2023

Regulation of immunological tolerance is central to maintain or restore homeostasis. Herein we show that Foxp3+regulatory T cells (Tregs) utilize Dickkopf-1 (DKK-1) to regulate T cell-mediated tolerance in T cell-mediated autoimmune colitis model. Tregs from DKK-1 hypomorphic doubleridge mice failed to control CD4+T cell proliferation, resulting in CD4 T-cell-mediated colitis. Foxp3+Tregs showed a robust expression of DKK-1 among other Wnt ligands. DKK-1 was markedly expressed in activated Tregs but not in naïve or effector CD4 T cells. Tregs expressed DKK-1 markedly in the cell membrane, co-expressing other well-known Treg markers in vivo. We found that T cell receptor stimulation is important to induce DKK-1 expression in vitro and in vivo. DKK-1 expression was regulated by the MAPK pathway and was dependent on de novo protein synthesis, but not by the canonical Wnt pathway, the mTOR or mevalonate pathway. Taken together, our results highlight that Tregs utilize DKK-1 to regulate immunological tolerance to prevent autoimmunity.
A novel image cytometric analysis method for T cell-mediated cytotoxicity of 3D tumor spheroids
Authors: Chan Leo , Humbert Laure , Cribbes Scott
Published: January 2023

Cell-mediated cytotoxicity assays have been frequently performed to characterize cytotoxic potential of immune cells, antibodies, and drug compounds. Traditionally, these assays are performed using release assays such as Cr51 (radioactivity), Calcein (fluorescence), or LDH (enzymatic). However, release assays have limitations such as the handling of hazardous material and the indirect measurement of cell death. These assays are commonly performed in 2D cultures, which have difficulty identifying qualifying drug candidates for clinical testing, thus there has been an interest of performing cytotoxicity assays in 3D tumor models. Traditional 3D spheroid analysis methods require the use of standard microscopy, which is time-consuming and subjective. In this work, we demonstrate a novel method of analyzing T cell-mediated cytotoxicity on 3D tumor spheroids in the presence of absence of ImmTAC molecules, which can promote higher T cell killing. In this experiment, MDA-MB-453 GFP expressing breast cancer cells are used to form tumor spheroids in an ultra-low attachment plate. The spheroids are then treated with primary T cells at different E:T ratios, and different concentrations of ImmTAC. The results showed a dose response effect of T cell killing with the addition of ImmTAC molecules by measuring spheroid size in GFP fluorescence and viability using propidium iodide. The captured bright-field and fluorescent images also clearly showed the cytotoxicity effect from the combination of T cells and ImmTAC. The ability to screen cytotoxic effects of immune cells, antibody, and drug compounds on 3D tumor spheroids can provide an alternative tumor model for identifying more qualified cancer drug candidates for drug discovery campaigns.