bluuebunny/crossref_metadata_2025_split huggingface dataset:
Charactering the immune signature of antigen presenting cells in the immune response to factor VIII in a murine model of hemophilia A using single-cell RNA sequencing
Authors: Batsuli Glaivy , York Elizabeth S , Krishnan Upaasana , Gill Surinder , Thomas Beena , Bhasin Swati 
Published: August 2023

Abstract
Deficiency of the procoagulant protein factor VIII (FVIII) is the defining characteristic for individuals with the inherited X-linked bleeding disorder hemophilia A. These individuals require intravenous infusions of FVIII to treat bleeding events, however ~30% develop neutralizing alloantibodies (i.e., inhibitors) against exogenous FVIII. Transcriptome analysis of FVIII-stimulated peripheral blood mononuclear cells in patients with hemophilia A and active inhibitors showed increased expression of innate immune modulators and proinflammatory pathways. Murine models of hemophilia A (FVIII KO) similarly develop anti-FVIII antibodies with FVIII exposure. Although the antibody response to FVIII is CD4-T cell dependent, the key antigen presenting cells (APCs) that mediate inhibitor formation is unclear. Bone marrow chimeras of transgenic mice with depleted dendritic cell (DC) subsets in FVIII KO suggest that type 2 classical DCs (cDC2), but not cDC1 or plasmacytoid DCs, mediate FVIII immunity. We then evaluated the evolution of splenic APCs and their immune cell signatures in FVIII KO mice injected with 0, 1, 3, or 5 doses of FVIII using flow cytometry and single-cell RNA sequencing (scRNAseq). FVIII KO injected with 5 doses of FVIII had similar frequencies of pDC, cDC1, and cDC2 subsets compared to mice injected with normal saline as a control. However, preliminary scRNAseq analysis demonstrated increased expression of phagocytic and interferon-related genes among DC and macrophage clusters, particularly CD8a−CD4+ Itgax− DCs. Further scRNAseq analyses of APC subclusters and differentially expressed genes will be assessed. Overall, these findings suggest that cDCs, specifically cDC2, are critical in FVIII immunity.
Supported by NIH K99HL150595 and Hemophilia of Georgia Clinical Scientist Development Grant.
X-Linked Chronic Granulomatous Disease in a Female Carrier: A Management Challenge
Authors: Sarkaria Sandeep , Hostoffer Robert W 
Published: August 2023

Abstract
Chronic granulomatous disease (CGD) is characterized by recurrent bacterial and fungal infections due to reduced nicotinamide adenine dinucleotide phosphate oxidase resulting in impaired production of reactive oxidant species, and aberrant inflammation. X-linked CGD (XL-CGD) is associated with a mutation in the CYBB gene. XL-CGD affects more males, although some females are affected affected due to skewed X-inactivation. Though management guidelines for CGD patients exist, they remain lacking for clinically affected XL-CGD female carriers. A 3 year old female with history of hematochezia, oral ulcers, and recurrent skin infections who presented with fevers and skin pustules. Pustule cultures were positive for B. cepacian, and colonoscopy was non-acute. Immunology had concern for XL-CGD with recent atypical infections and dihydrorhodamine (DHR) flow cytometry was abnormal, 17.7% (normal greater than 95%). DHR 2 months later showed 13% activation. Genetic testing showed a CYBB, c.45+2dup (intronic), heterozygous, VUS, gene mutation affecting the splice site, associated with XL-CGD, and a NOD2, c.3019dup, heterozygous, with a 2–4 fold increased risk for Crohn’s disease. She was started on bacterial prophylaxis only. She is awaiting gp91phox expression for gene sequencing.
XL-CGD carriers can be at higher risk for infections, and risk is inversely correlated with %DHR neutrophil counts, but only clinically apparent with %DHR less than 20%, and more as levels are less than 10%. Though our patient was started on Bactrim prophylaxis with her %DHR less than 20%, there remains the question of when and if she should be started on anti-fungal prophylaxis. There remain no clear guidelines on the management of XL-CGD carriers.
Identifying immune biomarkers associated with human trauma as potential therapeutic targets
Authors: Fathi Parinaz , Karkanitsa Maria , Rupert Adam , Ricotta Emily ,   Traumatic Injury Drug Prevalence Team, Sereti Irini , Sadtler Kaitlyn 
Published: August 2023

Abstract
Severe trauma can induce systemic immunosuppression, which makes understanding the biomarker profiles of trauma patients critical for therapeutic development and for predicting potential immune responses to treatments such as biomedical implants. Our goal was to establish a data set containing levels of a large array of biomarkers measured in blood from human trauma patients. Multiplex electrochemiluminescence assays were conducted to measure the levels of 59 biomarkers in 1000 blood samples collected from patients at the time of admission to trauma centers. Injury types were categorized as motor vehicle crashes (n=448), falls (n=221), gun wounds (n=78), stabbings (n=117), or other traumatic injuries. Patients ranged in age from 18 to 101, with a mean age of 42.7 for male patients (n= 725) and 49.8 for female patients (n= 272). There were no significant differences in biomarker expression levels between the different injury types. Differences in age distributions of male and female patients for each injury type were not found to be statistically significant, except in the case of falls, which had a higher average age for female patients compared to male patients. MIF, MIP-5, and YKL-40 had the highest expression, with concentrations of 554148 ± 62938 pg/mL, 17409 ± 919 pg/mL, and 98793 ± 10015 pg/mL (mean ± 95 % CI) respectively, at 1:100 dilution. Further experiments are currently being conducted to establish biomarker profiles for control uninjured human samples, and data comparing the profiles of trauma patients and uninjured controls will be presented. Overall, this work provides a resource for understanding immune responses to injury and potentially developing new therapeutics with these biomarker profiles in mind.
This work was supported by the National Institute of Biomedical Imaging and Bioengineering
Analysis of novel RAC2 variants associated with neutrophil dysfunction, lymphopenia, and primary immunodeficiency
Authors: Donko Agnes P , Sharapova Svetlana O , Marois Louis , Winterbourn Christine , Ashby Louisa , Martelius Timi , Seppänen Mikko , Andreae Doerthe A , Leiding Jennifer W , Gauthier Amélie , Campbell Nicholas , Ganesan Sundar , Hsiao Kuang-Chih , Holland Steven M , Falcone Emilia L , Hsu Amy P , Leto Thomas L 
Published: August 2023

Abstract
RAC2 is a member of Rho-family GTPases, abundant in neutrophils serving roles in NADPH oxidase activation and cytoskeleton dynamics. Patients with dominant, activating mutations in RAC2 (E62K, N92T, Q61R) are associated with neutrophil dysfunction, lymphopenia and immunodeficiency. We now characterize additional disease-related RAC2 variants with in-vitro analysis of RAC2 effector functions including NOX2-based superoxide production, regulation of PAK1-PDB binding, activation of AKT, protein stability, subcellular localization, and F-actin formation.
We identified both active and inactive variants associated with lymphopenia and immunodeficiency. Patients with dominant, active RAC2 variants with high protein stability (Q61R, Q61K) presented in the first days of life with severe combined immunodeficiency and absent lymphocytes. Activating variants with lower protein stability (I21S, E62K, N92K, N92S) presented as combined variable immunodeficiency with lymphopenia, detectable by newborn screening but clinically presenting later with recurrent sinopulmonary infections, reduced B-cells and hypogammaglobulinemia. A third class of mutations with unstable transcript or protein were phenotypic only in homozygosity (W56*, R68W). Severity of phenotype and initial clinical presentation was correlated with protein stability, with increased stability leading to neonatal presentation and decreased stability of dominant mutations having later clinical onset. Thus, heterologous expression and analysis of RAC2 variants is useful for deciphering the genetic and molecular mechanistic basis for the common underlying clinical phenotype related to lymphopenia and immunodeficiency.
Generation of thymic epithelial progenitor cells from induced pluripotent stem cells obtained from patients with thymic defects
Authors: Amini Kayla , Pala Francesca , Bosticardo Marita , Notarangelo Luigi D 
Published: August 2023

Abstract
Thymic epithelial cells (TECs) play a critical role in T-cell development as they provide essential cues to hematopoietic progenitors for their proliferation and differentiation in the thymus. Patients with genetic mutations affecting TEC functionality present with reduced thymus development and decreased T-cell output in the periphery. We have previously reported that T-cell development is blocked in patients with hematopoietic-intrinsic genetic defects using an artificial thymic organoid system, but this model does not allow testing of TEC defects. To address this, we have generated multiple induced pluripotent stem cell (iPSC) lines from patients carrying mutations on genes involved in thymic stromal development and/or function, including 22q11.2del (DiGeorge syndrome), CHD7, FOXI3, PAX1, AIRE, EXTL3, and TP63. We aim to differentiate these iPS lines into TECs and assess whether these mutations alter TEC generation. We are currently testing previously published protocols. However, most of these protocols focused on generating TECs from embryonic stem cells and were not optimized for human iPSCs. To address this, we are comparing and combining different methods to achieve a high efficiency in inducing definitive endoderm (DE), ventral pharyngeal endoderm (VPE) and finally thymic epithelial progenitors (TEP). We have already successfully reached the DE stage, as gene expression analysis by qPCR shows upregulation of genes associated with the DE and downregulation of iPS genes. Once we successfully generate TECs, we plan to incorporate them into our artificial thymic organoid system to create a model that will be solely based on human cells, obtaining a more accurate in vitro reflection of human thymic T cell development.
This work was supported by the Division of Intramural Research (DIR), NIAID, NIH
IL-21 or synthetic IL-2 treatment drives T cell IFNγ production or expansion, respectively, during adoptive cell therapy of melanoma
Authors: Walsh Michael , Zhangxu Kevin , Lim Birkley , Liu Samantha , Kureshi Rakeeb , Dougan Stephanie , Dougan Michael 
Published: August 2023

Abstract
Adoptive cell therapy (ACT) of tumor-infiltrating lymphocytes (TILs) can generate long-term durable responses in patients with melanoma and can even successfully treat patients that are refractory to anti-PD-1 checkpoint blockade. IL-2 is often used to expand TILs, however the administration of this cytokine can induce systemic toxicities in patients. Synthetic cytokine mimetics of IL-2 have been developed, including neoleukin-2 (Neo2), which has been shown to have enhanced therapeutic efficacy over IL-2, with reduced toxicity. IL-21 has also been used in TIL therapy to promote T cell expansion. Here, we tested the efficacy of Neo2 or IL-21 in treating mice bearing B16 tumors that received ACT with melanoma-specific T cells (TRP1High and TRP1Low; TCRs recognizing antigen with high or low affinity). Both Neo2 and IL-21 could slow growth of B16 tumors, curing several mice. We performed single-cell RNA-sequencing on these tumors to analyze cytokine therapy-induced changes in the microenvironment. Neo2-treated tumors had a signature primarily of T cell expansion, with few other associated transcriptional changes. IL-21 therapy generated a profound IFNγ signature, with an increase in T cells production of IFNγ and many cell types, including T cells, macrophages, and tumor cells responding with interferon-stimulated gene expression. Interestingly, IL-21 therapy did not induce profound T cell expansion as Neo2 did, but importantly both IL-21 and Neo2 therapy reduced levels of Tregs within the tumor. IL-21 also improved T cell production of granzyme B. Thus, IL-2 primarily expands T cells and IL-21 generates higher quality anti-tumor T cells. Therefore, IL-21 is a promising cytokine for improving ACT and other immunotherapies.
Overcoming immunotherapy resistance in melanoma defective of IFN-γ signaling
Authors: Shi Lewis Zhichang , Shen Hongxing , Ojo Gbenga , Bonner James A 
Published: August 2023

Abstract
Therapeutic resistance to immune checkpoint blockers (ICBs) in melanoma patients is a pressing issue. Initial efforts from others and us show that ICB resistance can derive from melanoma-intrinsic loss of the IFN-γ signaling, which occurs in ~20.9% of the 287 melanoma cases (CBioPortal), based on the cumulative frequency of damaging mutations of 12 core genes. However, how to overcome this resistance mechanism remains largely unexplored. Moreover, given the indispensable role of tumor-infiltrating T cells (TILs) in ICBs, little is known about how tumor-intrinsic loss of IFN-γ signaling (IFNγR1KO) impacts TILs. Here, we report that IFNγR1KO melanomas have reduced infiltration and function of TILs. Combining kinomics, RNA-seq, and proteomics, we unveil that Janus Kinase 1 and 2 (JAK1-2), key downstream kinases in the IFN-γ signaling cascade, are constitutively active, mediated by heightened mTOR. Importantly, mTOR inhibition with Everolimus induces a potent suppression of IFNγR1KO melanoma growth, particularly when combined with ICBs, despite its reported modest effects on WT tumors; similarly, targeting JAK1-2 with Ruxolitinib (Ruxo) selectively impedes the growth of IFNγR1KO but not WT melanomas, revealing effective “targeted” treatments for ICB resistance. Mechanistically, these therapeutics successfully reprogram “cold” TILs in IFNγR1KO melanoma to functional effectors (e.g., increased production of TNF), and deleting T cells or using TNFKO mice completely abrogates Ruxo efficacy. Altogether, our results highlight an active role of tumor-intrinsic IFN-γ signaling in shaping TILs and manifest therapeutic targets that can be harnessed to bypass ICB resistance of melanomas defective of IFN-γ signaling.
Supported by grants from NIH (1R21CA259721-01A1) and from DoD Idea Award (ME210108)
The effects of αPD-1 and αPD-L1 therapy on macrophage remodeling within the solid tumor microenvironment
Authors: Kwok Tina , Da Silva Ildefonso Alves , Dong Haidong , Lancaster Jessica 
Published: August 2023

Abstract
PD-1- and PD-L1-targeted immune checkpoint blockade therapy has shown great clinical potential. Unfortunately, this therapy has been ineffective in a majority of patients bearing solid tumors due to the highly immunosuppressive nature of the tumor microenvironment. As macrophages compose the most abundant myeloid cell subset within the tumor, current macrophage-targeted treatments aim to deplete macrophages within the tumor to reduce immune suppression. However, our goal is to determine whether αPD-1 and αPD-L1 therapy can harness macrophage potential by shifting tumor associated macrophages (TAMs) from a suppressive M2 to a pro-inflammatory M1 phenotype. Our preliminary data has shown that αPD-1/αPD-L1 treatment of mice bearing B16 murine melanoma leads to controlled tumor growth and this was accompanied by a higher M1:M2 ratio and CD8 T-cell abundance. Therefore, we hypothesized that αPD-1/αPD-L1 therapy may drive TAMs towards a pro-inflammatory phenotype and to become better activators of CD8 T-cells. By analyzing antigen presentation machinery on TAMs, we show that in the presence of tumor cells, αPD-L1 blockade upregulates expression of MHC1 and MHC2 on TAMs. Unexpectedly, direct αPD-L1 blockade on TAMs leads to reduced CD80 surface due to mechanisms still to be elucidated. Further, we analyzed co-cultures of αPD-L-treated TAMs loaded with ovalbumin and naïve OT1 cells, which led to greater proliferation of naïve CD8 T-cells compared to their isotype control counterparts. Our observations thus far support the ability of αPD-1/αPD-L1 therapy to induce an M1-phenotype shift among macrophages and also αPD-L1 blockade alone can drive TAMs to become better CD8 T-cell activators.
Mayo Clinic David and Margaret Grohne Cancer Immunotherapy and Mayo Clinic Immune Monitoring Core Pilot Awards
Hijacking Suppression: Anti-CD40 Converts Regulatory T Cells Into Type I Effectors
Authors: Maltez Vivien Ileana , Arora Charu , Sor Rina , Lian Qiaoshi , Vonderheide Robert H , Germain Ronald N , Byrne Kateyln T 
Published: August 2023

Abstract

Background
Pancreatic ductal adenocarcinoma (PDA) is an aggressive and heterogeneous cancer that is often refractory to current treatments. We found that a combination of anti-PD1, anti-CTLA-4, and anti-CD40 results in tumor regression. Thus, 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.

Results and Conclusions
In the TME of responsive tumors, our therapy severely depleted regulatory T cells. We found this Treg loss to be ADCC–independent, but anti-CD40–dependent, a surprising result given that anti-CTLA-4 was thought to mediate Treg depletion. Residual Tregs were restricted to the tumor periphery by 48 hours post anti-CD40 administration. We used tamoxifen-inducible Foxp3 lineage tracing mice to label all Foxp3–expressing cells prior to therapy initiation. Strikingly, we found that many of the lineage marked Tregs no longer expressed Foxp3. These ExTregs now had high levels of Tbet and IFNg, and had evidence of cognate antigen recognition, as assessed via imaging of NFAT1 nuclear translocation in situ. Single and combination treatments in the absence of anti-CD40 failed to induce this ExTreg phenomenon. Blockade of MHC-II, IL-12, or IFNg or tumor implantation into Batf3 KO, IL12p40 KO, or IFNg KO mice also ablated this phenomenon. Combined, these data reveal a unique mechanism by which the clinically relevant agonistic anti-CD40 amplifies the anti-tumor immune compartment through the conversion of immunosuppressive Tregs to an effector population within the tumor microenvironment.

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.
NSCLC patient-derived xenografts perpetuating tumor-associated, tumor-reactive T cells that respond to immunotherapy but do not cause GVHD in host mice
Authors: Radhakrishnan Vijay , Venkateshaiah Sathisha Upparahalli , Mahadevappa Chandrashekara Puthanapura , Okpasuo Onyekachi Juliet , Cheatham Christa , Nagaraj Suvilesh Kanve , Manjunath Yariswamy , Li Guangfu , Kaifi Jussuf T , Schrum Adam G , Gil Diana , Avella Diego M 
Published: August 2023

Abstract
Non-small cell lung cancer (NSCLC) is the leading cause of malignancy-related deaths worldwide. We wished to overcome a current major limitation of PDX modeling, by providing autologous human T cells suitable for immunotherapy responses, but notably, without graft versus host disease (GVHD). We have developed PDXs from NSCLC by implanting surgically resected patient tumors subcutaneously in NOD scid gamma (NSG) mice. We used a metastatic PDX model to test potential therapeutic efficacy of a novel T cell molecule, anti-human-CD3ɛ monovalent Fab fragment (Mono-OKT3-Fab) and compared tumor growth results with anti-human PD-L1 therapy. Overall engraftment rate of unique, primary patient tumors was 52%. Over multiple passages in vivo, PDX tumors retained expression of primary tumor-matched NSCLC-diagnostic pathological markers. Moreover, primary-tumor-associated T cells were present and passaged in mice in vivo, without producing any signs of GVHD. Immunotherapy showed significant response from a PDL-1+ tumor, consistent with functional tumor-specific T cell passaging in our system. In contrast, control experiments using patient-matched human PBMCs showed vigorous GVHD in the presence or absence of co-transplanted PDX tumor; this demonstrated that non-tumor-associated lymphocytes from these patients were capable of xenoreaction, even though tumor-associated T cells were not. We observed that PDX-NSG mice treated with either modality showed significantly reduced tumor burden and increased survival over control-Ig-treated mice. Overall, our study shows that PDXs implanted in NSG mice from surgically resected-NSCLC retain T cells sufficient for preclinical evaluation of novel T cell-targeting immunotherapies.
NIH, National Cancer Institute, IOTN(U01 CA244314)
NIH, NIAID, R01 AI097187
Mizzou Start-up funds
Understanding the cellular and molecular mechanism of anti-CTLA4 mediated tumor rejection
Authors: Liu Na , Kazmi Rida , Jin Wenwen , Gauthier Thierry , Patino Liliana C , Jin Wenwen 
Published: August 2023

Abstract
Although the clinical success of checkpoint inhibitors treatment is significant to prolong the patient survival, there is a substantial gap in our understanding of their mechanism in patients due to several technical and ethical limitations. Recent work suggests that the therapeutic effect of anti-CTLA-4 antibodies may not only block CTLA-4 interaction with its ligands, but also selectively deplete intratumoral Tregs (the highest expresser of CTLA-4) through ADCC. This releases the immunosuppressive state of the tumor microenvironment, thereby, promoting the expansion of effector CD4 and CD8, and increasing the secretion of IFN-g and TNF to reject the tumors. We observed a rapid Treg reduction characterized with fragile phenotype as early as 24 hours after anti-CTLA4 treatment. Using FOXP3-DTR mice, we confirmed that Treg depletion is the major contributor against tumor progression as anti-CTLA4 effect was abrogated in the DT-treated mice, and Treg function was maintained in the FcR KO mice. This suggested that FcR engagement is responsible for the Treg weakness. We hypothesized that there might be additional signal being transmitted when anti-CTLA4 is engaging with FcR which is capable of inducing Treg reduction. We made this hypothesis when we found the important receptor CD25 expressed on the Treg was significantly decreased after engagement. This receptor can also sense surrounding IL2 to maintain Treg cell survival, function, and proliferation. Interestingly, the CD25 expression was not inhibited when lacking FcR to induce CTLA4 engagement. Hence, there is still a need to study the underlying mechanism to see why Treg cells express lower CD25 after CTLA4 engagement.