bluuebunny/crossref_metadata_2025_split huggingface dataset:
Dual antigen transfer pathways mediate highly efficacious anti-tumor immunity induced by intravenously injected antigen-loaded monocytes
Authors: Huang Min-Nung , Nicholson Lowell , Batich Kristen , Kopin David , Prabhakar Vijay , Lipes Barbara , Sampson John , Gunn Michael 
Published: January 2023

Abstract
Dendritic cells (DC) have been key elements in developing effective cancer vaccines to induce potent anti-tumor immunity. However, the common approach adopted in clinical trials using ex vivo generated DC loaded with tumor antigens (Ag) has been challenged by its limited clinical response, complexity, and quality of the manufacturing process. Alternative efforts focused on in vivo Ag loading on endogenous DCs have not yet been well validated in their efficacy for cancer treatment, suggesting the efficiency of in vivo Ag transfer from Ag-carrying vehicles to DCs needs to be further improved. As an attempt to bypass the aforementioned problems, we are testing the hypothesis that Ag-loaded monocytes can induce efficacious anti-tumor T cell responses by directly presenting Ag to T cells through in vivo monocyte-to-DC differentiation. We find that intravenously (IV) injected monocytes loaded with either model or known tumor Ag induce robust Ag-specific CD4 and CD8 T cell responses in mice without triggering antibody responses, display anti-tumor efficacy superior to that seen with traditional adjuvants or RNA-pulsed DC vaccines, and can be combined with checkpoint blockade to increase their efficacy. Unexpectedly, monocyte-induced T cell responses do not require Ag presentation by monocytes themselves, but rather the transfer of Ag to endogenous splenic DC. This transfer occurs via gap junctions for CD8 T cell responses and via macrophages for CD4 T cell responses. These findings demonstrate the existence of efficient dual Ag transfer pathways between monocytes and splenic DCs and suggest that the administration of Ag-loaded monocytes may serve as a simple and efficacious immunotherapeutic platform for the treatment of human cancers.
PCI-based vaccination: a novel method for targeting of MHC class I antigen presentation and stimulation of antigen-specific cytotoxic T cells
Authors: Varypataki Eleni Maria , Kündig Thomas M. , Johansen Pål 
Published: January 2023

Abstract
Cytotoxic CD8+ T cells are key players in fighting cancer and infectious diseases, hence, the design of therapeutic vaccines focuses on ways to prime strong cytotoxic immune responses. Yet, current vaccines efficacy is limited mainly due to the default processing of exogenous antigens via the MHC class II pathway and the activation of CD4+ and not CD8+ T cells. Photochemical internalization (PCI) is a novel intracellular delivery approach, able to circumvent the MHC class II processing. Briefly, after endocytosis of antigen and photosensitizer by antigen presenting cells, light activation of the photosensitizer causes disruption of endosomal membrane and antigen release into the cytosol, resulting in stimulation of MHC class I-restricted CD8+ T cells.
Wild type and MHC class II-deficient mice were immunized intradermally with protein antigens and photosensitizer, followed by controlled light exposure for activation of the photosensitizer. The effectiveness of the PCI to stimulate CD8+ T cells was analyzed by measuring frequency, phenotype, and function of antigen-specific T cells in blood and spleens. PCI-treated mice showed a strong activation and proliferation of specific CD8+ T cells, with improved functions in terms of IFN-g production and cytotoxic capacity. The CD8+ T cell effector functions were not impaired in MHC class II-deficient or in CD4+ T cell-depleted mice, suggesting that the PCI-mediated antigen processing is independent of MHC class II and CD4+ T helper cells.
Our data demonstrate that PCI–based vaccination can stimulation strong and functional cytotoxic CD8+ T cells, making PCI a potential method for use in therapeutic vaccines against cancer and infectious diseases.
Humanized HLA-DQ8αβ transgenic mice treated with SEG/SEI superantigens exhibit long-term CD4+-mediated anti-tumor responses.
Authors: Knopick Peter Leo , Alvine Travis D , Biswas Nivedita , Nilles Matthew L , Terman David S , Bradley David S 
Published: January 2023

Abstract
SEG and SEI are primordial S. aureus superantigens (SAgs) notably unaccompanied by human neutralizing antibodies that have hampered the use of classic SAgs as cancer therapeutics. Here we show SEG/SEI presented from a HLA-DQ8αβ (HLA-DQA*0301 and HLA-DQB*0302) tg platform outperform C57BL/6 mice in achieving long term survival in vaccinated and established Lewis lung carcinoma (LLC) and B16-F10 melanoma. Vaccination of DQ8 tg mice with LLC or B16-F10 melanoma followed by SEG/SEI immunization and subsequent tumor challenge resulted in 100% and 83% survival for &gt;150 days respectively, compared to a median survival of 14 days in unvaccinated DQ8 tg controls (p&lt;0.001). Likewise, SEG/SEI treatment of established melanoma in DQ8 tg mice resulted in 100% survival for 70 days compared to a median survival of 20 days in untreated DQ8 tg controls. Similar vaccinations/challenges in C57BL/6 mice produced a median survival of 23–28 days. SEG/SEI-activated splenocytes from DQ8 tg mice displayed a TH-1/TH-17 cytokine phenotype with a threefold greater T cell proliferative and CD4+-mediated cytotoxicity responses, compared to C57BL/6 mice. These surprising data delineate a clear pathway to translation of an SEG/SEI-HLA-DQ8αβ tg platform for melanoma/lung cancer prophylaxis and treatment.
Probing changes to local and systemic immunity following intranodal injection
Authors: Andorko James I , Gammon Joshua M , Jewell Christopher M 
Published: January 2023

Abstract
Polymeric vaccine carriers protect and control delivery of encapsulated immune cues to target organs such as lymph nodes (LNs), tissues that coordinate adaptive immunity. Our lab recently established a non-surgical intranodal injection (i.LN.) technique allowing for control of the dose and combination of signals that reach inguinal LNs of mice. We discovered that after treating both inguinal LNs with a model antigen (Ovalbumin) and polymeric particles encapsulating a TLR3 agonist PolyIC (PolyIC MP), strikingly large increases to the percentage and number of activated dendritic cells and macrophages led to increases in B and T cells after 7 days. This treatment generated antigen-specific CD8+ T cells within the treated LNs (~3%) and blood (~13%) leading to memory phenotypes 28 days after vaccination. We used this system as a tool to study how local vaccine exposure and persistence impact systemic immunity by priming of one or more immune sites. Matching vaccine doses were deposited via i.LN. injection into one LN, two LNs, or with the antigen and adjuvant split into separate LNs. LNs receiving PolyIC MPs increased dendritic cell activation. Within the treated LNs, the highest percentage of antigen-specific CD8+ T cells occurred after treating one LN while treating two LNs resulted in half as many antigen-specific cells per LN. Interesting, while all treatments increased blood antigen-specific responses, the response to treating two LNs was double that of one LN treatment and six-fold higher than splitting the signals. These results indicate that the localization and kinetics of immune signals influences the magnitude of systemic immunity and could inform the design of future vaccine strategies to mount robust, antigen-specific responses.
Pro-inflammatory properties of orally delivered calcium-encapsulated CpG Oligodeoxynucleotides
Authors: Kayraklioglu Neslihan , Parker Katherine H , Scheiermann Julia , Klinman Dennis M 
Published: January 2023

Abstract
Synthetic oligodeoxynucleotides (ODN) containing unmethylated CpG motifs (CpG ODN) stimulate immune cells via TLR9. CpG ODN are being evaluated clinically for use as vaccine adjuvants and in cancer immunotherapy. CpG ODNs are generally delivered parenterally as they are susceptible to degradation by acids and nucleases in the GI tract if delivered orally. Wang et al. developed a method to protect ODNs from digestion by encapsulating them in calcium (ODNcaps). They reported that orally delivered ODNcaps could alter susceptibility to atopic dermatitis in mice. To understand the effect of orally delivered CpG ODNcap on local and systemic immune responses, their in vivo and in vitro activity was evaluated in comparison to free (unencapsulated) ODN. TLR9-expressing spleen cells stimulated with CpG ODNcaps in vitro responded by producing cytokines. Oral delivery of CpG ODNcaps increased cytokine production in the Peyer’s patches and mesenteric lymph nodes. Oral administration of CpG ODNcaps exacerbated disease severity in a murine model of dextran sulfate induced inflammatory colitis similar to oral CpG ODN. Oral CpG ODNcap had no detectable systemic effects: they induced no change in serum cytokine levels or spleen cell activation. However, oral CpG ODNcap did enhance host responsiveness to subsequent LPS challenge while reducing susceptibility to L. monocytogenes challenge. Of note, the protection conferred by CpG ODNcap did not exceed that provided when free CpG ODN were delivered orally. These findings indicate that ODNcaps retain biological activity but that calcium encapsulation does not improve their bioavailability or activity in vivo.
Modulation of the energy metabolism inhibits plasmacytoid dendritic cell activation and delays onset of autoantibody production in murine models of systemic lupus erythematosus
Authors: Qiu Connie Claire , Lee Michael Hweemoon , Gallucci Stefania 
Published: January 2023

Abstract
Despite the heterogeneity of systemic lupus erythematosus (SLE) clinical manifestations, an increased expression of type I interferon (IFN) regulated genes, termed the interferon signature, has been reported in most SLE patients. Long recognized to facilitate the progression of SLE, Type I IFNs can be produced in small amounts by most cell types, but the plasmacytoid dendritic cells (pDCs) are the primary producers. Since murine models of SLE have shown that depletion of pDCs stop the onset and progression of autoimmunity, we speculated that it is important to suppress pDC activities that go beyond the production of type I IFNs. We have confirmed that changes in fatty acid metabolism are essential for pDC activation. Using inhibitors of fatty acid oxidation, oxidative phosphorylation, and glycolysis, we have found that metabolic modulation prevents pDC activation upon stimulation with TLR ligands CpG and R848. This metabolic inhibition did not affect pDC viability. Moreover, in vivo treatment of mice with metabolic inhibitors delayed the onset of autoantibody production in murine models of SLE. Indeed, the inhibition of energy metabolism strongly reduced the production of anti-dsDNA and antichromatin autoanti-bodies in i) a model of lupus induced by Graft versus Host Disease (GvHD) and ii) in lupus-prone mice accelerated by TLR stimulation. Therefore, our findings suggest that the in vivo suppression of the major producers of type I IFN through inhibition of energy metabolism may therapeutically downregulate the production of autoantibodies in murine lupus. Our data raises the possibility of manipulating cellular metabolic pathways to regulate the detrimental type I IFN effects in SLE.
Rapid Immunoassay Development using the U-PLEX® Assay Platform.
Authors: Yatsula Victoria , Shelburne Christopher , Ning Qian , Krai Priscilla , Cheo David , Ranganathan Sripriya , Davydov Ilia V. , Liu Pu , Stewart David , Oberoi Pankaj , Wilbur James , Wohlstadter Jacob N. 
Published: January 2023

Abstract
In the expanding field of biomarker research, methods to identify immunoassay reagents and optimize their use can be rate limiting for product development programs. Flexible tools that accelerate assay development, from antibody screening and selection to assay feasibility, can accelerate these programs. Here we demonstrate the use of the MSD® U-PLEX platform to conduct early assay development steps in parallel in a multiplex format. This resulted in rapid identification of a multiplexed panel of compatible assays.
Unbiased pairwise screening of 59 antibodies was performed on MULTI-SPOT® U-PLEX plates using biotinylated capture antibodies and detection antibodies conjugated with SULFO-TAG™ label. Feasible antibody pairs were identified based on high signal and low background, then ranked using parameters such as dynamic range, sensitivity, specificity, sample recognition, and matrix tolerance. Within four weeks, two prototype immunoassays were developed and characterized. This screening time frame can range from days to weeks depending on the number of starting antibodies (typically 10–50) and the level of characterization required. Due to the flexible nature of the platform and the ability to mix and match reagents, the same conjugated reagents identified in screening were used to create and characterize multiplex assay panels. Parameters such as calibration curve, assay protocol, detection antibody concentration, and non-specific binding were characterized with up to ten assays simultaneously in a multiplex.
In conclusion, we demonstrate the utility of a flexible assay development system that allows rapid identification and optimization of assays for stand-alone use or in combination with existing MSD assays.
Suction blistering of vitiligo lesional skin provides insights into the pathophysiology of organ specific autoimmunity
Authors: Strassner James P , Rashighi Mehdi , Refat Maggi Ahmed , Richmond Jillian M , Harris John E 
Published: January 2023

Abstract
Vitiligo is an autoimmune disease of the skin in which CD8+ T cells destroy the pigment producing cells of the skin: melanocytes. To study the immune infiltrate that propagates vitiligo directly in human skin, we employed a novel suction blistering technique to study skin interstitial fluid by ELISA and flow cytometry. Consistent with previous publications demonstrating IFNγ signatures in vitiligo, CD8+ T cell number and CXCL9 protein concentration were elevated in vitiligo lesional skin compared to normal control skin. Receiver operator characteristic analysis revealed that these measurements may be used to distinguish active from stable disease. Phenotypic analysis of T cells in the skin interstitial fluid showed that the majority of CD4+ T cells in vitiligo skin express CXCR3, the receptor for CXCL9, whereas only 47% of CD4+ T cells expressing CXCR3 in the skin of healthy subjects. The frequency of CD8+ T cells expressing CXCR3 was high in both vitiligo patients and healthy donors. Higher CXCR3+ T cell recruitment via elevated CXCL9 expression may contribute to an observed increase in CD8+ T cell to Treg ratio observed active vitiligo skin (4.1 to 2.5 non-lesional). Additionally, all T cells in the skin microenvironment highly express PD-1. These results support the hypothesis that vitiligo is driven by IFNγ dependent signaling, and targeting components downstream of IFNγ may be useful in treating vitiligo. Moreover, suction blistering human skin is a powerful tool that can be used to aid the study of other inflammatory skin diseases.
Topology of multiple antigen expression in tonsillar lymphoid follicles and stroma analyzed by imaging mass cytometry.
Authors: Ornatsky Olga , Allo Bedilu , Talaei Nafiseh , Bouzekri Alexandre , Baranov Vladimir 
Published: January 2023

Abstract
Investigation of co-distribution of 35 biomarkers simultaneously (major cell type and immune-oncology, stromal and tissue architecture markers, as well as cell proliferation and nuclear markers) in normal lymphoid sections using Imaging Mass Cytometry (Qing et al, Cytometry: Part A, 2017) is presented. Tonsil tissue revealed primary lymph follicles as dense clusters of lymphocytes expressing B-cell markers, surrounded by CD3, CD4, and CD8 T-cells. Secondary lymph follicles could be distinguished by proliferating B-cells with high Ki-67 expression. Strong expression of Bcl-6 was detected in germinal-center B-cells. Tonsillar FoxP3 CD8 T-cells exhibit a Treg phenotype with high CTLA-4 and CD45RO. CD68+ mac/mono were found in germinal centers and rarely in stroma. Squamous epithelium of crypts was beta-catenin positive and infiltrated by PD-L1+ T-cells, while PD-1+ cells were also found within the follicle centers. Images acquired on the Hyperion™ Imaging System (Fluidigm Inc.) were compared to immunofluorescence (IF) of sequential sections stained with the same antibodies (Ab) conjugated to fluorophores (FL). For direct comparison of IF and IMC, dual tagged Abs were created by attaching a metal tag first (Maxpar®, Fluidigm Inc.), then conjugating to FL using click chemistry. CD3 and CD19 were labeled with metals and FL, and tested in combination with the full panel on 8 μm frozen tonsil sections. CD3+ and CD19+ lymphs were identified by IF first, followed by IMC analysis. Results show that IMC is comparable to IF images and allows identification, characterization and localization of cell populations and tissue architecture in the absence of autofluorescence, photobleaching, with low background for acquisition of up to 50 targets.
Platelet Detection in Unaltered Whole Blood
Authors: Bradford Jolene A , Ward Michael 
Published: January 2023

Abstract
Platelets are non-nucleated cellular fragments circulating in the peripheral blood, derived from megakaryocytes. They are critical in the maintenance of hemostasis. Alterations in their function are associated with a range of clinical conditions. Using flow cytometry, light scatter signals from platelets typically overlap signals from red blood cells (RBC). Since RBC outnumber platelets by more than an order of magnitude in whole blood, platelets are typically studied by depleting samples of RBCs by centrifugation methods or selective RBC cell lysis. These sample preparation procedures require manipulations that may affect platelet health and function, and result in loss of cells. Additionally, lysed samples require labeling of the platelets with a fluorescent marker to adequately resolve the platelets from debris/noise. We present a novel technique for resolving leukocytes and platelets from RBC in whole blood using orthogonal light scatter from 2 lasers. This method exploits the differences in light-scattering properties between RBCs and platelets and white blood cells (WBC). Hemoglobin readily absorbs 405 nm laser light, reducing the scatter signal from RBC relative to WBCs and platelets, resulting in a reproducible scatter pattern when analyzing human whole blood with both blue (488 nm) and violet (405 nm) side scatter. Using this approach, a distinct platelet population can be discerned in unaltered whole blood, requiring only dilution of whole blood in buffer. Combining this approach with acoustic cytometry allows great flexibility in determining optimal dilution and sample collection rates for minimal coincidence and rapid analysis. For research use only. Not for use in diagnostic procedures.
IL-17 plays a critical role in airway epithelial injury after transplantation
Authors: Xu Jiangnan , Zhang Yamei , Ochando Jordi , Li Liwu , Ding Yaozhong 
Published: January 2023

Abstract
Bronchiolitis obliterans syndrome (BOS) is the major obstacle limiting long-term allograft survival of lung transplantation. Airway epithelium is the primary target in obliterative airway diseases, and the degree of epithelial injury is closely correlated with the development of BOS. IL-17 plays a critical role in mediating inflammatory responses and is involved in transplant graft rejection. In this study, we performed murine orthotopic allogeneic trachea transplantations and examined the role of IL-17 in airway epithelial injury after transplantation. Expression of Th1 and Th17 cytokines, IFNγ, IL-6 and IL-17, was significantly increased in allografts of wild type recipients, and the airway pseudostratified epithelia of allografts changed into flat epithelia with inflammation and fibroblast proliferation in submucosa tissue. Deficiency in Th17 essential transcription factor, RORγt, completely restored normal airway epithelial morphology in trachea grafts, while absence of Th1 transcription factor, T-bet, resulted in exacerbated airway inflammation and epithelial injury with elevated IL-17 expression. Administration of anti-IL-17, anti-IL-6 or Stat3 activation inhibitor (C188-9), but not anti-IFNγ, improved airway epithelial integrity. Our results demonstrate that IL-17 is crucial to post-transplant airway inflammation and epithelial damage, and devoid of IL-17 production diminishes epithelial injury and prolongs graft survival. Our findings provide important implications to understand the role of IL-17 in lung transplant immunity and further indicate the necessity of suppressing IL-17 for airway epithelial protection and allograft survival after transplantation.
Cross-dressing of recipient Ag-presenting cells with donor exosomes trigger direct T-cell allosensitization in transplantation
Authors: Morelli Adrian E , Zeng Furong , Rojas-Canales Darling M , Divito Sherrie , Shufesky William J , Larregina Adriana T , Liu Quan 
Published: January 2023

Abstract
The idea that donor dendritic cells (DCs) from the graft present donor MHC Ag to naïve T cells has been challenged. Evidence suggests that donor DCs are undetectable or found are low numbers in graft-draining lymphoid organs. Thus, the aim of our study is to elucidate how donor MHC molecules are recognized so efficiently by recipient naïve T cells.

Results
After transplantation of CD45.2 BALB/c hearts in CD45.1 B6 mice, very few donor DCs were detected in the spleen, and no donor DCs were found in lymph nodes draining BALB/c skin grafted in B6 mice. In both cases, donor intact MHC molecules were detected by electron microscopy on exosomes attached to recipient conventional DCs in graft-draining lymphoid organs. The transferred MHC Ag induced proliferation and differentiation of CD8 T cells against the BALB/c H2Ld Ag. Cultures of BALB/c DCs with B6 DCs with inhibitors of exosome release (Rab27a siRNA) confirmed that passage of BALB/c MHC occurred via exosomes. Importantly, transfer of exosomes released by donor DCs (unlike other types of vesicles) promoted maturation of recipient DCs. By high resolution confocal microscopy, BALB/c DCs, engineered to release RFP+ exosomes and injected in CD11c-YFP B6 mice, transferred RFP+ exosomes to recipient YFP+ DCs in lymph nodes and spleen. Accordingly, depletion of recipient DCs in CD11c-DTR B6 recipients prevented presentation of donor intact MHC Ag to T cells and delayed heart allograft rejection.

Conclusion
Our results elucidate the ultrastructural basis of the still elusive semi-direct pathway of allorecognition, by demonstrating that donor exosomes (released by the graft or donor migrating DCs) cross-dress recipient APCs and promote the potent T-cell allosensitization seen in transplantation.