bluuebunny/crossref_metadata_2025 huggingface dataset:
Acute GVHD in liver and lungs is mediated by phenotypically-analogous but transcriptionally unique T cells.
Authors: Omdahl Kayleigh I , Bermea Rene , McGuckin Connor , Kaminski Jim , Lane Jennifer , Tkachev Victor , Kean Leslie
Published: January 2023

During acute Graft-versus-Host Disease (aGVHD), donor T cells invade host tissues and cause multi-organ damage. However, the organ-specific drivers of pathogenic alloimmunity are still poorly understood. To uncover both the universal and organ-specific mechanisms driving T cell-mediated damage in target tissues during aGVHD, we analyzed the flow cytometric phenotype as well as transcriptomic signatures of T cells in healthy control animals, in autologous hematopoietic cell transplant (HCT) controls, and during aGVHD after allogeneic HCT, using our established non-human primate aGVHD model. In this study we focused on 2 aGVHD target organs: the liver and lungs. Using both supervised and unsupervised flow cytometric analysis, we discovered that, during aGVHD, both liver- and lung-infiltrating T cells demonstrated similar phenotypes dominated by mature effector-memory CD8 T cells. In contrast, transcriptomic analysis revealed organ-specific gene expression programming. Through Weighted Gene Co-expression Network Analysis (WGCNA), differential expression (DE) analysis, and immune pathway analysis, we identified that the lung aGVHD T cell transcriptomic program was specifically associated with extracellular matrix-remodeling and chemotaxis pathways. In contrast, in liver aGVHD, we found enrichment for metabolic stress responses and cytokine signaling pathways. These results suggest that at the transcriptomic level, T cells infiltrating different aGVHD-target organs are uniquely programmed and could be amenable to organ-specific therapeutic targeting.

Supported by grants from the NIH (F31HL1562880)
PD-1H/VISTA expressed on host myeloid cells regulates acute graft-versus-host disease (aGVHD)
Authors: Hu Qianni , Hedgepath Carly , Hong Junshik , Huh Won Jae , Han Xue , Vesely Matthew , Chen Lieping , Kim Tae Kon
Published: January 2023

Programmed Death-1 Homolog (PD-1H, also known as VISTA) is a co-inhibitory molecule of the immunoglobulin superfamily and broadly found in hematopoietic cells. We previously demonstrated that PD-1H agonists prevent aGVHD in a murine MHC mismatched bone marrow (BM) transplantation model. aGVHD occurs when donor T cells are primed and activated by host antigen-presenting cells (APCs). PD-1H agonism suppressed donor allogeneic T cell activation leading to the suppression of aGVHD. However, it remains unknown whether PD-1H agonists also suppress host myeloid APCs, in which PD-1H is highly expressed. To test this, we adoptively transferred wildtype (WT) C57B/L6 (B6) BM into either PD-1H knock-out (KO) or WT BALB/c mice as hosts. PD-1H KO host mice had more severe aGVHD clinical scores and a worse survival rate when compared to WT host mice. To determine whether PD-1H agonism impacts host immune cells, we transferred B6 PD-1H KO BM into BALB/c WT host mice and treated host recipients with or without PD-1H agonists. Consistent with prior data, host mice treated with PD-1H agonists suppressed aGVHD and extended survival compared with isotype control treated mice. To test which cell subsets expressing PD-1H in host mice regulate aGVHD, we transferred WT BALB/c BM into myeloid lineage specific KO of PD-1H or WT littermate hosts. Interestingly, the myeloid lineage specific KO mice host had significantly worse survival rates and worse aGVHD symptoms compared with WT littermates. Our studies indicate that PD-1H expression on host myeloid cells regulates aGVHD in addition to donor T cell PD-1H. Finding new mechanisms of PD-1H agonism on aGVHD enables development of future therapeutic approaches for aGVHD treatment, such as myeloid APC suppression.

Supported by an Edward P. Evans Foundation Young Investigator Award, a Conquer Cancer Foundation American Society of Clinical Oncology Career Development Grant, and an American Cancer Society Clinician Scientist Development Grant
Dendritic cell-based amelioration of graft versus host disease in humanized mice
Authors: Kalinina Olga , Greene Anna , Knight Katherine L
Published: January 2023

Graft versus host disease (GVHD) is a severe, often lethal, complication of hematopoietic stem cell transplantation, and although prophylactic regimens are given as standard pre-transplantation therapy, up to 60% of these patients develop aGVHD, and require additional immunosuppressive intervention. Previously we showed that treatment of mice with a purified probiotic molecule, exopolysaccharide (EPS) from Bacillus subtilis, significantly increased survival of GVHD mice compared to control PBS-treated mice (70% vs 10%, respectively), with EPS-treated mice surviving more than 100 days after aGVHD induction. To test if EPS can also ameliorate GVHD in humans, we used humanized NSG-HLA-A2 mice and induced GVHD by i.v. injection of HLA-A2neg human PBMCs. After deriving dendritic cells (DC) from CD34+ cord blood cells, we pretreated them with EPS or PBS, and injected them together with PMBCs into the NSG-HLA-A2 mice. We found that EPS-treated DC significantly prolonged the survival of these mice, compared to PBS-treated DC. We also found that EPS-treated DC downregulated expression of activation markers (CD80, CD86), upregulated expression of inhibitory molecules (PD-L1, PD-L2), and inhibited activation of alloreactive T cells in MLR. We conclude that EPS induces tolerogenic human DC, which ameliorate GVHD in humanized NSG-HLA-A2 mice, likely by inhibiting alloreactive T cell response.

Supported by NIH R41 AI155281
IL-10 Signaling in T Cells is Essential for Transplant Tolerance Induction
Authors: Raimondi Giorgio , Lozano Marcos Igleasias , Komin Alexander , Chicco Maria
Published: January 2023

Costimulation blockade (CoB)-based immunotherapy is a promising approach for transplant recipients yet to be realized. Elucidating which factors impact the efficacy of CoB is a necessary step to enable clinical applicability. In this regard, pro- and anti-inflammatory cytokines are being recognized as having an impact on T cell activation beyond effector differentiation. We then aimed at elucidating the role that direct IL-10 signaling in T cells has on the outcome of CoB.

We employed a mouse skin transplt model were Balb/c skin was transplanted to wt C57BL/6 (B6) mice or to B6 with T cell-restricted expression of a dominant negative IL-10 receptor (10R-DN). Recipients received peri-transplant donor specific infusion and three weekly anti-CD154 mAb doses. Unmanipulated 10R-DN recipients rejected their transplant with dynamics identical to wt B6. However, differently from wt B6, graft survival in 10R-DN could not be promoted by CoB (MST 105d vs 30d in the latter) revealing a novel and important effect of IL-10 on T cells. This accelerated rejection correlated with increased production of TNF-α, IFN-γ and IL-17 by T cells in draining lymphoid tissues. MLR experiments indicated that despite IL-10 signaling is not involved in the ability of anti-CD154 to modulate alloreactive T cell proliferation, absence of this pathway increased the differentiation of TH1 effector cells. Moreover, the same experiments indicated that lack of IL-10 signaling in T cells impaired induction of Tregs.

Overall, our results reveal previously unappreciated roles of IL-10 signaling in T cells that represent potential “Achille’s Heels” in the successful implementation of CoB and suggest complementary interventions for the actuation of robust immunoregulation.

Supported by a grant from JDRF (2-SRA-2016-304-S-B)
Oral alloantigen exposure promotes donor-specific tolerance in a mouse model of minor mismatched skin transplantation
Authors: Wang Peter , Chen Luqiu , McIntosh Christine M , Lane Jorden I , Li Rena , Xie Stephen Z , Sattar Husain , Esterhazy Daria , Chong Anita S F , Alegre Maria-Luisa
Published: January 2023

Oral tolerance is a powerful, non-invasive route to induce immune tolerance, yet it has been unsuccessful to promote graft acceptance in rodent models of transplantation. To this end, we developed a mouse model where the model antigen ovalbumin (OVA) was introduced orally, in a regimen designed to improve bioavailability, duodenal lymph node drainage and prolong duration of antigen exposure, prior to transplantation with skin grafts constitutively expressing membrane-bound OVA. We found that oral OVA treatment pre-transplantation promoted permanent graft acceptance as well as linked tolerance to skin grafts expressing OVA coupled to the additional model antigen 2W. Tolerance was donor-specific, as secondary donor-matched, but not third-party grafts were spontaneously accepted in the absence of additional immunosuppression. Treatment with this oral OVA regimen promoted an anergic phenotype in OVA-reactive CD8+ T cells, generation of iTregs, and dysfunction of OVA-reactive CD4+ T cells pre-transplantation. The tolerance induced by oral OVA was robust, as anti-CD25/anti-PD-L1 at the time of transplantation failed to prevent skin graft acceptance. Mechanistically, we revealed a critical role for the proximal gut draining lymph nodes (gLNs) in mediating this effect, as an intestinal infection that drains to the proximal gLNs prevented the induction of transplantation tolerance. Our study extends previous work applying oral antigen exposure to transplantation and serves as proof of concept that the systemic immune mechanisms supporting oral tolerance are sufficient to promote long-term graft acceptance. Regimens that improve bioavailability and duration of oral alloantigen administration may hold promise in the clinic.

This work was supported by a NIAID grant to ASC and MLA (P01AI-97113). Peter Wang was supported by undergraduate research grants from the Katen Scholars Program and the Dean’s Fund for Undergraduate Research.
Graft-Matched Pregnancy Imparts Profound Epigenetic Changes onto Alloreactive Memory T Cells to Enforce a Phenotypic and Transcriptional State of Tolerance
Authors: Pollard Jared , Yin Dengping , Mandal Malay , Gounari Fotini , Alegre Maria-Luisa , Chong Anita
Published: January 2023

Pregnancy is an immunological paradox, where the maternal immune system develops tolerance to a semi-allogeneic fetus, yet pregnancy is a major sensitizing event in clinical transplantation. We previously reported that pregnancy robustly tolerizes fetus-specific T cells, allowing for the spontaneous acceptance of fetus-matched allografts when B cells and alloantibodies are absent at the time of transplantation(1). In this study, we report that mice sensitized to 2WOVA.B/c allogeneic skin grafts (Memory) underwent successful pregnancies comparable to Naïve mice when mated with 2WOVA.B/c males. We utilized a high-dimensional multi-omics approach to characterize post-partum (PP) fetus-specific T cells from Naïve or Memory mice after semi-allogeneic pregnancies. Strikingly, pregnancy induced fetus-specific naïve and memory T cells to achieve a nearly identical phenotype, characterized by upregulation of co-inhibitory molecules FR4, CD73, PD1, and CTLA4 in CD4+FoxP3- Tconvs and upregulated PD-1, FR4 and Lag3 in CD8+ T cells. We confirmed that naïve and memory PP CD8+ T cells acquired similar transcriptional profiles via RNA-Seq; unexpectedly, pregnancy induced profound epigenetic modifications in memory, but not naïve, CD8+ T cells. In vitro studies indicated reduced TNF aproduction by memory PP CD4+ and CD8+ T cells, and experiments examining their response to fetus-matched allografts are ongoing. Our studies suggest that significant epigenetic imprinting is necessary to constrain memory T cells to preserve fetal viability during semi-allogeneic pregnancy. These findings may provide insights into controlling antigen-specific memory T cells, a major barrier to clinical transplant success
Evaluation of checkpoint inhibitor therapies using a mixed lymphocyte reaction (MLR) assay
Authors: Trigg Jasmine E , McBain Kirsty , Liu Zhaoping , Dale Timothy , Szybut Clare
Published: January 2023

Checkpoint inhibitors block the ‘off’ signals that are engaged when T cells interact with antigen-presenting cells, such as dendritic cells (DCs). This increases T cell activation and enhances the anti-tumor response. Here we demonstrate the use of advanced flow cytometry to evaluate these immunotherapies in vitro by co-culturing immune cells from two donors in an MLR assay.

Assays were set up as either one-way or two-way MLR co-cultures. One-way assays combined isolated DCs from one donor with CD4+ T cells from another. Two-way assays mixed PBMCs from two donors. Cells and checkpoint inhibitors were plated in 96 or 384-well formats for 3–6 days. Samples taken during the time course were analyzed for cell subsets and cytokine concentrations using the iQue® Advanced Flow Cytometry Platform.

Expression of activation marker CD25 on T cells was 3-fold greater after 6 days in co-culture with DCs (62 ± 2 %) compared to T cells in monoculture (22 ± 9 %). The addition of an anti-PD1 checkpoint inhibitor induced a further, concentration dependent increase in CD25 expression. This was accompanied by increased production of cytokines indicative of activation and inflammation, such as TNFα and CCL2. Throughout the study, there were large differences in sensitivity to drug response between donor pairs. For example, IFNγ release increased 13.5-fold upon addition of anti-CTLA4 (17 ng/mL) with one donor pair, compared to only 1.3-fold with another. This highlights the need to characterize drug activity using multiple immune cell donor pairs.

These data exemplify the use of advanced flow cytometry to generate pharmacological data for checkpoint inhibitor activity on T cells in MLR, with the potential to profile libraries of novel therapeutics in minimal time.
Sialylation of CD44 by ST8sia6 is required for recognition by the inhibitory receptor Siglec-7
Authors: Chen Yin , Friedman David J , Saraswat Mayank , Shapiro Michael J , Wilfahrt Drew , Pandey Akhilesh , Shapiro Virginia Smith
Published: January 2023

Glycans decorate almost all proteins and lipids on the cell surface, and they play a critical role in cell-cell interactions. Terminal sialic acid addition to glycans helps the immune system distinguish between “self” and “non-self.” The enzyme ST8sia6 adds alpha-2,8 disialic acids to cell surface receptors, and this sialic acid addition promotes binding to the inhibitory receptor Siglec-E. We have previously shown ST8sia6 overexpression in tumors accelerates tumor growth in a Siglec-E dependent manner. Proteomic analysis in murine tumor lines identified CD44 as a target for ST8sia6. CD44 expression on tumors is associated with metastasis and chemoresistance. Since humans and mice have different sialic acid structures, we examined whether ST8sia6 makes CD44 a ligand to the ortholog of Siglec-E in human cells, Siglec-7. When HEK293T cells overexpress ST8sia6 and CD44 together, Siglec-7 binding was significantly increased compared to ST8sia6 or CD44 overexpression alone. Thus, ST8sia6 has a conserved role from mouse to human in decorating CD44 with sialic acid. Our study proposes that ST8sia6 sialylation of CD44 could suppress immune responses and enables cancer progression in human.

Supported by grant: 2T32AI007425-23 to DJF and R01 CA243545-01S1 to V.S.S.
Organoid models for high-grade endometrial cancer to dissect tumor immune microenvironments
Authors: Chung Charlie , Nizam Aaron , Goldberg Gary L , Beyaz Semir
Published: January 2023

Inactivation of antigen processing and presentation (APP) pathways is one of the major mechanisms for immune evasion in many cancer types including high-grade endometrial cancer (HGEC). Impairments in APP pathways may contribute to the low immunotherapy response rate among the patients. However, lack of pre-clinical models for HGEC poses a significant challenge in studying critical therapeutic aspects of the cancer such as cancer-immune cell interactions. To our knowledge, we have established the largest endometrial biobank comprising primary tissues, patient-derived organoids (PDO), and their matching immune cells from all existing endometrial cancer types, enabling us to establish the interaction between cancer and immune cells in vitro.

Using a subset of our biobank, we performed RNA-seq on 68 PDOs and found that genes involved in APP pathways are downregulated in HGEC organoids compared to their matching normal pairs. We found that MHC-I and -II could be upregulated by IFNg and Tazemetostat, an EZH2 inhibitor. We further assessed the effects of enhanced MHC-I and -II expression mediated by the two molecules on cancer-immune cell interactions by setting up syngeneic organoid-immune cell co-cultures. We found that HGEC organoids that were pre-treated with IFNg or Tazemetostat displayed a greater immune cell co-localization and immune cell-mediated apoptosis during the co-culture period.

Our proof-of-principle experiments demonstrate the utility of our co-culture system in studying the cancer-immune cell interactions in vitro. Our system could be used to test the efficacy of current therapeutics or to screen potential drugs which enhance immune responses against HGEC, which lacks effective treatment options.

This research was supported by grants from The Mark Foundation for Cancer Research (20-028-EDV630), NCI CancerCenter Support Grant (5P30CA045508),and CSHL-northwell transitional research fund.
Turning immunologically cold tumors into hot ones by activating hepatoma-intrinsic FADD/NF-κB/CCL5 pathway
Authors: Lu Jiahuan , Chan Anthony Wing Hung , To Ka Fai , Zhou Jingying
Published: January 2023

Lymphoepithelioma-like hepatocellular carcinoma (LEL-HCC) is a distinct variant of HCC that displayed immunologically hot tumor features with prominent tumor-infiltrating CD8+ T lymphocytes (TILs). Our whole exosome sequencing data pointed out an increased prevalence of chromosome 11q13.3 amplification in LEL-HCC, in which 13/17 genes located in 11q13.3 were upregulated. Among these genes, fas-associated death domain (FADD) displayed a strongest correlation with differentially upregulated immune regulatory genes in HCC with 11q13.3 amplicon. In addition, CD8+ T cells preferred to migrate into tumors with higher FADD expression in vitro and in vivo. Mechanistically, FADD activated nuclear factor-κB (NF-κB) that in turn promoted T cell trafficking chemokine C-C motif chemokine ligand 5 (CCL5) production. Furthermore, Fadd expression was positively correlated with Ccl5 and TIL proportions, leading to increased immune checkpoint blockade (ICB) responsiveness in orthotopic HCC mouse models. As LEL-HCC patients also exhibited a concordant higher expression of FADD and CD8, our findings suggested that activation of FADD may turn cold tumors into hot ones and improve HCC ICB responsiveness.

This project is supported by grants from Health and Medical Research Fund (07180556) and General Research Fund 2019/20 (14108219).
Ovarian cancer cell glucocorticoid receptor activity modulates cytokine secretion promoting infiltration of immunosuppressive cells into the tumor microenvironment
Authors: Taya Manisha , Gonzalez Janet , Bennett Lynda , Conzen Suzanne D
Published: January 2023

Myeloid-derived suppressor cells (MDSCs) have been identified as a heterogeneous cell population that expand during tumor progression and infiltrate the tumor, peripheral blood, as well as ascites of ovarian cancer patients. MDSCs contribute to tumor immune tolerance via inhibition of T-cell proliferation/activation. It is also known that high tumor cell glucocorticoid receptor (GR) expression is associated with a relatively poor prognosis in ovarian cancer patients. We hypothesized that ovarian tumor cell GR expression and activity may modulate tumor cell cytokine secretion thereby leading to increased MDSC generation and recruitment, thus creating an immunosuppressive tumor microenvironment. Our preliminary data confirms GR-mediated secretion of pro-tumorigenic cytokines including G-CSF, TGF-b, and MCP-1 in high-grade ovarian cancer models. We also observed GR-mediated downregulation of IL-2 and IFN-g, cytokines required for anti-tumor T-cell function. Importantly, treatment of cells with a competitive GR antagonist reversed this immunosuppressive secretome effect. Moreover, culturing healthy peripheral blood mononuclear cells (PBMCs) with tumor conditioned media from GR activated ovarian cancer cells demonstrated that secreted immunomodulatory factors have the capacity to promote differentiation of human MDSCs from PBMCs. Based on the insight provided by these data, we propose to determine whether ovarian tumor cell-intrinsic GR activation and resulting MDSC generation has an inhibitory effect on cytotoxic T-cell function, contributing to earlier relapse of GR-positive ovarian cancers, and eventually whether this mechanism may be targeted to improve patient outcomes in this subset of ovarian cancers.

Supported by grant from CPRIT
Transcriptome analysis identifies networks and key drivers in tumor microenvironment for metastatic castration-resistant prostate cancer
Authors: McKinney Lawrence P , Singh Rajesh , Jordan I King , Varambally Sooryanarayana , Dammer Eric B , Sanda Martin G , Lillard James W
Published: January 2023

Prostate cancer (PCa) is the second most cancer-related cause of death in men. The most lethal form of PCa is metastatic castration-resistant prostate cancer (CRPC) that has progressed to the bone. The molecular mechanism underlying PCa progression to bone has not been fully elucidated. Here, we identify gene networks and characterize the cell types that contribute to the heterogeneity and complexity of CRPC associated with bone metastasis. Networks were investigated by using a novel weighted gene network co-expression analysis (WGCNA) method and examining overrepresentation of upstream regulators and signaling pathways within co-expressed transcriptome modules across clinically annotated transcriptomes from mCRPC patients (N=60). Functional Enrichment analysis was used, including Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment, and protein-protein interaction analysis to identify biological functions of related hub genes overrepresented in our module of interest. WGCNA identified significant modules that are associated with CRPC bone metastasis. KEGG and GO analysis results revealed genes in these modules were mainly related to collagen formation, cell signaling peptides, and bone regulation processes. Genes positively correlated with bone metastasis exhibited the following biological pathways: PI3K-Akt signaling, ECM-receptor interaction, and protein digestion and absorption pathways. This study provides novel insights into the biological pathways associated with CRPC metastasis to the bone and its tumor immune microenvironment (TIME) niche. The modules associated with bone metastasis and overall survival represent both known and novel pathways.

Supported by NIH/NCI Grant: MSM/Tuskegee U/UAB Comp. Cancer Center Partnership Name & Number of Grant Award: NCI - U54CA118638
Multiplex microscopy reveals unique spatiotemporal effects of cancer immunotherapies
Authors: Maltez Vivien Ileana , Byrne Katelyn T , Germain Ronald N
Published: January 2023

Pancreatic ductal adenocarcinoma (PDA) is an aggressive and heterogeneous cancer that is often refractory to current treatments. To address this issue, we employ a mouse PDA-derived tumor clone library where individual clones elicit a spectrum of unique immune cell infiltration profiles in the tumor microenvironment (TME). We found that a combination of anti-PD1, anti-CTLA-4, and anti-CD40 results in tumor regression only in tumor clones with a high number of infiltrating T cells. The aim of our study is to gain mechanistic understanding into this therapeutic combination. To that end, we leverage quantitative high multiplex microscopy, enabling us to decipher the complexities of cellular behaviors, interactions, and phenotypes in an intact TME. We found that our therapy distinctively alters the TME of both responsive and non-responsive tumors. Unique to non-responsive tumors, we found discrete myeloid clusters scattered through the TME that increase in frequency and size post therapy. Additionally, we witnessed prominent cellular movement within myeloid clusters using 2-photon intravital imaging. In contrast, in the TME of responsive tumors our therapy severely depleted, reprogrammed, and restricted regulatory T cells to the tumor periphery within 48 hours post anti-CD40 administration. We found this to be ADCC–independent, but anti-CD40–dependent, with the mechanistic factors driving the fate of these cells under detailed investigation. Combined, these data reveal mechanistic insight into anti-CD40 combination therapies and provide a platform for investigating the factors driving the formation and maintenance of an immunosuppressive TME.

This work was supported by (1) Intramural Research Program of NIAID, NIH, (2) Postdoctoral Research Associate Training (PRAT) Fellowship of NIGMS, NIH, (3) Parker Institute for Cancer Immunotherapy, and (4) Bench to Bedside Award.
Identification of immunogenic peptide neoantigens expressed in sarcomas and their therapeutic potential
Authors: Sedlacek Abigail L , Osei-Hwedieh David , Agyekum-Yamoah Archibald , Mena Luis , Iyer Swati , Weiss Kurt , Binder Robert J
Published: January 2023

T cells recognize tumor neoantigens (TNag) in the context of MHC and eliminate cells which express these antigens, leaving those lacking immunodominant epitopes. Central to investigating tumor immunoediting is the ability to define immunogenic TNag. Differential aggretope index (DAI) is a measure of the difference in MHC binding affinity between mutated and unmutated peptides and has emerged as a highly efficient way of identifying immunogenic TNag. Our previous studies have demonstrated that in mice, TNag with a DAI >8 are considered immunogenic and elicit T cells that lead to the tumor elimination. Thus, tumors expressing TNag of DAI >8 are only found in immunodeficient mice. Given these observations, we investigated the mutational burden and immune phenotype of bone proximal tumors in 122 patients. Tumors included are of bone, surrounding tissue and distal metastases, and were analyzed by flow cytometry, whole exome and TCR-Vβ sequencing. We hypothesized that tumors with high DAI TNag would be immunoedited in patients with robust T cell responses as measured by T cell clonality. Similar to observations in mice, this correlation was only observed in TNag with DAI >8. In contrast, there was no correlation between DAI and frequency of CD8 TILs indicating that the repertoire of TILs is a better indicator of immune reactivity than their frequency. Among tumor types analyzed, osteosarcomas were the least immunoedited, suggesting greatest potential for immunotherapy. We confirmed this potential in a mouse model of osteosarcoma, where we show that therapeutic HSP-mediated responses, augmented by PD-1 blockade, conferred the best survival. These studies provide compelling rationale for novel immunotherapies in patients with osteosarcoma.

Supported by NIH R01 CA233803 and an HCC UPMC Developmental Award
RNA-protein Co-detection using spatial analysis to profile tumor-infiltrated immune cells
Authors: Dikshit Anushka , Basak Sayantani , Doolittle Emerald , Bunting Michaeline
Published: January 2023

Interrogating complex tumor microenvironment requires a multi-omics approach. Detecting target immune cell markers using immunohistochemistry/Immunofluorescence (IHC/IF) and visualizing cytokine expression with in situ hybridization (ISH) can provide comprehensive information about the activation states of immune cells. Here, we demonstrate a newly developed integrated ISH and IHC/IF workflow compatible with manual and automated platforms with improved RNA-protein co-detection.

We demonstrate the use of our RNA-Protein Co-detection assay in combination with the automated and manual RNAscope Multiplex Fluorescent assay as well as the automated RNAscope Chromogenic Duplex assay to detect T cell markers, macrophage markers and checkpoint markers in the tumor microenvironment by using a microarray with different tumor samples.

We identified CD4+ helper T cells, CD8+ cytotoxic T cells and determined their activation states by visualizing IFNG, GZMB and IL-2 expression. We were also able to identify macrophages detected by CD68 protein expression and differentiate the M1 and M2 subtypes by using M2-specific marker, CD163. We could also delineate tumor-stroma border in the samples by using the Pan-CK probe which distinctly marks the tumor cells and visualize expression of immunoregulatory receptors PD-L1 and CTLA4 in the tumor.

This assay is enables for multiplexing by combining with our fluorescent assays or enables target detection while retaining morphological context when combined with our chromogenic assays. Overall, the new RNAscope-ISH-IHC/IF co-detection workflow and reagents enable simultaneous visualization of RNA and protein targets by enhancing the compatibility of antibodies with minimal optimization.
Thymic stromal lymphopoietin (TSLP) receptor signaling controls a tumorigenic Treg to promote colorectal cancer
Authors: Obata-Ninomiya Kazushige , Ziegler Steven F
Published: January 2023

Regulatory T cell (Treg) is a hallmark of progression of cancer. However, the subset of Tregs and the factor regulating Tregs in colorectal cancers (CRCs) have not been defined. We identified a novel subset of Tregs expressing TSLP receptor (TSLPR) and ST2 was predominantly associated with CRC in human and mouse. Mef2c, a transcription factor, exclusively expressed on this Treg controlled the expression of CTLA-4 on this Treg under TSLPR signaling. To study the role of TSLP signaling on this Treg, we examined mice lacking TSLPR on Tregs (TSLPRΔTreg) in AOM/DSS-induced CRC model. The results showed that the number and the expression of CTLA-4 on this Treg were lower in TSLPRΔTreg mice and the size and number of tumors were reduced compared to control mice, concomitant with increase of Th1 cells. In addition, in human CRCs, the number of TSLPR+ ST2+ Tregs is negatively correlated with the number of Th1 cells. These findings suggested that TSLPR+ ST2+ Tregs play a pivotal role in progression of CRC through suppression of anti-tumor immunity in CRCs in human and mouse. Furthermore, to address the cure of CRC, we administrated mice with neutralization antibody for TSLP after development of colorectal tumors. Blockage of TSLP signaling reduced size and number of tumors via reduction of TSLPR+ ST2+ Tregs, indicating that neutralization of TSLP inhibits progression of CRCs. Taken together, and TSLP signaling on tumorigenic TSLPR+ ST2+ Treg can be good therapeutic targets for CRC.
Cloning ospA of Borrelia Burgderferi for Plant Expression
Authors: Browne Quinton Ambrose , Puthoff David P , Taylor Rebekah
Published: January 2023

Borrelia burgdorferi (Lyme disease) is a vector-borne disease that is both very common in the Mid-Atlantic region and which causes long term negative health effects. It is primarily spread to humans by deer ticks, and because one of the main ways that it is passed between ticks is by previously infected mouse blood, our goal is to design an oral vaccine which decreases infection rates in mice. Our goal is to do this by inserting outer surface protein A (ospA) of B. burgdorferi into a plant vector: pCambia 1201. Agrobacterium tumefaciens was used as a means of introducing the vector (bearing B. burgdorferi ospA) into Arabidopsis thaliana. The pCambia vector was introduced into A. tumefaciens competent cells after being purified from E. coli colonies which had undergone a similar transformation process, as E. coli is more suitable for cloning. The successfully electroporated A. tumefaciens was then plated and colonies were picked and grown as liquid cultures. Those liquid cultures transformed with both the ospA bearing pCambia and a control pCambia were used for floral dip of Arabidopsis thaliana (both ospA bearing and pCambia only control plants). These dipped plants’ seeds have been tested for successful transformation, and one plant has been found to contain the sequence for ospA. Continued floral dips will be undertaken to increase the number of positive plants, and to develop a proper control for testing efficacy as a vaccine. Any additional seeds of positive plants will be planted and crossed to ensure use of only plants which express pCambia in later testing, and to ensure that the process has been successful.

Supported by Frostburg State University Department of Biology and FSU Foundation
A recombinant Chlamydia muridarum MOMP encapsulated PLGA 85/15 nanovaccine induced antibodies elicited cross-recognition of serovar-D elementary bodies
Authors: Sahu Rajnish , Verma Richa , Sharma Homa Nath , Tadjuidje Vanella , Joshi Sameer , Forte Othreniel A , Singh Shree R , Dennis Vida A
Published: January 2023

Chlamydia trachomatis (Ct), caused by various serovars, remains the leading sexually transmitted bacterial infection worldwide. An efficient delivery system is critical to developing whole or subunit vaccines against Ct, and vaccine developmental efforts are rapidly targeting biodegradable nanoparticles based vaccines with encapsulated antigens. We have developed a nanovaccine employing the recombinant major outer membrane protein (rMOMP) of C. muridarum (Cm) encapsulated in poly (lactic acid-co-glycolic acid) (PLGA) 85/15 (PLGA-rMOMP). We previously compared the humoral responses of priming routes; subcutaneous (SC) or intramuscular (IM-p) followed by two sc-boosts to evaluate rMOMP specific serum antibodies [Th1 (IgG2a, IgG2b), Th2 (IgG1)] produced by PLGA-rMOMP immunization of female BALB/c mice. Here we explored the ability of systemic antibodies produced in immunized mice to recognize UV-inactivated whole elementary bodies (EB) of Cm and one of the prevalent human serovar D (Ct-D) using antigen-specific ELISA. Serum from phosphate-buffered saline (PBS) or PBS encapsulated (PLGA-PBS) groups of mice served as a negative control to validate the EB recognition by immunized sera. We observed higher EB specific total IgG (4-fold) and isotypes IgG2b (16-fold), IgG1 (2-fold) antibodies recognizing Ct-D compared to Cm for the SC and IM-p immunization groups. Evaluation of the Th1 (IgG2b)/Th2 (IgG1) antibody titer ratio revealed a higher Th1 antibody recognition of Cm (4-fold) and Ct-D (16-fold), for the SC and IM-p immunization routes. Our data show that mice immunized with PLGA-rMOMP produced Th1 systemic antibodies against EB, but more importantly, there was robust serological cross recognition of the human serovar D.

This research was supported by the National Institute Of Allergy And Infectious Diseases of the National Institutes of Health under Award Number R21AI111159, NIH-NIGMS-RISE (1R25GM106995-01) and the National Science Foundation (NSF)-CREST (HRD-1241701) and NSF-HBCU-RISE (HRD-1646729) grants.
The use of outer membrane vesicles as novel, mucosal adjuvants against intracellular bactiera
Authors: Harrell Jaikin , Morici Lisa A , McLachlan James B
Published: January 2023

Many pathogens first enter the body via mucosal surfaces where they can then invade and disseminate systemically to cause disease. Despite this, most vaccines are given parenterally and are unable to induce mucosal immunity. Immunizing directly at the mucosa could solve this problem, however delivering vaccines at these surfaces often doesn’t invoke robust immunity. One way to alleviate this is to use adjuvants that can evoke an immune response. Most adjuvants, like aluminum salts, are unable to induce mucosal immunity and so novel adjuvants must be employed. Outer membrane vesicles (OMVs) from Burkholderia pseudomallei are potent immune mediators and have been shown to have adjuvant capabilities. The goal of this study is to highlight the role of OMVs as a novel adjuvant that can be used in the next generation of mucosal vaccines. To test this, we created an OMV-adjuvanted inactivated whole-cell vaccine against two intracellular pathogens – Salmonella Typhimurium and Francisella holarctica LVS that could be delivered mucosally. An oral vaccine against S. Typhimurium adjuvanted with OMVs showed protection against lethal challenge in addition to evoking antigen specific CD4 T cells, B cells, and anti-Salmonella antibodies. These antibodies induced greater bacterial killing in macrophages. We are currently exploring an OMV-adjuvanted oropharyngeally delivered vaccine against F. holarctica LVS. Immunity against Francisella requires both CD4 and CD8 T cells and we are determining how an OMV-adjuvanted vaccine will influence these immune cell populations. This study represents a novel approach to mucosal vaccines using OMVs as adjuvants.

Supported by NIH U01 AI124289 NIH BAA HHSN72201800045C
Mechanisms of impaired muscle repair following influenza infection in aged mice.
Authors: Cadar Andreia , Keilich Spencer R , Martin Dominique E , Torrance Blake L , Haynes Laura , Bartley Jenna M
Published: January 2023

Older adults are susceptible to infectious diseases including Influenza (flu), due to widely accepted declines in immune response with age. Flu infection in older adults commonly results in hospitalization and catastrophic disability, leading to loss of independence. Previous findings demonstrate that flu infection induces severe and prolonged muscle injury in aged mice. We aimed to explore the effects of flu infection on skeletal muscle gene expression and immune cell populations to elucidate mechanisms of flu-induced disability in older adults. Young and aged C57BL/6 male mice were infected with a sublethal dose of PR8 (H1N1) influenza. Gastrocnemius muscles were harvested for RNA sequencing, confocal microscopy, and flow cytometry. Our findings showed differential gene expression changes between young and aged mice, including downregulation of muscle regeneration and organization genes and upregulation proinflammatory cytokine and migratory cell pathways in aged muscle. Pathway analysis revealed significant increases in T cell activation pathways in the aged muscle. Confocal microcopy confirmed increased leukocytes (CD45+) and T cells (CD3+) intramuscularly following flu infection. Flow cytometric analyses confirmed that aged mice have increased infiltrating CD8 T cells during infection compared to young mice, while young muscle have increased infiltrating regulatory T cells (Tregs) compared to their aged counterparts. Further analysis suggested that differences in IL-33 levels between young and aged mice influence these infiltrating populations. Future research is necessary to determine if IL-33 supplementation may prevent or ameliorate flu-induced disability in older adults.

Supported by NIH/NIA R01 AG051647 (Co-I) NIH/NIA R01 AR075346 (Co-I) NIH/NIA R33 AG061456 (Co-I) NIH/NIA R21 AG071292 (Co-I) American Federation for Aging Research Reboot (PI)
Identification and Characteristics of HLA-restricted HMPV-specific CD8+ T cells
Authors: Lan Jie , Parks Olivia , Zhang Yu , Walters Sara , Sojati Jorna , Williams John V
Published: January 2023

Human metapneumovirus (HMPV) is a leading cause of respiratory tract infection in pediatric, elderly, and immunocompromised populations. All individuals have been exposed to HMPV by the age of 5, but humoral immunity does not fully protect against reinfection of adults. Despite the clinical burden, there are currently no FDA-approved vaccines or therapeutics for HMPV. T cell responses are important for protection and viral clearance. The aim of this study was to better understand the human T cell response to HMPV and guide vaccine development. To accomplish these goals, we sought to identify HLA class I-restricted viral epitopes. In this study, 5 transgenic mouse strains that express human HLA-A*01:03, HLA-A*24:01, HLA-B*35:01, HLA-B*15:02, or HLA-Cw*07:01 were used to map HMPV-specific epitopes and characterize HMPV-specific CD8+ T cells. We used ELISpot screening of overlapping peptides and predictopes to discover several epitopes and generate MHC-I tetramers for each genetic background. We found that human CD8+ T cells of subjects expressing the same HLA types could recognize viral epitopes by ELISpot and tetramer staining. Our results suggest that the transgenic mouse is a useful model to identify HLA-restricted viral-specific epitopes and novel targets for vaccination against HMPV.

Supported by grants from NIH (R01 AI085062)
Induction of blood brain barrier disruption through reactivation of virus antigen specific brain resident memory T cells
Authors: Ayasoufi Katayoun , Namen Shelby L , Wolf Delaney , Tritz Zachariah Peter , Goddery Emma , Pfaller Christian K , Gulbicki Lauren , Fain Cori E , Jin Fang , Hansen Michael , Johnson Aaron J
Published: January 2023

The contribution of antiviral resident memory T cells (TRM) to blood-brain barrier disruption has not been defined despite the implication of this lymphocyte in clinical encephalitis. We therefore evaluated the capacity of TRM cells to induce CNS vascular permeability in mice which resolved Theiler’s murine encephalomyelitis (TMEV) infection. Following TMEV infection of the brain, persistent populations of virus antigen specific resident memory T cells are induced within the brain. This population of resident memory T cells can be further reactivated at 60 dpi through administration of the cognate immunodominant virus peptide antigen, VP2, inducing blood brain barrier disruption in the process. We determined that brain resident memory T cells expand following reactivation with administered VP2 peptide. This reactivation is associated with infiltration of T cells, and inflammatory myeloid cells into the brain. Importantly, using depletion and T cell sequestrating strategies with low dose anti CD8 antibodies and FTY720, we determined that TRM induced blood-brain barrier disruption is independent of peripheral T cells. We conclude that reactivation of brain TRM cells during peptide administration occurs without dependency on peripheral responses, highlighting the importance of this cell type in inducing neuroinflammation and underlying neuropathology.
Exploring the role of c-Myc in defining the fate of exhausted CD8 T cells
Authors: Toumi Ryma , Deo Rucha , Bhise Shruti , Sarkar Surojit , Kalia Vandana
Published: January 2023

c-Myc is a major transcription factor expressed upon activation of CD8 T cells that drives their clonal expansion and metabolism. The role of c-Myc in regulating the outcome of exhausted CD8 T cells during chronic infection and cancer remains poorly defined. In this study, we used a unique model of c-Myc conditional ablation following antigen priming of CD8 T cells to investigate whether c-Myc exerts a role in CD8 T cell exhaustion in chronic antigenic settings. Our studies show that targeted deficiency of c-Myc in antigen-specific CD8 T cells resulted in decreased cell numbers but promoted the generation of stem-cell-like TCF-1+ less exhausted CD8 T cells. These cells are key therapeutic target for overcoming CD8+ T cells exhaustion by providing the proliferative burst after PD1 checkpoint blockade immunotherapy (CBI). We, therefore combined anti-PD-L1 therapy and c-Myc inhibition to determine whether c-Myc inhibition might synergize to improve the therapeutic outcome. Our data using c-Myc inhibitor alone revealed an increased frequency of TCF-1+ CD8 T cells. However, the number of antigen specific CD8 T cells and the production of effector cytokines (IFN-γ and TNF-α) remained identical to untreated group. Notably, the combination of c-Myc inhibitor and PD-1 CBI significantly promoted the proliferation of antigen-specific CD8 T cells and the production of IFN-γ and TNF-α. Overall, findings support a role for c-Myc in defining the fate of exhausted CD8 T cells, and illustrate a potential pathway for the optimization of check point blockade immunotherapy of chronic infections and cancer.

Supported by NIH R21AI154363 NIH R01AI32819
Persistence of SARS-CoV-2 in long COVID-19 patients
Authors: Goh Denise , Lim Jeffrey Chun Tatt , Fernandez Sonia Bilbao , Lee Justina Nadia , Joseph Craig Ryan , Guerrero Sílvia , Neo Zhen Wei , Lau Mai Chan , Yeong Joe Poh Sheng
Published: January 2023

The World Health Organization has defined long COVID-19 (LC) as a condition where patients exhibit persistent symptoms over time after its acute phase, which cannot be explained by alternative diagnosis. Since we have previously reported residual viral antigens in tissues of convalescent patients, we now aim to assess the presence of such antigens in post-convalescent tissues. Here, we established the presence of residual virus within the appendix and breast tissue of 2 patients who exhibited LC symptoms, 163 to 462 days upon positive diagnosis, using immunohistological techniques. We observed positive staining for viral nucleocapsid protein (NP) in the appendix, and tumour-adjacent region of the breast, but not within the tumour. Multiplex immunohistochemistry further detected colocalization of viral NP with macrophage marker CD68, in support of immune cells being direct targets. Notably, with RNA scope, both positive-sense and negative-sense (replicative intermediate) viral RNA were detected. As a single-stranded virus, SARS-CoV-2, have to produce a replicative intermediate as a template to synthesize new genomic RNAs. Thus, the detection of negative-sense viral RNA suggests ongoing viral replication. While viral RNA and antigen from gastrointestinal and stool samples of convalescent patients has been extensively reported, we believe this is the first study to detect viable virus. Furthermore, our positive finding in the breast tissue also corroborated with recent reports that immunocompromised patients had also experienced LC symptoms and persistent viral replication. Overall, our findings, along with emerging LC studies, question the possibility of the gastrointestinal tract functioning as a reservoir.

Supported by A*STAR Career Development Award (C21112056)
Homeodomain protein Pbx1 regulates regulatory T cell development, stability and suppressive function
Authors: Choi Seung-Chul , Park Yuk Pheel , Roach Tracoyia A , Morel Laurence
Published: January 2023

Pre-B cell leukemia transcription factor 1 (Pbx1) regulates multiple processes in various cell types. The Pbx1 gene is located in the Sle1a1 lupus susceptibility locus in the NZM2410 lupus-prone mouse model. The Pbx1-d dominant negative isoform lacking the DNA binding domain is more frequent in the CD4+ T cells from NZM240 mice as well as lupus patients as compared to healthy controls. In addition, the transgenic expression of Pbx1-d in CD4+ T cells reproduced the phenotypes of Sle1a1 mice, with impaired Foxp3+ Treg cell homeostasis as well as increased inflammatory CD4+ T cells. To study the role of Pbx1 in Treg cells, we generated Foxp3-specific Pbx1 knock-out mice (Foxp3-Pbx1 KO), which were bred to CD4-Pbx1-d-Tg mice to generate Pbx1-d Tg-KO mice that have Treg cells solely expressing Pbx1-d. Foxp3-Pbx1 KO mice showed reduced numbers of Treg cells with impaired suppressive activity. The numbers of natural Treg (nTrg) cells are decreased in thymus but not in spleen of Foxp3-Pbx1 KO mice, while the number of induced Treg (iTreg) cells are increased in the thymus and decreased in the periphery, suggesting that Pbx1 regulates Treg cell development and stability. The results were enhanced in Pbx1-d Tg-KO mice compared with Foxp3-Pbx1 KO mice, leading to autoimmune phenotypes, suggesting that the expression of Pbx1 functional isoform is necessary for the maintenance and function of Treg cells. RNAseq analyses with Pbx1-KO Treg cells indicated that Pbx1 regulates cell cycle, apoptosis and migration. Overall, our results suggest that Pbx1 regulates Treg cell homeostasis, stability and their suppressive functions and that Pbx1-d promotes lupus development by decreasing Treg cell development and functions.

Supported by a grant from the NIH (R01 AI045050)