bluuebunny/crossref_metadata_2025 huggingface dataset:
Disrupting CXCR2-mediated MDSC trafficking enhances antitumor efficacy of vaccinia virotherapy
Authors: Tan Zhiwu , Chiu Mei Sum , Yue Ming , Song Youqiang , Man Kwan , Chen Zhiwei
Published: August 2023

Oncolytic virotherapy aims to potentiate antitumor responses via multiple mechanisms, including direct tumor lysis, increasing immune cell infiltration, modulating innate and adaptive immune interactions, and vascular modification. In an ideal situation, the oncolysis leads to release of tumor antigens and danger signals, to attract immune cells particularly dendritic cells (DC) into the tumor microenvironment (TME) and to recruit functional cytotoxic T lymphocytes (CTL). However, inflammation in the TME also has detrimental effects to promote cancer progression through reducing intratumoral T cell viability, promoting cancer cell proliferation and angiogenesis, and attracting immune suppressive cells. This complex balancing act dictates the outcome of oncolytic virotherapy and is yet fully understood. In this study, we demonstrated that in situ vaccinia virotherapy primarily recruited polymorphonuclear myeloid derived suppressor cells (PMN-MDSC) into the TME where they exhibited strong suppression of T cell effector function in a ROS-dependent way. Single-cell RNA sequencing analysis confirmed the heightened suppressive activity of PMN-MDSC at the transcriptomic level and identified CXCR2 as a therapeutic target expressed on PMN-MDSC. Abrogating PMN-MDSC trafficking by CXCR2-specific small molecule inhibitor during in situ vaccinia virotherapy exhibited enhanced antitumor efficacy in three syngeneic cancer models, through manipulating MDSC and T cell populations in the TME, activating CTL and skewing suppressive TME into an antitumor environment. Our results warrant clinical development of CXCR2 inhibitor in combination with oncolytic virotherapy.

Hong Kong Research Grant Council (TRS: T12-703/19-R and T11-709/21-N; CRF: C1134-20G and C7156-20G); Hong Kong Health and Medical Research Fund (HMRF 05162326), the Hong Kong Pneumoconiosis Compensation Fund Board (PCFB).
Inhibition of Ly-49/LILRB-MHC-I interactions by anti-MHC-I augments innate and adaptive immunity
Authors: Panda Abir Kumar , Natarajan Kannan , Sinha Surajit , Boyd Lisa F , Jiang Jiansheng , Teke Martha E , Hernandez Jonathan M , Margulies David H , Shevach Ethan M
Published: August 2023

Mouse NK cells recognize MHC-I antigens via stochastically expressed inhibitory Ly49 receptors. The loss of MHC-I expression on tumor cells (“missing self”) abrogates inhibitory signals, resulting in NK activation. We have identified an anti-mouse pan MHC-I mAb (M1/42), which blocks Ly49-MHC-I interactions but not TCR-MHC-I interactions. Administration of M1/42 in vivo markedly activated IFNg-producing NK cells that further drove the proliferation of NK cells and memory phenotype (MP) T cells and profoundly augmented adaptive immunity against viral infection and cancer metastasis. In contrast to mouse Ly-49 antigens, human killer cell inhibitory receptors bind to HLA at a site overlapping the TCR binding site and blocking of KIR binding to MHC-I would inhibit TCR recognition. However, two anti-pan-human MHC-I abs (DX17 and W6/32) markedly induced NK cell proliferation and IFNg production in cultures of human PBMC. DX17 potently blocked the interaction of leukocyte Ig-like inhibitory receptors LILRB1, B2, B3 and B5 with human MHC-I but had no effect on TCR-MHC-I interactions. The crystal structure of a DX17 Fab/MHC-I complex reveals that the footprint of DX17 overlaps the LILRB1 binding site on MHC-I. Administration of W6/32 Fab to humanized mice (PBMC reconstituted NSG or NK cell reconstituted NOG-IL-15) induced human NK and MP T cell proliferation. Marked reduction of tumor size was seen when NOG-hIL-15Tg mice were reconstituted with CD3−cells and then treated with W6/32 Fab. DX17 Fc silenced mAb (DX17 LALAPG) activated human tumor infiltrating lymphocytes and monocytes. These results strongly suggest that inhibition of LILRB-MHC-I interactions by anti-MHC-I in humans may result in marked augmentation of anti-tumor immunity.

This work was supported by the Intramural Research Program of NIAID, NIH.
Investigating molecular mimicry by microbial peptides in the induction of experimental autoimmune encephalomyelitis
Authors: Schwegman Lance , Bartsch Brenden , Abdul-Baki Nawal , de Oca Arena Maria Montes , Negron Austin , Forsthuber Thomas
Published: August 2023

Multiple sclerosis (MS) is an incurable neuroinflammatory autoimmune disease in which a patient’s immune system targets the myelin sheath of neurons. This impairs neuronal signaling to the periphery, manifesting as severe pain, loss of vision, and partial paralysis. Several environmental and genetic risk factors such as the DRB1*1501 haplotype have been identified, but the exact etiology of this disease remain unclear. It is thought that MS is primarily mediated by myelin-reactive CD4+ T cells, however, the autoantigen epitopes targeted are diverse and vary between patients. Furthermore, certain peptides from these autoantigens share amino acid sequence similarities with those of microbial peptides. This is the basis for a phenomenon known as “molecular mimicry”, in which these similarities are sufficient to activate autoreactive CD4+ T cells. Previous work in our lab using the mouse model of MS, experimental autoimmune encephalomyelitis (EAE), found that immunization of DRB1*1501 mice with a peptide from M. avium generates both M. avium- and myelin basic protein (MBP)-reactive CD4+ T cells, though the presence of these cells failed to induce disease. We have previously demonstrated that immunization with suboptimal amounts of multiple myelin-derived peptides, which would not induce EAE individually, result in EAE if combined. We therefore hypothesize that there is a threshold effect in which the concurrent presence of multiple microbial peptides with structural similarity to myelin can contribute to induction of EAE in transgenic DRB1*1501 mice. With the data from this study, we hope to clarify the role of microbial infection in the pathogenesis of MS.

This work was supported by grant RG1602 from the National Multiple Sclerosis Society (T.G.F.), support by the Jesse H. & Mary Jones Gibbs Endowed Chair (T.G.F.), support by grant G12MD007591 from the National Institute on Minority Health and Health Disparities, and NIH/NIBIB ESTEEMED grant R25EB027605 (LS), support by NIH grant R01NS117742 (T.G.F.).
DM functions differently in thymic antigen presentation cell subsets in development of autoreactive CD4+ T cells
Authors: Zhou Zemin , Wang Xiaomin , Cordero Bethanie , Evavold Brian D , Jensen Peter E
Published: August 2023

Autoreactive CD4+ T cells broadly exist in healthy individuals and potentially cause autoimmune diseases; however, why they can escape from thymic negative selection is still controversial. We observed that the activity of DM, a peptide epitope editor in MHC class II antigen presentation pathway, is differentially regulated in different thymic antigen presenting cells (APC). It is high in cTEC that involved in cortical positive selection, but low in mTEC, B cell, pDC and cDC that mediate negative selection in medulla, while it remains high in all types of peripheral APC. The molecular mechanism of differential regulation is still unknown. It is not directly inhibited by DO, a negative regulator of DM that directly competes DM catalytic site with peptide-MHC class II complex, since DM editing activity remains low in those thymic medulla APC in DO−/− mice. We further confirmed that presentation of autoantigen epitope MOG35–55 for thymic positive selection and in vitro activation of autoreactive CD4+ T cells in EAE, a mouse model of human multiple sclerosis disease, is DM dependent. This is also true for presentation of insulin epitope B9–23 to activate autoreactive CD4+ T cells restricted to type 1 diabetes associated HLA-DQ8. Our findings highlighted a fundamental mechanism that immune system differentially regulates DM activity in thymus to balance the efficiency of negative selection to minimize autoimmunity and the maximum of T cell repertoire to defend infection; however, the DM-dependency property of certain autoantigen epitopes accidently caused the failure deletion of autoreactive CD4+ T cells.
Investigating the requirement of IL-17A and IFNg in EAE disease development using HLA-DR3.IL-17A−/−.IFNg−/− transgenic mice
Authors: Turner Ti-Ara J , Peterson Stephanie , Shahi Shailesh , Mangalam Ashutosh K
Published: August 2023

Multiple sclerosis (MS), an autoimmune disease of the central nervous system, is thought to be mediated by autoreactive CD4+ T cells producing IL-17A and IFNg. However, it is unclear if these cytokines are essential for disease development, progression, or both. We have shown that neither IFNg nor IL-17A, alone, are required for the development of disease in experimental autoimmune encephalomyelitis (EAE), a mouse model of MS, but the absence of either results in different disease phenotypes. Specifically, HLA-DR3(DRB1*0301) (DR3).IFNg−/− mice develop severe atypical EAE characterized by increased levels of IL-17A, while DR3.IL-17A−/− mice develop mild, classical EAE. These data suggest that these cytokines may have compensatory roles. To further investigate the roles of these cytokines in EAE, we generated HLA-DR3.IFNg−/−.IL-17A−/− double knockout (DKO) mice and characterized their immune profiles, gut microbiomes, and disease phenotype. Our data show that DKO mice harbor distinct myeloid and CD4+ T cell profiles, myelin antigen-specific immune responses, and gut microbiota. Interestingly, DKO mice develop atypical EAE mimicking the disease phenotype of IFNg−/− mice. Thus, our data suggest that factors other than IL-17A and IFNg mediate atypical EAE susceptibility. Further characterizations of disease are ongoing in DKO mice to better define the precise roles of IL-17A and IFNg in the pathology of MS/EAE.

NIAID/NIH (1R01AI137075-01) and a generous gift from Margaret Heppelmann and Michael Wacek.
MHV-68 induces central nervous system autoimmunity in an MBP-specific TCR transgenic mouse model of multiple sclerosis
Authors: Deffenbaugh Joshua L , Lovett-Racke Amy E
Published: August 2023

Multiple Sclerosis (MS) is a demyelinating autoimmune disease of the central nervous system. Latent infection with Epstein-Barr virus (EBV) has been identified as a necessary yet insufficient factor in the development of the disease. Several groups have demonstrated that the murine EBV analogue, MHV-68, exacerbates disease severity in experimental autoimmune encephalomyelitis (EAE). However, the induction of EAE requires immunization and does not fully replicate the clinical features of MS. We sought to develop a virus-inducible model that accurately represents MS etiology and does not rely on classical EAE immunization. Using a transgenic mouse line with a myelin basic protein-specific T cell receptor, we found that MHV-68 induces demyelinating disease in 72% of infected mice. These mice exhibit clinical features such as optic neuritis, incontinence, and head tilt, which are commonly observed in MS patients but not in mice with EAE. This phenomenon was specific to MHV-68 and was not seen with other viral pathogens including PR8 influenza. Our work demonstrates that latent gammaherpesvirus infection can drive CNS autoimmunity in genetically predisposed mice without prior EAE induction, thus providing a more natural murine model of MS.

National Institute of Health R01AI140741-04
Exhausted CD8 T cell progenitors are sustained with TCR engagement during anti-tumor responses
Authors: Lan Xin , Mi Tian , Youngblood Ben A
Published: August 2023

T cell exhaustion is a major contributor to tumor immune invasion. The durability of an anti-tumor response is in part determined by the persistence of exhausted CD8 T cell progenitors (Tpex) that reconstitute the effector T cell pool. While it has been reported that Tpex are able to survive without antigen, the role of TCR engagement in regulating Tpex self-renewal during tumor progression remains to be fully explored. Here, we used a Lewis lung carcinoma model where a robust or dampened TCR signal was elicited resulting in bona fide tumor-infiltrating Tpex. Longitudinal analysis of tumor-specific CD8 T cells revealed that robust TCR stimulation was required for the formation and maintenance of the Tpex reservoir in tumor-draining lymph nodes (tdLNs). Replenishment of tumor-infiltrating Tpex was abrogated by suboptimal priming despite a generation of central memory-like T cells in tdLNs. Moreover, attenuated TCR engagement substantially accelerated the terminal differentiation of optimally primed Tpex. Chromatin accessibility and whole genome DNA methylation profiling revealed that Tpex-associated epigenetic programs were significantly enriched in optimally engaged T cells in tdLNs. Additionally, tumor-infiltrating sub-primed Tpex gained more Ets1 motifs while Tpex established from robust TCR stimulation acquired greater chromatin accessibility at Tcf-1 targeting loci. These Tcf-1 targets were further validated to be Tpex-specific when compared to those in naive T cells. Collectively, our results highlight the importance of TCR engagement in sustaining Tpex during tumor progression.
Early T cell infiltration correlates with anti-CTLA4 treatment response in the P815 mastocytoma model
Authors: Almonte Andrew Allan , Cavic George , Neeman Teresa , Fahrer Aude M
Published: August 2023

Immune checkpoint blockade (ICB) antibodies are a revolutionary class of cancer treatment, but only approximately 30% of patients receive a lasting benefit from therapy. Pre-clinical studies using animals from the same genetic backgrounds, challenged with the same cancer models, also show non-uniform responses. Most mouse studies that have evaluated tumour-infiltrating leukocytes (TILs) after ICB therapy cannot directly correlate their findings with treatment outcomes because terminal methods were used to acquire immune infiltrate data. In the present study, we used fine-needle aspiration (a non-terminal sampling method) to collect multiple aspirates over several days from subcutaneously implanted P815 mastocytomas treated with ICB antibodies. These aspirates were then analysed with flow cytometry to directly correlate nine TIL populations from both the innate and adaptive arms of immunity with treatment success. We found that the P815 model responds well to anti-CTLA4 but not anti-PD1. Mice that regressed following anti-CTLA4 therapy showed a significant increase in CD8+ T cells on days 3, 5, and 7 and CD4+ T cells on days 5 and 7, and a simultaneous decrease in macrophages and monocytes on days 3, 5, and 7 post-treatment. We also found that P815 tumours sometimes regressed in response to non-specific isotype control antibodies, likely due to their binding with Fc receptors on the cancer cells. Our study is the first to directly correlate early tumour infiltration of T cells with anti-CTLA4 treatment success, thus providing a mechanistic clue toward understanding why alloidentical mice challenged with identical tumours do not respond uniformly to ICB therapies.

This project was funded by the Australian Government Research Training Program, the Lea Chapuis Memorial Fund, The Lionel & Yvonne Spencer and William G Maxwell Trusts administered through Perpetual’s IMPACT Philanthropy, and the generosity of Tim and Margaret Bourke.
Extracellular vesicles derived from a specific strain of commensal bacteria promote anticancer immune responses and synergize with anticancer therapies
Authors: Kim You-Me , Bae Jihong
Published: August 2023

Commensal microbiota regulates cancer development and influences the efficacy of anticancer therapies. To develop a novel microbiota-based anticancer therapy, we screened human intestinal bacteria for their ability to induce IL-12 production in dendritic cells (DCs). A specific strain of Lactobacillus bacteria and their extracellular vesicles (EVs) induced a high level of IL-12 production without a concomitant induction of IL-10 secretion. In addition, DCs stimulated with the Lactobacillus EVs promoted differentiation of Th1 and Tc1 cells in vitro. In mice, oral or intravenous administration of the Lactobacillus EVs reduced tumor growth in both subcutaneous tumor cell implantation and spontaneous colon cancer models by enhancing the production of IFN-γ and cytotoxic effector molecules in tumor-infiltrating T cells. The EVs also enhanced anticancer efficacy of an anti-PD-1 antibody and oxaliplatin treatment. The tumor growth inhibition by the Lactobacillus EVs was abrogated when mice were treated with IL-12-blocking antibody, confirming that the anticancer effect of the EVs depends on their ability to induce IL-12 production. By biochemical and genetic analyses, we identified that bacterial DNA within the Lactobacillus EVs is the main factor inducing IL-12 production in DCs via stimulation of TLR9. Accordingly, DNA purified from EVs of the specific Lactobacillus strain, but not of a closely related other Lactobacillus strain, was able to stimulate IL-12 secretion. Moreover, the EVs did not show IL-12 induction and cancer suppressive function in TLR9-deficient mice. Our results suggest that a rational selection of commensal bacteria-derived EVs can be a strategy to develop a new cancer immunotherapy.

Supported by grants from the National Research Foundation of Korea (2017M3A9F3047085, 2020R1A2C2011307)
A novel population of CD103-expressing CD56+ regulatory ILCs suppresses intratumoural T cells and are associated with poor prognosis in patients with ovarian carcinoma
Authors: Chung Douglas , Vakharia Jehan , Warner Kathrin , Jacquelot Nicolas , Sayad Azin , Han SeongJun , Ghaedi Maryam , Garcia-Batres Carlos , Elford Alisha R , Matthews Jessica A , Wang Ben X , Nguyen Linh T , Shaw Patricia A , Clarke Blaise A , Bernardini Marcus Q , Ferguson Sarah E , Crome Sarah Q , Ohashi Pamela S
Published: August 2023

Regulatory innate lymphoid cells (ILCregs) are an emerging family of immunoregulatory cells that suppress innate and adaptive immunity in the context of diseases, including cancer. However, there is currently a lack of lineage-defining surface markers or transcription factors (TFs) that distinguishes ILCregs from other subsets of ILCs. Moreover, the functional role of ILCregs within the tumour microenvironment (TME) has not been well established. In this study, we uncovered a novel population of CD103-expressing CD56+ ILCregs within the TME that correlated with poor recurrent-free survival (RFS) in patients with epithelial ovarian carcinoma (EOC). CD103+ ILCregs expressed distinct surface markers and TFs that distinguishes them from canonical CD56+ natural killer (NK) cells. Moreover, intratumoural CD103+ ILCregs expressed markers (i.e., CD39, CD69, CD101, TIGIT) and transcriptomic profiles that are shared with Tregs, suggesting that they may have immunoregulatory properties. We found that ascites-derived TGF-β drives NK cell conversion into CD103+ ILCs that can suppress autologous CD4+ T cells in vitro. Furthermore, intratumoural CD103+ ILCregs suppressed autologous CD8+ T cells in vitro. Finally, we found that the abundance of CD103+ ILCregs in the TME were associated with reduced levels of both activated CD4+ T cells and CD8+ T cells. Overall, we have identified CD103+ ILCregs as distinct immunoregulatory cells that suppresses T cells within the TME of patients with EOC. Further untangling of the complex networks of immune regulation, including ILCregs, may give rise to novel therapeutic targets to enhance anti-tumour immunity and improve clinical response rates to immunotherapies.

This work was supported by a CIHR foundation award (CIHR FDN #143220), a TRANSCAN CIHR grant (CIHR – TRN #184713), a TRI grant from the Ontario Institute for Cancer Research (TRI OICR GCS #108796), and the Terry Fox Research Institute (TFRI-iTNT #1060). This project was also generously funded in memory of Mr. JIM JoTak under the Canadian Cancer Society (CCSRI #706152).
TIGIT mediates experimental ischemia reperfusion and nephrotoxic acute kidney injury in mice
Authors: Noel Sanjeev , Lee Kyungho , Gharaie Sepideh , Kurzhagen Johanna T , Arend Lois J , Kuchroo Vijay K , Cahan Patrick , Rabb Hamid
Published: August 2023

Background

We unexpectedly found upregulated expression of T cell immunoreceptor with Ig and ITIM domains (TIGIT) on kidney CD4 T cells after ischemia reperfusion (IR) using RNA-Seq. However, TIGIT role in kidney T cells and during acute kidney injury (AKI) is poorly understood.

Methods

Effect of TIGIT on murine kidney T cells was assessed at baseline and after IR injury by flow cytometry and single cell RNA sequencing (scRNA-Seq). B6 wild type (WT) and TIGIT knockout (KO) mice were subjected to IR and cisplatin-induced AKI. TIGIT expression was assessed in AKI patients using the Kidney Precision Medicine Project (KPMP) scRNA-Seq dataset.

Results

We found increased kidney CD4+TIGIT+ central memory (10.0±2.3 vs 0.9±0.1%, p=0.001) cells compared to CD4+TIGIT− cells in WT mice. TIGIT KO mice had reduced serum creatinine (SCr) after IR (24h Scr, 1.1±0.2 vs 2.7±0.1 mg/dL; p≤0.001) and cisplatin (72h Scr, 0.8±0.1 vs 1.4±0.1 mg/dL; p=0.0002) injury compared to WT mice. TIGIT KO kidneys had reduced necrotic tubules in outer medulla after IR (48.8±4.7% vs 73.0±1.5%; p=0.001) and cisplatin (11.5±3.0% vs 31.3±4.0%; p=0.008) injury than WT kidneys. Gene set enrichment analysis of scRNA-Seq data showed enrichment of inflammatory genes in Th17 cells from WT kidney and oxidative phosphorylation and mTORC1 signaling genes in TIGIT KO kidney. Human KPMP data demonstrated significant TIGIT expression in kidney T/NK cells from AKI patients compared to controls (p≤0.0001).

Conclusions

TIGIT mediates both IR and nephrotoxic AKI in mice and its expression increases in kidney T cells from AKI patients. TIGIT is a promising novel therapeutic target for AKI, and also relevant due to increasing efforts to develop TIGIT blockade to treat cancers.

American Society of Nephrology, Johns Hopkins University Ed Kraus Award, National Kidney Foundation of Maryland and Delaware
Inducing cell-intrinsic dysfunction in pregnancy-sensitized alloreactive B cells
Authors: Durgam Samarth S , Wang Qiang , Yin Dengping , Alegre Maria-Luisa , Chong Anita S
Published: August 2023

Objective

Pregnancy is an immunological paradox where the maternal immune system develops tolerance to semi-allogeneic fetus yet is a sensitizing event that is detrimental to subsequent organ transplantation. Humoral sensitization by semi-allogeneic pregnancy in WT mice prevents anti-CD154-mediated tolerance to fetus matched allograft. In contrast, pregnancy in mice lacking secreted immunoglobulins (mIgM−/−AID−/−; SIgKO) permitted anti-CD154 mediated transplant tolerance, suggesting that fetus-specific antibodies (FSA) were mediating resistance to anti-CD154. Here, we tested whether pregnancy-induced memory B cells could be desensitized to acquire a state of cell-intrinsic dysfunction.

Methods

Pregnancy-sensitized SIgKO or WT mice were subjected to fetus-matched heart transplant/anti-CD154 (PP-tolerant B) or not (PP-memory B). Enriched B cells were adoptively transferred (AdTr) into secondary hosts (MD4 mice) and challenged with fetus-matched splenocytes. Germinal center (GC) allo-reactive B cells from MD4 mice were identified with MHC tetramers, and Fas+GL7+.

Results

While SIgKO PP-memory B cells differentiated into GC-B cells in secondary hosts; this was significantly decreased by 7–10-fold in SIgKO PP-tolerant B cells. In contrast, WT PP-memory B cells and PP-tolerant B cells comparably differentiated into GC-B cells.

Conclusions

In the absence of FSA, PP memory B cells can acquire a cell-intrinsic hypo-functional state. This suggests possible B cell desensitization for multiparous women, who are currently disadvantaged by pregnancy-induced humoral sensitization.

R01AI42747, P01AI097113
Loss of autoreactive B cell tolerance and production of autoantibodies in chronic kidney allograft rejection in mice
Authors: Sayin Ismail , Oien Jacqueline , Ghosh Deepjyoti , Yin Dengping , Durkee Madeleine S , Sage Peter T , Clark Marcus R , Chong Anita S F
Published: August 2023

Loss of autoreactive B cell tolerance and production of autoantibodies have been observed in patients undergoing chronic rejection. However, how autoreactive B cells lose their tolerance, and the role of autoreactive antibodies in chronic rejection remains an enigma. Recent observations from our laboratory that autoreactive B cells with a transcriptome profile of innate B cells accumulate in transplanted kidneys diagnosed with antibody-mediated rejection led us to hypothesize that (i) the loss of autoreactive B cell tolerance occurs primarily in the inflamed kidney and (ii) autoantibodies are contributing to graft rejection. In this study, we developed acute and chronic mouse kidney transplant models to test these hypotheses. Donor-specific antibody responses were assayed using donor MHC coated multiplex beads. Autoantibodies were detected with Hep-2 autoantibody detection kit and quantified with an image analysis pipeline in CellProfiler. The presence of autoantibody secreting cells was confirmed by graft tissue culture, and accumulation of autoreactive B cells was confirmed by ex vivo Nojima cultures. We found that autoantibodies were produced during chronic and acute kidney allograft rejection, even when donor MHC-specific antibody was inhibited with CTLA-4Ig. Remarkably, autoantibodies specific for cytoplasmic, nuclear and nucleolar antigens dynamically changed over time, suggesting rapid epitope spreading. Furthermore, there was an enrichment of autoreactive B cells and autoantibody secreting cells in the graft. Taken together, our mouse models complement data from rejecting biopsies, demonstrating a loss of autoreactive B cell tolerance and accumulation of autoantibodies within rejecting allografts.

Supported by grant from NIH R01AI148705
Effects of temperature on success of scale autografts and allografts in Poecilia reticulata
Authors: Redmond Sarah , Aneli Kiana , Benson Aaliyah , Bridge Aidan , Brown David , Cardenas Andrew , Kennedy Jonah , Redden Alex , Spencer Sierra , Twum Analisa , Wolfe Miranda , Aguilar Marcella , Akers Ashanti , Alshami Duha , Christman Zach , Cruz Alex , Daniel Mark , Dean Sierra , Ennis Ashton , Rodriguez Stefani Guendulay , Harmon Tessa , Johnson Zach , Kravchick Rebecca , Krumtum Rachel , Locklear Abby , Martinez Jazmin , Matthews Chad , Mendola John , Rea Scout , Reed Cierra , Rose Austin , Rupert River , Simpkins Brooke , Sisk Katie , Smith Caleb , Swallow Austin , Tall Ilana , Tamanjah Mamata , Theo Kalina , Thomas-Todd I’adore , Wheeler Katie
Published: August 2023

Acceptance or rejection of transplanted tissues is dependent upon both genetic and environmental factors. We used guppy fish, Poecilia reticulata, as a model for studying those factors impacting the immune response to transplanted scale tissues. Scale tissue acceptance is observed when blood has begun to circulate throughout the grafted tissue and rejection can occur at different time intervals due to the degree of genetic difference between donor and recipient. Individual fish were genotyped for MHC class I (UBA) and MHC class II (DAA and DAB) similarity and underwent both autografts and genetically distinct allografts, followed by a recovery period with different environmental temperatures. Fish exhibit behavioral fever under infection and stress models, and were expected to show different levels of inflammation and graft success at different environmental temperatures. We set tank temperatures at 20, 23, and 26 degrees Celsius (n = 5 fish per tank) and monitored subjects for five weeks following the graft procedure to observe for signs of chronic rejection. Typically, chronic rejection takes two to three weeks to become evident, fish were monitored longer to confirm acceptance or rejection of the grafts. We observed signs of rejection, including necrosis and loss of pigmentation, at higher rates in allografts than in autografts.
The effects of microplastics on autografts and allografts in Poecilia reticulata
Authors: Redmond Sarah , Aneli Kiana , Benson Aaliyah , Bridge Aidan , Brown David , Cardenas Andrew , Kennedy Jonah , Redden Alex , Spencer Sierra , Twum Analisa , Wolfe Miranda , Aguilar Marcella , Akers Ashanti , Alshami Duha , Christman Zach , Cruz Alex , Daniel Mark , Dean Sierra , Ennis Ashton , Rodriguez Stefani Guendulay , Harmon Tessa , Johnson Zach , Kravchick Rebecca , Krumtum Rachel , Locklear Abby , Martinez Jazmin , Matthews Chad , Mendola John , Rea Scout , Reed Cierra , Rose Austin , Rupert River , Simpkins Brooke , Sisk Katie , Smith Caleb , Swallow Austin , Tall Ilana , Tamanjah Mamata , Theo Kalina , Thomas-Todd I’adore , Wheeler Katie
Published: August 2023

Over half of the microplastics found in the ocean are polystyrene - this impacts naturally harvested fish and shellfish in those environments. Recent studies have shown that, in fish, polystyrene microplastics affect growth, development, chemical responses, and digestive contents. Microplastics are not directly immunogenic, but can increase general inflammatory signaling due to tissue damage - fish are particularly sensitive to this type of damage because their respiratory and digestive systems are in constant interaction with the aquatic environment. This study addresses the effects of varying concentrations of polystyrene microplastics on the immune response to scale autografts and allografts in Poecilia reticulata (guppy fish). Tank conditions with microplastic concentrations of 0 particles/m3, 2,500 particles/m3, 10,000 particles/m3, and 80,000 particles/m3 were used to represent the range of natural environments with 4 fish per treatment group. Each individual fish underwent bilateral scale grafts, with autografts on one side and genetically distinct allografts on the other side. Genetic differences were confirmed based on genotyping the UBA locus - representative of MHC class I. Post-surgical graft monitoring used necrosis and pigment loss as markers of chronic rejection and vascular development as a marker of graft success. Chronic rejection based on genetic differences is observable in the differential success of autografted and allografted scale tissues.
Microbiota regulation of thymic microbiota-specific T cell development
Authors: Ruiz Daniel Fernando Zegarra , Chen Amanda , Acosta Monica , Kim Dasom V , Callaghan Ryann C , Saldana-Morales Fatima B , Matthews Dean , Stanford Nikketa , Longman Randy S , Bettini Matthew L , Diehl Gretchen E
Published: August 2023

While exposure to microbial antigens in peripheral sites causes expansion and differentiation of antigen-specific T cells, a role for antigen-specific expansion had not been identified during T cell development in the thymus. We recently found that intestinal colonization induced migration of dendritic cells carrying bacterial antigens to the thymus, driving thymic expansion of microbiota-specific T cells early in life. In contrast, colonization in adulthood led to expansion of peripheral but not thymic microbiota-specific T cells. Thymic microbiota-specific T cells expanded early in life could cause intestinal inflammation in colitis models upon antigen reencounter. We next sought to understand microbiota regulation of this developmental window and define if thymic expansion of microbiota-specific T cells could be restarted in adulthood. Remarkably, we found that microbiota manipulation reestablished expansion of microbiota-specific T cells in the adult thymus. In contrast to early life, adult thymic microbiota-specific T cells ameliorated intestinal inflammation in colitis models. In both early life and adulthood, development of thymic microbiota-specific T cells was driven by the same intestinal dendritic cell population. Our data suggests that intestinal colonization controls the development of thymic microbiota-specific T cells which can modulate inflammatory processes.

NIH (K99AR080207)
Bioengineering Advanced Artificial Antigen Presenting Cells for Immunotherapy
Authors: Shannon Sydney R , Hsieh Zoe , Green Jordan , Schneck Jonathan P
Published: August 2023

Promising cytotoxic (CD8+) T cell-based cancer therapies have invigorated recent efforts to develop highly tunable, robust, and cost-effective artificial antigen presenting cells (aAPCs) for T cell stimulation both ex vivo and in vivo. Natural APCs, such as dendritic cells (DCs), can be licensed to deliver optimal activation signals (peptide-MHC as S1, costimulatory S2, and cytokine S3) to CD8+ T cells with CD4+ T cell help. While numerous studies demonstrate that “un-helped” CD8+ T cells mount defective responses, many existing therapies overlook this and handicap the function/persistence of the resulting cells. The objective of this study is to synthesize a tri-signal aAPC that couples the CD4+ T cell/DC interaction to stimulate anti-tumor CD8+ T cells.

Using novel blends of poly(lactic-co-glycolic acid) (PLGA) and cationic poly(beta-amino-ester) (PBAE) polymers, we synthesized double emulsion microscale aAPCs containing S3 (murine IL-2) encapsulated into the particle core, with S1 (anti-CD3) and S2 (anti-CD28) conjugated to the particle surface. Dynamic Light Scattering measurements show particles of approximately 2 μm in diameter and conjugation efficiency assays showed similar amounts of bound S1 (0.858 μg protein/mg particles) and S2 (0.939 μg protein/mg particles). CD8+ T cell stimulation shows 20-fold proliferation when aAPCs are incubated with CD8+ T cells.

Thus, biodegradable PLGA/PBAE aAPCs can recapitulate natural immune cell interactions for optimal CD8+ T cell anti-cancer efficacy. Currently, we are characterizing additional aAPC physicochemical and biological properties. Our novel biomimetic aAPC has both translational relevance and may also shed light on basic T cell biology.
Intracellular Monitoring by Dendritic Cells – a New Way to Stay Informed – From Simple Scavenger to Active Gatherer
Authors: Herbst Christopher H , Bouteau Aurélie , Igyarto Botond Z
Published: August 2023

The ability of dendritic cells (DCs) to acquire antigen from their environment is critical for the induction of an adaptive immune response and the maintenance of tolerance. Despite its importance, it is tacitly assumed that DCs acquire antigen by scavenging extracellular material via endocytosis or macropinocytosis. Here, using labeled RNA and time lapse imaging, we observe DCs siphoning material directly from live neighboring keratinocytes. This contact dependent mechanism of antigen uptake – which we term intracellular monitoring – accounts for a large percentage of RNA transferred to DCs, and cannot be blocked by inhibitors of known antigen uptake processes. It is exclusive to DCs, as other cell types do not efficiently siphon RNA from neighboring cells, and common among most DCs subsets tested. Langerhans cells with a conditional gene deletion can successfully overcome their deficiency by siphoning RNA and protein from their neighbors, but only if the deleted protein is expressed in the donating cell. Such behavior reframes our understanding of DCs from mere scavengers to highly informed supervisors with extensive access to the cytosol of surrounding cells. Intracellular monitoring may therefore play important roles in the maintenance of tolerance, defense against infection, and detection of cancerous cells.
Visualization and characterization of the elusive MHC I intermediate states by x-ray crystallography and in silico studies
Authors: Bouvier Marlene , Peng Xubiao , Li Lenong
Published: August 2023

Background

Studies have suggested that MHC class I (MHC I) molecules fluctuate rapidly between conformational states as they sample peptides for potential ligands. It is the molecular features of these intermediates that are recognized by tapasin and TAPBPR at the C-terminus of the groove. To date, however, MHC I intermediates remain elusive molecules due to their transient nature. Moreover, whether the N-terminus of the groove has a role in MHC I maturation, and if it also serves as a conformationally sensing region, is not well characterized.

Methods

We designed the HLA-B8-restricted 20mer (RA)6FAKKKYCL and (RA)6FVKKKYCL peptides and corresponding 8mer FAKKKYCL and FVKKKYCL controls. We determined the x-ray crystal structures of HLA-B8E76C presenting these four peptides. Molecular dynamics (MD) simulations of the complexes were also performed.

Results

The structures of FA and FV 20mers revealed significant conformational heterogeneity at the N-terminus of the groove. Long stretches of N-terminal MHC I residues were missing in the electron density maps creating an unstructured and widely open-ended groove. Our structures also showed highly unusual features in MHC I and peptides at the N-terminus of the groove, and in MHC I-peptide interaction. MD simulations showed that these complexes have varying degrees of mobility in a manner consistent with our structures. We suggest that our 20mer structures represent conformational substates explored by MHC I during peptide selection.

Conclusion

Our study advances our understanding of peptide-induced conformational adaptation and functional dynamics in MHC I, and brings into focus the N-terminus of the groove as an important region in mechanisms of high-affinity peptide selection.

NIAID, National Institutes of Health (R01 AI114467, R01 AI108546, and R21 AI173863)
Characterization of the HLA-F ligandome in T-cell lymphoma
Authors: Mota Ines Frutuoso , Osean Jaspreet , Song Eric , Clement Cristina C , Lenz Jack , Miller Danny E , Inghirami Giorgio , Montagna Cristina , Bouvier Marlene , Iwasaki Akiko , Santambrogio Laura
Published: August 2023

Identifying tumor-specific antigens across different types of malignancies and HLA haplotypes remains challenging. Recently, the expression of tumor-specific Human Endogenous Retroelements emerged as a potential new class of cytotoxic T-cell response mediators (1). HLA-F, a non-classic HLA-I molecule, has been hypothesized to present peptides to T-cells and to regulate immunity through interactions with distinct NK-cell receptors (2). HLA-F low genetic diversity and limited peptide repertoire represent an unexplored avenue in cancer immunotherapy. HLA-F-bound peptides were eluted from five patient-derived T-cell lymphoma cell lines and total T-cells purified from ten healthy donor PBMCs; Retroelement peptides were identified in the HLA-F ligandome. To quantify and validate their expression, RNA-sequencing data from the same samples were screened using ERVmap database (3). Lastly, to characterize their expression, we incorporate genome-wide characterization of DNA methylation patterns. We identified a set of retroelement peptides bound to HLA-F shared by patient-derived T-cell lymphoma cell lines but not total T-cells isolated from healthy donor PBMCs, suggesting a potential role of HLA-F in cancer-immune responses. Among those we characterized four tumor-specific retroelement peptides, validated by the presence of their RNA transcripts as well as by DNA integration and chromosomal location. Tumor-specific HLA-F-bound retroelement peptides hold great promise for the discovery of previously unknown tumor-specific markers and therapeutic targets. More importantly, the characterization of their immunogenicity may yield insights broadly applicable to a greater number of patients due to HLA-F monomorphism.
mRNA-adjuvanted Influenza A Vaccine Can Induce High Cellular Immunity and Cross Reactivity in Mice
Authors: Wei Lai
Published: August 2023

In recent years, the great success of mRNA-based vaccines for SARS-CoV-2 revealed a potential future for safe and effective mRNA vaccine designs. Given the flexibility and accessibility of mRNA designs, adjuvants in mRNA forms may be applied to enhance immune response and generate better cross-protection. In this study, we fabricated an Influenza mRNA lipid nanoparticle (LNP) vaccine with Influenza A/Aichi/2/1968 (Aichi, H3N2) Hemagglutinin (HA) mRNA and cytokine-adjuvant mRNA (GIFT4 or CCL27). We found that two doses of immunization with mRNA-adjuvanted LNPs induced increased antigen-specific antibody titer and effective protection against Influenza A viruses in mice. The supplement of cytokine mRNAs (GIFT4 or CCL27) provided more robust immunogenicity, including more IL-4 and IFN-γ secreting T cells in the spleen and more IgG and IgA-secreting B cells in the bone marrow. The cytokine mRNA adjuvants also induced early activation of T and B cells in germinal centers. Thus, GIFT4 and CCL27 mRNAs are potential adjuvants for Influenza A mRNA vaccine to elicit higher and broader immune responses in mice.
Army Liposome Formulation Augments the Immunogenicity of HIV-1 AIDSVAX® B/E Protein
Authors: Kim Jiae , Allam Atef , Peachman Kristina K , Onkar Sayali , Matyas Gary R , Sinangil Faruk , Alving Carl R , Rao Mangala
Published: August 2023

Adjuvants play a critical role in activating innate cells, which can shape the subsequent adaptive immune response including the durability. The RV144 HIV-1 Vaccine Phase 3 Trial tested ALVAC® DNA (prime) and HIV-1 gp120 subtype B/subtype E (AIDSVAX® B/E) protein as the boost. Although, the vaccine regimen showed a modest 31.2% efficacy with antibodies to gp120 V1V2 region as one of the correlates of protection, the immune responses were not durable. Therefore, to enhance durability, we mixed AIDSVAX® B/E which contained aluminum hydroxide (AH) as the adjuvant with lyophilized Army Liposome Formulation containing monophosphoryl lipid A (MPLA; ALF). BALB/c mice were immunized 1, 2 or 3 times at 3-week intervals with AIDSVAX® gp120 B/E (AH adjuvant) or with AIDSVAX® gp120 B/E-ALF (AH and liposomal MPLA adjuvant; ALFA). Spleens were collected on days 3, 5 (after 1 or 2 immunizations), and week 10 (after 3 immunizations), and analyzed by flow cytometry for plasma cells (PC) and T follicular helper cells (Tfh). Serum samples were analyzed for antibody responses by ELISA. ALFA adjuvanted protein induced 2–8-fold higher antibody titers against gp120 V1V2 region at the time points tested compared to AH adjuvanted protein. ALFA induced a higher proportion of PC (B220−IgD−CD138+PNA−GL7−) and IL-21 producing Tfh cells as early as days 3, and 5. At week 10, PC and Memory Tfh cells (CD4+CD44hi CXCR5+PD-1hiBCL-6hi) were also increased in the ALFA group with a higher expression of the activation marker CD40 on germinal center B cells. Microarray analysis of splenic PCs showed increased expression of signaling pathways involved in PC proliferation and antibody secretion. These factors likely contributed to the robustness of ALFA as a vaccine adjuvant.
Development of swine immune reagents for swine immunity and biomedical research
Authors: Lunney Joan K , Loving Crystal , Kenney Scott , LaBresh Joanna , Suresh Raksha , Byrne Kristen , Walker Kristen , Aquino Jovan , Renukaradhya Gourapura
Published: August 2023

The USDA-NIFA Swine Immune Toolkit Initiative has been involved in generating priority immune reagents for swine immunity and biomedical research and pipelining them for marketing. With the help of commercial partners we expressed each target immune molecule as a soluble protein using yeast expression systems and then produced panels of monoclonal antibodies (mAbs) against each target. Most recently we generated panels of mAbs reactive with porcine IL-5, IL-6, IL-13, IL-21, IL-28B, CXCL10, and BAFF and screened their reactivity in multiple immune assays. We determined mAb reactivity with orthologous proteins for most panels of mAbs. A sensitive sandwich ELISA is now available for IL-13 and CXCL10; other targets are being screened for best mAb pairs. Reactivity tests of labeled α-CXCL10, α-IL-6 and α-IL-13 mAbs for intracellular staining of porcine immune cells using flow cytometry assays are underway using different cell stimulation conditions. Immunohistochemistry analyses for binding of α-CXCL10 and other mAbs on formalin fixed pig lymph nodes and spleen tissues are in progress. Further, analysis of a large panel of commercial α-human CD antigen mAbs for cross-reactivity with porcine cells is ongoing. For each target, our goal is to provide the veterinary community with new commercial reagents and standardized assay techniques for their research efforts. Finally, efforts are proceeding to develop SLA tetramers for antigen presentation studies. Tools and reagents generated by this project will undoubtedly advance our understanding of swine immune responses to disease and vaccine and use for biomedical research efforts.

Supported by USDA-NIFA AFRI grant # 2019-67015-29815 and USDA ARS project 8042-32000-117.
Evaluating immunological outcomes associated with histotripsy treatment in canine osteosarcoma: a comparative human oncology model
Authors: Wagner Alayna , Ruger Lauren , Vickers Elliana , Coutermarsh-Ott Sheryl , Patwardhan Manali Nitin , Gannon Jessica , Allen Irving C , Vlaisavljevich Eli , Tuohy Joanne
Published: August 2023

Osteosarcoma (OS), the most commonly occurring primary bone tumor in dogs and humans, has deficient treatment options and dismal survival outcomes. Spontaneously occurring OS in dogs shares numerous similarities with humans and has greater prevalence, allowing the dog to serve as an informative comparative oncology model. The objective of this study was to investigate the non-thermal focused ultrasound technique histotripsy as an innovative treatment option for OS. Histotripsy has the potential to both ablate the primary tumor and stimulate an antitumor immune response to mitigate metastatic disease, the leading cause of death in OS. To investigate the immunological outcomes associated with histotripsy and OS, we delivered histotripsy to client-owned canine OS patients (n=29) and utilized an in-vitro model for cross species comparisons between human, dog, and mouse. Histotripsy ablation led to a significantly greater expression of activation markers CD80 and CD62L on circulating monocytes, at 1- and 5-days post histotripsy (DPH), respectively. We observed a significantly greater production of TGFβ in circulating monocytes at 3 DPH compared to baseline. On an independent patient basis, we observed a greater population of CD5+ CD4+ and CD5+ CD8+ cells within the treated region of the tumor compared to untreated region and changes in genetic signature also indicated immune activation. Across species in-vitro we observed macrophage activation (CD80, CD62L, and CD86), and upregulation of genes associated with immune cell chemotaxis and pro-inflammatory response. Our results indicate that histotripsy ablation leads to immune activation and has strong potential to target both primary OS and metastatic disease.

NIH (1R21 EB030182-01), Focused Ultrasound Foundation (PLL7GBVK and FUSF-RAP-823R1), and American Kennel Club (Canine Health Foundation No. 02773)