bluuebunny/crossref_metadata_2025 huggingface dataset:
Smad signaling determines the fate of activated CTLs via multiple intersecting signaling pathways
Authors: Chandiran Karthik , Suarez-Ramirez Jenny E , Hu Yinghong , Kaech Susan , Cauley Linda S
Published: January 2023

Immune responses to an infection are controlled by various factors including antigens, co-stimulatory molecules and cytokines present in the tissue micro-environment. These factors modulate the differentiation of effector CD8 T cells and memory subsets. TGFβ plays a pivotal role in fate determination of activated CTLs. In TGFβ pathway, Smad proteins are critical for downstream signaling. Using transgenic animal models and pharmacological inhibitors, we investigate the role of TGFβ and Smad proteins in fate determination of activated CTLs. We identified that TGFβ and Smad proteins play important roles in regulating the expression of several homing receptors on effector CD8 T cells (KLRG1) and memory subsets (CD62L, S1PR1 and CD103). In addition, targeted-ablation of different Smad proteins alter the expression levels of different transcription factors that participate in fate determination, including Eomes and KLF2. We found that while Smad proteins regulate the gene expression in canonical TGFβ pathway, they also have TGFβ independent functions in determining the fate of activated CTLs. Particularly, we show that Smad4 is required for the expression of KLRG1 and acts as a suppressor of CD103, independent of TGFβ. This study reveals a novel role for Smad signaling cascade in guiding the fate decisions of activated CTLs and tissue localization via multiple intersecting signaling pathways.
The utilisation of clinically relevant Immunological research examples for undergraduate Medical sciences education
Authors: Sika-Paotonu Dianne , Gu Vivian , Williams Grace , Pinfold Esther , Whyte Julia , Motu Lakin , Anitelea Toni , Yuhoi Jessica , Laing Bridie
Published: January 2023

Immunology education supports the clinical understanding and contextualisation of important cellular processes and mechanisms. To support effective learning and teaching within undergraduate Medical sciences courses, the integration of clinically relevant Immunological course elements and related research teaching examples are needed. The aim was to incorporate a clinically relevant Immunological based research example, presented in class to 4th year undergraduate Medical sciences students. A partial focus of this work was also to help raise awareness of current health issues and priorities facing Māori and Pacific population groups living in New Zealand (NZ) and the Pacific Region. The autoimmune condition known as Acute Rheumatic Fever (ARF), caused by untreated Group A Streptococcal (GAS) infection of the throat (and skin) was discussed in class within the context of regular administration of Benzathine Penicillin G (BPG) that prevents subsequent ARF attacks. Untreated ARF can trigger permanent cardiac damage called Rheumatic Heart Disease (RHD). Rates of ARF in NZ, Australia and the Pacific Region are among the highest in the world, particularly within Māori, Pacific and Indigenous Aboriginal population groups. Student feedback indicated that in-depth interest and understanding was demonstrated using the ARF and RHD Immunological based teaching example. In addition, students were motivated to engage in Summer Scholarship placements focused on the ARF/RHD and BPG based research work. These observations highlight the successful incorporation of a clinically relevant Immunological teaching example that supported the learning and application of scientific research content for undergraduate Medical students.
Characterizing the BCG induced T cell independent mechanisms for defense against Mycobacterium tuberculosis
Authors: Bickett Thomas , Creissen Elizabeth , Angulo Fabiola Silva , Izzo Antonio , Izzo Linda , Izzo Angelo A
Published: January 2023

Mycobacterium bovis Bacille Calmette Guérin (BCG) is a potent immune stimulator that activates innate and adaptive immunity. In the C57BL/6 mouse model of tuberculosis, BCG stimulated immunity causes a significant reduction of M. tuberculosis burden after pulmonary infection. The majority of work has focused on BCG induced T cell immunity as CD4+ T cells play a significant role in protection against tuberculosis. We hypothesized that BCG also induced a T cell independent mechanism that was sufficient to significantly restrict mycobacterial growth. BCG robustly activates innate immunity and our preliminary experiments showed that BCG induced a significant reduction in colony forming units (CFU) in the lungs of mice vaccinated 7 days before pulmonary infection. Our studies indicated that although BCG was administered through subcutaneous inoculation, increased numbers of Ly6C+ monocytes were observed in the lungs within 7 days. Selective depletion also determined that neutrophils played a role in priming innate immunity. Others have identified a role for innate immunity during BCG vaccination termed trained innate immunity. However, our results suggested that at day 7 post BCG vaccination trained innate immunity did not cause the CFU reduction, as the NOD2 receptor or NK cell activation were not absolute requirements, despite being linked to trained immunity. Furthermore, in vivo killing of mycobacterium was dependent on BCG being viable and was monocyte derived, highlighting a novel mechanism. Taken together our data suggest that an early innate mechanism induced by viable BCG is responsible for reducing the mycobacterial burden and precedes trained innate immunity.
The Ca2+ sensor STIM1 regulates the type I interferon response by retaining the signaling adaptor STING at the endoplasmic reticulum
Authors: WOO JIN SEOK , Srikanth Sonal , Wu Beibei , El-Sherbiny Yasser M , Leung Jennifer , Chupradit Koollawat , Rice Laura , Seo Gil Ju , Calmettes Guillaume , Ramakrishna Chandran , Cantin Edouard , An Dong Sung , Sun Ren , Wu Ting-Ting , Jung Jae U. , Savic Sinisa , Gwack Yousang
Published: January 2023

Stimulator of interferon genes (STING) is an endoplasmic reticulum (ER) signaling adaptor that is essential for the type I interferon response to DNA pathogens. Aberrant activation of STING is linked to the pathology of autoimmune and autoinflammatory diseases. The rate-limiting step for the activation of STING is its translocation from the ER to the ER-Golgi intermediate compartment. Here, we found that deficiency in the Ca2+ sensor stromal interaction molecule 1 (STIM1) caused spontaneous activation of STING and enhanced expression of type I interferons under resting conditions in mice and a patient with combined immunodeficiency. Mechanistically, STIM1 associated with STING to retain it in the ER membrane, and coexpression of full-length STIM1 or a STING-interacting fragment of STIM1 suppressed the function of dominant STING mutants that cause autoinflammatory diseases. Furthermore, deficiency in STIM1 strongly enhanced the expression of type I interferons after viral infection and prevented the lethality of infection with a DNA virus in vivo. This work delineates a STIM1-STING circuit that maintains the resting state of the STING pathway.
Lysophosphatidylcholine activates caspase-1 by bringing about release of ATP and sensitization of purinergic receptor
Authors: Ismaeel Sana , Qadri Ayub
Published: January 2023

Lysophosphatidylcholine (LPC) is one of the major damage associated molecular patterns (DAMP) released during inflammation and infection. It generates inflammatory responses from different cell types including macrophages and neuronal cells, and causes demyelination in experimental models of neuroinflammation. LPC activates cellular responses through GPCR dependent and independent signaling. Recently, it has been shown to bring about release of IL-1β through inflammasome-mediated activation of caspase-1. However, the exact mechanism by which LPC stimulates inflammasomes has not been investigated. In this study, we show that LPC triggers release of ATP from cells and activates purinergic signaling, which produces cytotoxicity and caspase-1 – dependent production of IL-1β. LPC activates caspase-1 in a G-αi independent manner. Treatment with apyrase or inhibition of P2X7 receptor reduces LPC - mediated release of LDH and IL-1β. These results identify induction of ATP-release as the critical first trigger by which LPC brings about caspase-1 – dependent inflammatory response.
Novel monoclonal antibodies for the detection of human TLR9
Authors: Acuff Nicole V , Oida Takatoku , Divekar Anagha A , Yang Xifeng
Published: January 2023

Human TLR9 (CD289) is a well characterized mediator of antiviral immunity. TLR9 is primarily activated by unmethylated CpG sequences leading to the production of type I interferons and other inflammatory cytokines. Recently, changes in TLR9 expression in cancer have also been reported. Here we describe novel monocloncal antibodies for the detection of human TLR9 by flow cytometry. These clones recognize intracellular TLR9 in B cells and plasmacytoid dendritic cells (pDCs), as well as on HEK293 cells transfected with human TLR9, and are compatible with multiple fixation and permeabilization buffers. We also show surface expression of TLR9 on pDCs, which is reported to be upregulated following stimulation. In response to CpG-B stimulation, two clones enhance and inhibit TLR9 signaling in a concentration dependent manner leading to alterations in cytokine secretion. Reported here are multiple human TLR9 clones with greater specificity than other commercially available clones.
Induction of innate antiviral immunity through a novel pattern recognition receptor, the RNA helicase DHX16, is regulated by unanchored K48-linked poly-ubiquitin chains
Authors: Hage Adam , Bharaj Preeti , Rajsbaum Ricardo
Published: January 2023

The type-I interferon (IFN-I) pathway mediates antiviral responses and is regulated to balance pathogen clearance while mitigating host damage. Unanchored K48-linked poly-ubiquitin (K48 poly-ub) chains, which associate with targets through non-covalent interactions, have been shown by our group to be a critical regulatory signal in IKKe kinase-mediated antiviral immunity. To identify other components requiring unanchored poly-ub, we developed a novel method to isolate these chains from influenza virus (IAV)-infected mouse lungs. Using this approach combined with mass spectrometry analysis we identified DHX16, an RNA helicase and pre-mRNA splicing component. The role of DHX16 in innate immunity and regulation of its activity have not been described. We found that during IAV infection, the levels of unanchored K48 poly-ub that associate with DHX16 are significantly increased and these DHX16-interacting chains are synthetized by the E3-ubiquitin ligase TRIM6, which we have previously shown to be critical in IFN-I and antiviral responses. Next-Generation sequencing identified a number of IFN-stimulated genes whose expression was significantly downregulated in DHX16 knockdown cells. Consistent with this, overexpression of DHX16 induced IRF3 phosphorylation, which can be further enhanced upon IAV infection. Furthermore, the ATPase activity of DHX16 is enhanced upon addition of unanchored K48 poly-ub only in the presence of viral RNA suggesting DHX16 first recognizes viral RNA and unanchored poly-ub regulate its activity. Together these data suggest DHX16 acts as a novel pattern recognition receptor involved in the IFN-I pathway and its activity is regulated by unanchored K48 poly-ub.
NME4 regulates TLR3 activation and type I IFN production through autophagy-dependent TRAF6 degradation
Authors: Sun Bing , Zhao Xin Hao , Zhang Rong Hua
Published: January 2023

Nucleoside diphosphate kinase (NDPK)/Nm23 family proteins play multiple roles in energy metabolism, proliferation, differentiation, inflammatory responses and tumour invasion. Among these proteins, only NDPK-D (NME4) is located in the mitochondrial luminal space and the inner mitochondrial membrane. NME4 shows high affinity for cardiolipin (CL) and facilitates its redistribution to the outer mitochondrial membrane (OMM), which leads to mitophagy and inflammatory regulation. Here, we demonstrate that NME4 can regulate the type I interferon pathway by modulating the TLR3-TRAF6-IFN axis. NME4 deficiency promotes the degradation of TRAF6 and impairs the phosphorylation of p65 and p38. Overexpression of NME4 significantly upregulates the interferon production induced by a TLR3 ligand. However, when the NEM4 signal peptide for mitochondrial localization is deleted, the regulation of IFN production is abolished. We demonstrate that NME4 deficiency-mediated suppression of interferon production is rescued when ATG5 is knocked down following TLR3 stimulation, suggesting that NME4 is able to regulate interferon production through the autophagy pathway. Furthermore, influenza A virus-infected NME4 knockout mice showed higher mortality, which was associated with reduced interferon production compared to that in wild-type mice. Taken together, our results indicate that NME4 plays an important role in regulating TLR3-mediated interferon production.
Mutation of Tgfbr1 leads to non-hematopoietic defects that drive early onset eosinophilic inflammation in the esophagus
Authors: Laky Karen , Kinard Jessica L , Guerrerio Anthony , Li Min Jenny , Guerrerio Pamela
Published: January 2023

Eosinophilic esophagitis is a chronic disease characterized by tissue-restricted eosinophilia, epithelial basal cell hyperplasia, and esophageal dysfunction. There are three endotypes of eosinophilic esophagitis which are classified based upon the age of disease onset, degree of fibrostenosis, and gene expression patterns. Genetic studies have identified variants that correlate with an increased likelihood to develop eosinophilic esophagitis. Among the top ten variants, nearly all occur in genes expressed by epithelial cells, and approximately one-third encode proteins involved in the transforming growth factor beta (TGFβ) signaling pathway. The pathophysiologic mechanisms underlying these genetic risk factors are poorly understood. Using mice engineered to have an autosomal dominant loss-of-function mutation in the kinase domain of Tgfbr1 we demonstrate that altered TGFβ signaling in non-hematopoietic cells is necessary and sufficient to cause disease that clinically, immunologically, histologically, and transcriptionally recapitulates human eosinophilic esophagitis endotype 2. Tgfbr1 mutation impairs esophageal epithelial cell homeostasis resulting in tissue-restricted increases in proteins that act directly, indirectly, and synergistically to drive accumulation and activation of eosinophils, mast cells, and type 2 innate lymphoid cells (ILC2). These data suggest new ways to approach the prevention and treatment of eosinophilic esophagitis.
Comparison of the suppressive function of alloantigen-reactive Treg cells and non-stimulated Treg cells in allogeneic skin transplantation
Authors: Kim Yong-Hee , Shevach Ethan M.
Published: January 2023

Adoptive cellular therapy with Treg cells is being evaluated in numerous clinical trials in GvHD and organ transplantation. Cellular biotherapy with Treg cells requires ex vivo expansion. While alloantigen stimulated Treg cells have been used in several studies, very few studies have directly compared the efficacy of alloantigen-reactive with non-stimulated Treg cells in vitro and no studies have compared their efficacy in vivo in organ transplantation. In this study, we obtained enriched population of alloantigen-reactive Treg by stimulating proliferation dye-labeled C57BL/6 CD4+Foxp3+CD25+ Treg cells with allogeneic H-2bm12 dendritic cells in the presence of IL-2. After 5~7 days, the alloantigen-reactive Treg cells and resting Treg cells were separated by FACS. Treg cells that had proliferated in response to alloantigen stimulation were more potent suppressors of the reactivity of C57BL/6 CD4+Foxp3− T cells to H-2bm12 stimulators than non-reactive Treg cells. In contrast, both Treg populations equally suppressed the response of C57BL/6 CD4+Foxp3− T cells to 3rd party BALB/c dendritic cells. Both alloantigen-reactive and non-reactive Treg cells were co-transferred with C57BL/6 CD4+Foxp3− T cells (1:2 ratio) to immunodeficient recipients followed by transplantation of allogeneic H-2bm12 and 3rd party BALB/c skin. Recipients of alloantigen-reactive Treg cells demonstrated delayed rejection (26 days -> 49 days) of H-2bm12 skin, but normal rejection of 3rd party skin. Taken together, this protocol offers a simple, rapid method for enrichment of alloantigen-reactive Treg with enhanced antigen-specific suppressive function in vitro and in vivo.

This research was supported by the Intramural Research Program of NIAID, NIH.
p40 Homodimers Indirectly Induce Endogenous Donor-Reactive Memory CD8 T Cell Proliferation within High-Risk Allografts via IL-15 signaling
Authors: Fairchild Robert L , Valujskikh Anna , Tsuda Hidetoshi
Published: January 2023

Increasing cold ischemic storage (CIS) time from 0.5 to 8 hrs prior to transplant promotes the increased proliferation and effector functions of endogenous donor-reactive memory CD4 and CD8 T cells infiltrating mouse cardiac allografts 24–48 hrs after transplant. This increased mCD8 T cell proliferation within high-risk allografts requires mCD4 T cell help via CD40-CD154 interactions with graft dendritic cells to induce IL-12p40 homodimer (p40 HD) production required for this mCD8 T cell proliferation. This study tested if p40HD directly or indirectly stimulate the mCD8 T cell proliferation within high risk allografts. While endogenous mCD8 T cells within highly ischemic allografts expressed both IL12Rβ1 and β2, most proliferating CD8 T cells did not express the IL12Rβ1 needed for p40 HD mediated signaling. Moreover, endogenous mCD8 T cells isolated from allografts did not proliferate ex vivo when cultured with p40 HD. qPCR analysis indicated increased mRNA expression of IL2Rα (CD25), β(CD122) and IL15 Rain infiltrating mCD8 T cells purified from allografts subjected to prolonged vs. minimal CIS on day 2 post-transplant. ELISA indicated longer CIS time markedly increased IL-12p40 and IL-15 protein in the allografts and these increases were dependent on recipient CD4 T cells. Blocking of p40 or IL-15 signaling with anti-p40 or anti-CD122 mAb inhibited endogenous mCD8 T cell proliferation within high-risk cardiac allografts and extended the survival of the high-risk allografts from day 18 to day 54 in CTLA-4Ig conditioned recipients. These results suggest that the p40 HD stimulate proliferative cytokines such as IL-15, which directly stimulate endogenous donor-reactive mCD8 T cell proliferation within the higher-risk allografts.
Exopolysaccharide from a commensal bacterium improves outcome of graft versus host disease by decreasing donor T cell activation
Authors: Kalinina Olga , Talley Sarah , Paik Wonbeom , Campbell Edward M , Knight Katherine L
Published: January 2023

We previously showed that exopolysaccharide (EPS) produced by a commensal bacterium, Bacillus subtilis, induces alternatively-activated, anti-inflammatory M2 macrophages that protect mice from T cell-dependent inflammatory disease, induced by the enteric pathogen Citrobacter rodentium. Here, we show that EPS induces clinical improvement in another T cell-mediated disease, graft versus host disease (GVHD). GVHD is a common complication of allogenic bone marrow transplantation and is caused by the activation of alloreactive donor T cells. To monitor the inflammatory response of allogeneic bone marrow cells in recipient mice, we used donor cells from transgenic mice that express a luciferase-based biosensor that is cleaved by caspase-1, a protein activated during inflammation. We found that donor inflammatory responses and T cell activation was decreased in EPS-treated mice. Our previous data indicate that EPS does not have a direct effect on T cells. To identify the cell type affected by EPS, we tested the activity of EPS on bone marrow-derived dendritic cells (BMDCs) and found that these cells have upregulated expression of the inhibitory molecule, PD-L1, and the cells inhibited alloreactive T cell activation and proliferation in mixed lymphocyte reactions (MLR). We also found that in vivo injection of EPS resulted in the upregulation of PD-L1 expression on splenic dendritic cells. We conclude that EPS ameliorates clinical manifestations of GVHD by decreasing the activation of donor T cells, and we suggest that EPS affects T cells by inducing inhibitory dendritic cells.
Human monocytes educated with exosomes from TLR4 primed mesenchymal stem cells treat acute radiation syndrome by promoting hematopoietic recovery
Authors: Forsberg Matthew H , Kink John , Hematti Peiman , Capitini Christian M.
Published: January 2023

Total body irradiation is often used as a conditioning regimen for bone marrow transplants but can cause life threatening damage to host tissues especially the bone marrow. Developing a cellular therapy that can protect the bone marrow from acute radiation syndrome and stimulate hematopoiesis is a priority for patients exposed to therapeutic or even accidental radiation injury. In this study, exosomes derived from MSCs stimulated with the TLR4 agonist lipopolysaccharide (LPS) were used to alternatively activate human monocytes, termed LPS EEMos, as a potential novel radioprotective cellular therapy. LPS EEMos expressed higher levels of PD-L1 (p<0.0001), and lower levels of CD16 (p<0.01), CD86 (p<0.01), and CD206 (p<0.0001) by flow cytometry compared to monocytes educated with exosomes from unstimulated MSCs (EEMos). Using qPCR, increased gene expression in LPS EEMos of IL-10 (p<0.05), IDO (p<0.001), FGF2 (p<0.05), IL-15 (p<0.05), and IL-6 (p<0.0001) were detected compared to EEMos. Using a xenogeneic radiation injury model, infusion of human LPS EEMos 4 hours after lethal radiation led to reduced clinical scores and an increased survival at 40 days post-infusion, as compared to infusions of PBS, EEMos, and monocytes alone, all of which led to worse clinical scores and 0% survival with uniform death by 20 days (p<0.05). Complete blood cell counts in LPS EEMo recipients showed leukocyte, erythrocyte and platelet counts equivalent to non-irradiated mice, demonstrating complete restoration of hematopoiesis. Infusion of LPS EEMos may be a useful strategy to protect the bone marrow from acute radiation syndrome by expression of anti-inflammatory molecules and cytokines that promote hematopoiesis/engraftment.
Extracellular mitochondria Released from Hepatic Ischemia Reperfusion Induce Macrophage activation both in vitro and in vivo
Authors: Hu Qianni , Haidl Ian D. , Marshall Jean S , Alwayn Ian P.J.
Published: January 2023

Background

During liver transplantation ischemia and reperfusion injury (IRI), an immune driven inflammatory response provoked by cellular oxygen deprivation, is unavoidable. The inflammatory responses, resulting from acute oxidative stress and consequent hepatocellular death during the early reperfusion phase, cause the release of endogenous danger signals. Recent studies show that the release of free mitochondria (FM) from deceased organ donors is directly linked with functionally important immune activation and early allograft dysfunction (Pollara, et al., 2018). Organs from donors after cardiocirculatory death (DCD) undergo a prolonged warm ischemia process compared with organs from donors after neurological determination of death (NDD), and therefore have more severe IRI. Herein, we investigated the roles of FM in liver IRI, particularly as related to macrophages.

Methods

Liver biopsies were assayed by TEM to assess for FM release. FM were co-cultured with macrophages (cell line, bone marrow derived macrophages, and primary Kupffer cells), and injected i.p. in vivo to explore the recruitment and activation of macrophages. Inflammatory mediators were tested by luminex.

Results

FM levels were significantly higher in IRI. Co-culture of FM with macrophages significantly increased inflammatory mediator release but this process was not Formyl Peptide Receptor 1 dependent. The injection of FM induced significant accumulation of monocyte-derived macrophages and activation of macrophages in the peritoneum.

Conclusion

Our results indicate that IRI can elicit significantly increased FM, and that FM from hepatic IRI can attract and activate macrophages, which may influence the outcome of liver transplantation.
Two ICD Inducers are better than one for induction of anti-tumor immunity in cold carcinomas
Authors: Jessup J. Milburn , Kabbout Mohamed , Henderson Paul T. , Hewitt Stephen , Mattoo Abid R.
Published: January 2023

Background

Cytotoxic agents and viruses cause Immunogenic Cell Death (ICD), which initiates a primary immune response in tumor-bearing hosts by releasing cellular antigens with immune chaperones. We postulate that two ICD inducers are better than one in immune competent mouse models of colorectal and breast cancer.

Methods

Approximately 6 mm diameter CT26 rectal or 4T1 breast carcinomas were established in groups of 5–7 BALB/c mice and injected once intratumorally (IT) with oxaliplatin (OX) mixed with a human adenovirus AdV optimized for calreticulin externalization in 3-D spheroids of human carcinomas. Tumor volumes were measured every other day and rate of growth analyzed by Rate-Based T/C Ratio at 19–21 days. Tumor-Infiltrating Lymphocytes (TIL) and nodal cytotoxicity were assessed 11 days after treatment.

Results

In 3 experiments with OX-AdV IT in PBS buffer the T/C ratios were 0.17–0.29 those of the volume of PBS IT injected mice. The T/C Ratio for OX-AdV IT in D5W (used to stabilize the OX) was 0.10 of the IT control volume with 0.32–0.40 for OX or AdV alone and with all groups different by P<0.05 by 2-way ANOVA. OX-AdV IT increased TIL by 4-fold and decreased CD8+PD-1+EOMES+ from 79% to 19% with a dramatic influx of CD8+PD-1-Eomes- T cells compared to PBS IT controls. Draining node lymphocytes were cytotoxic to CT26 cells from OX-AdV IT mice while lymphocytes from PBS IT mice were not. OX-AdV IT in D5W caused 40% of mice to be complete responders that later rejected a contralateral challenge of CT26.

Conclusions

The buffer for IT treatment with OX is critical. The two ICD inducer formulation with D5W is promising as a checkpoint therapy sensitizer for carcinomas that lack TIL or resist checkpoint inhibitor therapy like colorectal carcinoma.
Optimization of the T2 HLA-A2 shift assay for testing of the biological activity of immunotherapies
Authors: Tram Cynthia , Hrytsenko Olga , Stanford Marianne
Published: January 2023

T2 is a lymphoma-derived cell line that express low amounts of HLA-A2 on the cell surface due to TAP deficiency and can only present exogenous peptides. Binding of exogenous peptides to HLA-A2 stabilizes the HLA-A2- peptide complexes and can be detected using immunofluorescence staining. The T2 cell line is commonly used for identification and analysis of novel HLA-A2-restricted CTL epitopes in the assay known as T2 shift. In this study we evaluated parameters that may influence the T2 shift assay performance and considered new applications of this method for testing of the biological activity of peptide-based immunotherapies. Method optimization was performed using HLA-A2-restricted peptide SurA2.M alone and in the context of a peptide-based immunotherapy drug, DPX-Survivac. DPX-Survivac is a lipid nanoparticle-based formulation that contains five peptides aiming to elicit a CTL response against tumor cells presenting the survivin peptides on HLA class I molecules. Results indicated that performance of the T2 shift assay was highly sensitive to cell passaging intervals, pH variations, cell concentrations and β2-microglobulin concentrations. Discrimination of the T2 cells by either size or granularity did not affect the results for the T2 shift assay. We determined the linear response range for the SurA2.M peptide to be 30-110 μg/mL in the T2 shift assay. The T2 shift assay can detect SurA2.M within the DPX-Survivac matrix. In conclusion, we found that optimal culturing conditions of T2 cells are critical for the T2 shift assay reproducibility and linearity and should be carefully considered if the assay is to be used for quantitative or semi-quantitative evaluation of the peptide activity alone or as a part of an immunotherapeutic drug.
Vasoactive intestinal peptide signaling – a novel immune checkpoint in pancreatic ductal adenocarcinoma
Authors: Ravindranathan Sruthi , Li Yiwen , Wang Shuhua , Zaidi Mohammad , Lesinski Gregory B , El-Rayes Bassel , Waller Edmund K
Published: January 2023

Pancreatic ductal adenocarcinoma (PDAC) is projected to become the second leading cause of cancer related death in the U.S. by 2030. Being completely refractory to checkpoint therapies, management of PDAC remains an unmet clinical need. Intriguingly, the human protein atlas (THPA) shows elevated mRNA expression of vasoactive intestinal peptide (VIP), a neurotransmitter in exocrine pancreatic cancers. Interestingly, VIP also has immunosuppressive properties leading to decreased T cell proliferation and enhanced expansion of myeloid derived suppressor cells (MDSCs) and regulatory T cells (Tregs). Thus, we are investigating the role of VIP in PDAC progression and the effect of inhibiting VIP signaling for improved responsiveness to immunotherapy.

We found that consistent with THPA, cell culture supernatants collected from murine PDAC cell lines had significantly higher levels of VIP as compared to melanoma and breast cancer cells. Similarly, elevated levels of VIP were observed in mice bearing PDAC tumors, and in plasma from human PDAC patients, when compared to naïve mice and healthy volunteer samples, respectively. Treatment of immune competent mice bearing PDAC tumors (MT5 or KPC cell lines) with the combination of a VIP antagonist (VIPhyb) and anti-PD1, produced complete and durable regression of tumors in 20% of the mice and an overall delayed tumor progression in both tumor models. Upon analysis of the tumor tissues sections via immunohistochemistry, significant infiltration of CD8+ T cells was observed in mice treated with the combination of VIPhyb and anti-PD1. Further experiments are underway to understand the mechanism of action of VIP antagonist and to devise methods to improve its efficacy in-vivo.
DeepTM IL-12 Priming enhances tumor-specific T cell efficacy and inflammatory activity in tumors without eliciting systemic toxicities
Authors: Sauer Karsten , Chang De-Kuan , Ahmad Gulzar , Nardozzi Jonathan , Sackton Kate , Lyons Jesse , Andresen Thomas , Jones Douglas
Published: January 2023

Interleukin-12 (IL-12) is a potent pro-inflammatory cytokine that augments anti-tumor immune responses by promoting CD4 T cell Th1 differentiation, increasing CD8 T cell and NK cell cytotoxicity, inducing MHC expression on antigen presenting cells, and reprogramming myeloid-derived suppressor cells. However, the clinical utility of IL-12 has been limited by systemic toxicities. DeepTM IL-12 is a fusion protein between IL-12 and a Fab antibody against CD45, an abundant surface receptor on T cells. This enables tethering of IL-12 onto T cells prior to adoptive cell transfer (ACT). We report here the superior safety and efficacy of ACT with tumor-specific Deep IL- 12 Primed T cells which carry surface-tethered IL-12.

The PMEL/B16-F10 cancer model utilizes adoptively transferred PMEL CD8 T cells that contain a TCR specific for the gp100 antigen expressed in B16-F10 melanoma cells. Deep IL-12 tethering to PMEL T cells significantly improved tumor growth inhibition and survival in tumor-bearing mice compared to PMEL T cell therapy alone or combined with systemically delivered IL-12. Doses up to 40 million Deep IL-12 Primed T cells did not result in overt toxicities. Thus, in contrast to systemically delivered free IL-12, T cell tethered IL-12 is well-tolerated. Deep IL-12 Primed PMEL T cells additionally resulted in elevated and sustained induction of IFNγ in the tumor microenvironment. In contrast, IFNγ induction in serum was transient and returned to baseline levels within four days of ACT. Further toxicological analyses will be discussed.

In conclusion, ACT with Deep IL-12 Primed T cells delivers a favorable therapeutic profile by promoting T cell function selectively in the tumor microenvironment while limiting systemic IL-12 exposure.
Impact of HIV-1 infection and antigen class on T follicular helper (TFH) cell responses to pneumococcal polysaccharide-protein conjugate vaccine (PCV-13)
Authors: Jha Vibha , Nicholson Lindsay K , Gardner Edward M , Rahkola Jeremy T , Pratap Harsh , Janoff Edward N
Published: January 2023

Pneumococcal infections are common and serious complications of HIV-1 disease. Prevention has been compromised by the limited magnitude and quality of antibody responses to T cell-independent (TI-2) pneumococcal capsular polysaccharides (PPS). The PCV-13 vaccine conjugates PPS to a T cell-dependent (TD) protein (diphtheria toxoid; DT). We investigated the differential response to PPS and DT by antibody-secreting B cells (ASC) after immunization with PCV-13 in newly-diagnosed healthy HIV+ and control adults. The numbers of PPS-specific IgG ASC increased significantly and similarly in HIV+ and controls 1 week after vaccination. However, DT-specific IgG ASC increased significantly in controls but not HIV+ subjects. To determine the cellular basis of these disparate responses to DT and PPS, we characterized the frequency and activation of T follicular helper (TFH) cells, the predominant T cell subset providing B cell help. The percentages of and intracellular IL-21 in TFH cells were unchanged with vaccination. However, expression of ICOS, which sustains TFH function and phenotype, increased significantly after vaccination among controls, but not the HIV+ group. Increases in ICOS+ TFH correlated with changes in TD DT-specific IgG ASC in controls but not in HIV+. In contrast, ICOS expression did not correlate with TI-2 PPS-specific ASC in either group. Levels of TFH differentiating factors IL-12 and IL-6 increased post vaccination only in controls. In summary, although TI-2 PPS-specific responses were similar in both groups and independent of TFH activation, TFH ICOS-expression was most closely related to responses to TD DT, which were compromised even in recently diagnosed healthy HIV+ adults.
Skin recirculating B cell subsets in a model of lymph cannulation in sheep
Authors: Hunka Julia , Riley John T. , Debes Gudrun F.
Published: January 2023

The skin is a key barrier organ and cells of the skin immune system are integral to skin immunity and inflammation. While the trafficking of skin T cells is well defined, less is known about B cells that reside in and traffic through skin. Here we studied skin-associated B cells and their homing receptors using lymph vessel cannulation in sheep. Afferent lymph draining the skin was collected and lymph-borne B cells were compared to B cells from skin, blood, skin and mesenteric lymph nodes, mesenteric efferent lymph, and spleen using flow cytometry. The skin-homing molecule α4β1-integrin as well as activated β1-integrin were upregulated in B cells in skin-draining lymph and skin compared with lymph nodes, suggesting that α4β1 integrin is key to B cell recirculation through and/or homing to skin. Of all tissues analyzed, B cells from skin exhibited the highest frequency in skin-homing molecule E-selectin ligand expression. Among afferent lymph B cells, the CD21− B cell subset showed the highest expression of E-selectin ligand. As recent studies showed a functional endocannabinoid system in the skin, we assessed chemotaxis of skin-recirculating B cells toward 2-arachidonoylglycerol (2-AG), a ligand for cannabinoid receptor 2 (CB2). Interestingly, CD21+ B cells from skin draining lymph were highly enriched in responsiveness to 2-AG as well as CCL20, a skin-expressed chemokine. We propose that α4β1 integrin is integral to all B cell homing to skin and that CD21 expression delineates skin-recirculating B cell subsets with distinct trafficking receptor usage, while CD21− B cells use E-selectin ligand, CD21+ B cells employ receptors for 2-AG and CCL20 to recirculate through skin.
Aerosol vaccination with Bacille Calmette-Guerin induces a trained innate immune phenotype in calves
Authors: Guerra-Maupome Mariana , McGill Jodi L.
Published: January 2023

M. bovis Bacillus Calmette-Guérin (BCG) is a live attenuated vaccine for use against tuberculosis (TB). BCG is known to reduce childhood mortality from infections other than TB. The unspecific protection induced by BCG has been associated with the induction of memory-like traits identified as ‘trained’ immunity. In humans and mice, in vitro and in vivo BCG training leads to enhanced production of proinflammatory cytokines in response to secondary unrelated pathogens. While BCG nonspecific protective effects have been studied extensively in humans and mice, it has not been defined in agricultural species. Here, we show that in vitro BCG training induces increased transcription of proinflammatory cytokines upon restimulation with the TLR agonists. Importantly, in vivo, aerosol BCG vaccination of calves induced a ‘trained’ phenotype in PBMCs, that lead to a significantly enhanced TLR-induced proinflammatory cytokine response compared to PBMCs from unvaccinated calves. Similar to the long-term training effects of BCG reported in humans, our results suggest that in young calves, the effects of BCG induced innate training last for at least 3 months in circulating immune cells. Interestingly, however, aerosol BCG vaccination did not ‘train’ innate cells at the mucosal level, as alveolar macrophages from aerosol BCG vaccinated calves did not mount an enhanced inflammatory response to secondary stimulation, compared to cells isolated from control calves. Together, our results suggest that, like mice and humans, the innate immune system of calves can be ‘trained’; and that BCG vaccination could be used as an immunomodulatory strategy to reduce disease burden in juvenile food animals before the adaptive immune system has fully matured.
A comprehensive map of the human dendritic cell HIV-response transcriptional network
Authors: Johnson Jarrod S , De Veaux Nicholas , Rives Alexander W , Lahaye Xavier , Lucas Sasha Y , Pérot Brieuc , Luka Marine , Amon Lynn M , Watters Aaron , Aderem Alan , Manel Nicolas , Littman Dan R , Bonneau Richard , Ménager Mickaël M
Published: January 2023

Transcriptional programming of the innate immune response is pivotal for host protection. However, the transcriptional mechanisms that link pathogen sensing with innate activation remain poorly understood. During infection with HIV-1, human dendritic cells (DCs) can detect the virus through an innate sensing pathway leading to antiviral type I interferon and DC maturation. Here, we have developed an iterative experimental and computational approach to map the innate response circuitry during HIV-1 infection. By integrating genome-wide chromatin accessibility with expression kinetics, we have inferred a gene regulatory network that links 542 transcription factors (TFs) with 21,862 target genes. Through genetic perturbation and drug treatments we identify PRDM1 and RARA as essential regulators of the interferon response and DC maturation, respectively. Our work provides a resource for interrogation of regulators of HIV replication and innate immunity, highlighting the complexity and cooperativity in the regulatory circuit controlling the DC response to HIV-1 infection.
Crosseactivity of flaviviruses specific CD8+T cell responses across different viral species
Authors: Grifoni Alba , Voic Hannah , Sidney John , de Silva Aruna D , Durbin Anna , Diehl Sean A , Harris Eva , Sette Alessandro , Weiskopf Daniela
Published: January 2023

Several flaviviruses, including Dengue Virus (DENV), Zika Virus (ZIKV), Japanese Encephalitis Virus (JEV), West Nile Virus (WNV), and Yellow Fever Virus (YFV), share significant sequence homology and often circulate in the same geographical regions. Significant levels of cross-reactivity could in turn result in pre-existing T cell immunity modulating T cell responses to subsequent flavivirus infections or vaccination. Whether and to what extent cross-reactivity at the level of CD8 responses is detected is currently unclear. Thus we designed pools of epitopes and predicted HLA binding peptides derived from DENV, ZIKV, JEV, WNV and YFV. We then used PBMC of individuals vaccinated with DENV or YF to test their potential to recall antigen specific CD8 memory T cell response in an Intracellular Cytokine Staining (ICS) assay. Significant cross-reactivity of CD8 T cell responses against several of the pools was observed both in the case of DENV and YF vaccinees, but the extent of cross-reactivity varied as a function of the flavivirus species considered, and the cross-reactive responses were significantly lower than the responses to the autologous virus. Phenotypic analyses showed a suboptimal expression of activation markers in cross-reactive responses. We are currently characterizing cross-reactive responses at the single epitope level, and in PBMCs from donors naturally exposed to flaviviruses. Characterization of the extent and functionality of CD8 cross-reaction across different flaviviruses will contribute to the understanding of immunity in the natural infection, and has particular implications for vaccine efficacy and safety in endemic settings.
Targeted protein degradation via NEDD8 conjugation does not enhance direct MHC class I antigen presentation
Authors: Vijayasimha Kartikeya , Leestemaker-Palmer Amy L. , Gibbs James S. , Yewdell Jonathan W , Dolan Brian P
Published: January 2023

MHC class I molecules present peptide antigens derived from proteins synthesized and degraded within the antigen presenting cell. An understanding the protein degradation pathways that contribute to peptide generation is necessary for developing next generation immunotherapies. We recently discovered that conjugation of the ubiquitin-like molecule NEDD8 to the N-terminus of a protein can result in the rapid degradation of the target protein by either the ubiquitin-proteasome system (UPS) and the autophagy pathway. To determine if protein NEDDylation increased peptide presentation, we fused NEDD8 to the N-terminus of a cytosolic form of ovalbumin (OVA) and measured presentation of the SIINFEKL peptide via the murine MHC class I molecule Kb using a monoclonal antibody specific for the Kb-SIINFEKL complex. Like other constructs we have characterized, NEDDylated OVA was rapidly degraded by the UPS and autophagy pathways. While proteasome inhibition prevented presentation of SIINFEKL peptides, inhibiting autophagy did not affect antigen presentation. We then compared presentation of NEDDylated OVA to ubiquitin conjugated OVA and found that ubiquitin conjugation was far more effective at generating antigenic peptides. These data demonstrate that rapid degradation per se of an antigenic protein is not sufficient to enhance antigen presentation but rather the precise manner in which a protein is degraded governs the efficiency of peptide generation. Our findings are consistent with the idea that protein degradation is organized in some way to optimize generation the immunopeptidome for immunosurveillance.
Isoflavone Rich Diet Ameliorates Experimental Autoimmune Encephalomyelitis (EAE) through Modulation of Gut Bacteria Depleted in Multiple Sclerosis Patients
Authors: Freedman Samantha N , Shahi Shailesh K , Zarei Kasra , Gupta Arnav , Guseva Natalya V , Bossler Aaron D , Legge Kevin L , Mangalam Ashutosh K
Published: January 2023

Multiple sclerosis (MS), an autoimmune disease of the central nervous system, has been shown to be influenced by gut bacteria. We and others have shown that MS patients have depletion of isoflavone-metabolizing bacteria compared to healthy individuals. We hypothesize that gut bacteria-mediated isoflavone metabolism induces an anti-inflammatory state and reduction/depletion of these gut bacteria might predispose to MS development and/or severity. To test this, we placed mice on a diet with or without isoflavones and induced EAE, an animal model of MS. Mice on an isoflavone diet developed milder disease compared to those on an isoflavone-free diet. This phenomenon was dependent on the presence of isoflavone-metabolizing gut bacteria as confirmed by antibiotic depletion and bacterial reconstitution. Mice on an isoflavone diet exhibited altered peripheral immune responses, CNS cellular infiltration, and CNS pathology post EAE induction compared to mice on an isoflavone-free diet. Additionally, the gut microbiome of mice on an isoflavone diet and an isoflavone-free diet closely resembles the gut microbiota of healthy individuals and MS patients, respectively. Thus our results suggest that dietary isoflavone metabolites produced with the help of gut bacteria can influence the disease through modulation of the immune system.
A newly discovered dual expresser lymphocyte that clonally expanded in Type 1 diabetes (T1D) patients secretes a public antibody that recognize public TCR in T1D
Authors: Ahmed Rizwan , Omidian Zahra , Giwa Adebola , Karakus Kagan E , Majety Neha , Yang Angela , Zhang Hao , Rabb Hamid , Jie Chunfa , Donner Thomas , Hamad Abdel R.A.
Published: January 2023

PURPOSE

We have recently discovered a new lymphocyte that co-express BCR and TCR (Ahmed et al, Cell, 2019: 177:11583) and referred as X cell to denote its crossover phenotype. Importantly, X cells express a public BCR that also encodes a potent autoantigen in its CDR3 sequence that is 10 fold more potent than native insulin peptide (InsB:9–23) in binding to DQ8 and activating autologous CD4 T cells. The x-autoantigen cross-activate insulin specific CD4 T cells as a peptide in the context of HLA-DQ8 molecules or as a soluble intact mAb (x-mAb). The goal of this study is to characterize autoreactive CD4 T cells that are responsive to x-mAb to determine their phenotype, cytokine profile and TCR repertoire and whether they express public TCRs.

METHODS

We used EBV-lymphoblastoid X cell clone as a source of x-mAb (IgM) and FACS based protocol to identify IgM reactive CD4 T cells (referred as IgMpos) and their functional properties. ImmunoSEQ assay used to characterize TCR repertoires.

RESULTS

Preliminary data show that frequency of IgMpos CD4 T cells is significantly higher in T1D as compared to Healthy subjects. In addition, IgMpos CD4 T cells exhibit an activated phenotype as compared to autologous IgMneg CD4 T cells, including expression of CD45RO, CD44, and CD69. Analysis of TCRVβ repertoire shows that IgMpos CD4 T cells are enriched for public clonally expanded TCRs as compared to IgMneg counterparts.

CONCLUSIONS

X cells in T1D patients are predominated by a single public BCR and that the secreted version of this BCR (x-mAb) is autoreactive against a specific subset of CD4 T cells that predominated by few clonotypes that express public TCRs. Our results are revealing previously unknown mechanism that appears to be a play critical role in pathogenesis of T1D.