bluuebunny/crossref_metadata_2025_split huggingface dataset:
High-Content CD14+ Cells in Apheresis Products negatively affects the Transduction and Expansion of CAR T cells during Large-scale cGMP Manufacturing
Authors: Riviere Isabelle , Qu Jinrong , Stefanski Jolanta , Borquez-Ojeda Oriana , Hack Anniesha , Wang Xiuyan 
Published: January 2023

Abstract
Adoptive transfer of chimeric antigen receptor (CAR) engineered T cells is a promising strategy to treat patients with cancer. Apheresis products are commonly used as starting material for large-scale T cell selection using CD3/28 magnetic beads. At the time of collection, the composition in various cell lineages is highly variable. We have encountered manufacturing challenges with apheresis products containing high proportion of CD14+ cells. As an illustration, we investigated the transduction and expansion of T cells using an apheresis product derived from a patient with ALL and containing 70% CD14+ cells. 350 × 106 CD3+ cells were selected from a cryopreserved apheresis product using CD3/28 magnetic beads. A short adherence procedure decreased the CD14+ cell composition from 70% to 45%. On day 3, 350×106 vs 20 ×106 total cells were recovered from each group without and with the depletion step, respectively. Selected cells from both groups were transduced using the same dilution of vector stocks at a cell concentration of either 0.4×106/mL or 0.2×106/mL in tissue culture bags. We observed a drastic difference in transduction efficiency between the group without depletion (4.5%) and the group in which the CD14+ cells were depleted (38%). Cells from the depleted group expanded 350 fold from day 3 to day 10 (100% CD3+). Our results suggest that high CD14+ cell content poses a manufacturing challenge when the selection of the CD3+ cells in the apheresis product is performed with CD3/CD28 magnetic beads. The CD14+ cell threshold for successful manufacturing without depletion will be discussed and additional samples will be analyzed.
Mitochondrial Membrane Potential Identifies Cells with Enhanced Stemness for TCR and CAR based Adoptive Immunotherapy of Cancer
Authors: Sukumar Madhusudhanan , Restifo Nicholas 
Published: January 2023

Abstract
Long-term survival and anti-tumor immunity critically depends on their metabolic fitness but approaches to select metabolically robust T cells for T cell based immunotherapy remains unknown. We utilized a lipophilic cationic dye (tetramethylrhodamine methyl ester) to identify and isolate metabolically robust T cells based on mitochondrial membrane potential (ΔΨm). Cells with lower membrane-potential (low ΔΨm) had a molecular profile of memory precursors and displayed an enhanced ability to enter the memory pool as compared to cells displaying higher mitochondrial potential (high ΔΨm) characteristic of short lived effectors. Interestingly, we also found that multiple distinct negative inhibitory receptors such as PD1, CTLA4, BTLA, LAG3 and TIM3 were enriched in the high ΔΨm subset compared to the low-ΔΨm subset. Comprehensive metabolomics profiling demonstrated global features of metabolic fitness (increased lipid oxidation) in low ΔΨm sorted CD8+ T cells. Transfer of low ΔΨm T cells was associated with superior long-term in vivo persistence as evidenced by 100 fold increase in the frequency of T cells 300 days after adoptive transfer, augmented autoimmunity and an enhanced capacity to eradicate established cancer compared with high ΔΨm cells. Mechanistically, we found that the IL2 driven mTOR activation caused increased mitochondrial oxygen consumption and accumulation of higher level of ROS. Surprisingly, use of Ψm to enrich for cells with superior metabolic features was observed within central memory, effector (Tc17, Th1, and Th17) T cells and long term hematopoietic stem cells (LT HSC). This novel metabolism based approach may be applicable to therapies involving transfer of lymphocytes for treatment of advanced cancer.
Models of TCRαβ/CD19-depleted hematopoietic stem cell transplantation
Authors: Baiu Dana C , Otto Mario 
Published: January 2023

Abstract
Accelerating the reconstitution of protective immune T cell populations and immune function, while avoiding graft-versus-host disease (GVHD), is crucial for improving clinical outcomes following hematopoietic stem cell (HSC) transplantation. We developed and validated a clinical HSC graft engineering technique that selectively depletes αβ TCR T cells and CD19+ B cells from the graft, thereby minimizing GVHD and lymphoproliferative disease while maintaining HSC and important immunoprotective cell populations in the graft. The enrollment of the first patients in a University of Wisconsin conducted phase I clinical trial using this TCRαβ/CD19-depletion method in haploidentical HSC transplantation is to begin in the current year. In parallel, we developed preclinical models to explore the post-transplant HSC persistence and differentiation, and to evaluate the role of accompanying cell populations in facilitating HSC engraftment and immune recovery. We evaluated the efficiency of both CliniMACS and AutoMACS αβ TCR T cell depletion procedures by flow cytometry. Threshold numbers of contaminating αβ TCR T cells capable of inducing acute GVHD were determined after adoptive cell transfer in immunodeficient NOD.Scid.gamma mice. In vivo post-transplant persistence of αβ TCR and of γδ TCR T cells, NK cells and CD34+ engraftment were determined by flow cytometry at early (1–4 week) and late (5–12 week) time points. In addition to monitoring the post-transplant animal health status as a measure of graft alloreactivity/xenoreactivity, T cell activation markers (CD197, CD45RA) were assessed by flow cytometry. The murine models we developed will be instrumental in determining the effect of drug therapies on T cell reconstitution.
Temporal changes in tumor-associated immune cell populations in subcutaneous Tu-2449SC tumors transduced with Toca 511 following treatment with 5-fluorocytosine
Authors: Robbins Joan , Mitchell Leah , Yagiz Kader , Espinoza Fernando Lopez , Mendoza Daniel , Jolly Douglas J 
Published: January 2023

Abstract
Toca 511 (vocimagene amiretrorepvec), a gamma retroviral replicating vector encoding cytosine deaminase, with 5-fluorocytosine (5-FC), kills tumor by local production of 5-fluorouracil (5-FU), and induces local and systemic immunotherapeutic response resulting in long-term survival after cessation of 5-FC. This work identified the immune cell populations that change over time following administration of 5-FC to mice bearing Tu-2449SC tumors expressing cytosine deaminase.
Tu-2449SC cells (2% pretransduced with Toca 511) were injected subcutaneously in B6C3F1 mice. 5-FC or PBS treatment was initiated once tumors were palpable. Tumor burden was significantly reduced in mice that received 5-FC vs. PBS control. At 3, 6, 9, and 14 days after 5-FC start, spleens and tumors were harvested for immunophenotyping. 3 days after treatment initiation, B cells and CD4+ T cells in the tumor were significantly reduced with 5-FC. Of the CD4+ T cells that remained in 5-FC treated tumors, a greater number expressed markers of activation (41BB, CD40L, and PD-1), were defined as effector T cells, and fewer were naïve.
At day 6 post 5-FC initiation, CD4+ T cell percentages were no longer reduced with 5-FC vs. PBS. Tumor associated macrophages (TAM), myeloid derived suppressor cells (MDSC), and monocyte populations were significantly reduced in tumors 6 days after 5-FC start. Myeloid cell depletion correlates with previous work with systemic 5-FU.
At 14 days post 5-FC, TAM and MDSC remained reduced in tumors of treated animals, and both CD4+ and CD8+ T cells were significantly increased.
Toca 511+5-FC treatment results in reduced tumor burden and creates a tumor microenvironment that is more permissive to immune activation and anti-tumor immune response.
T cell exhaustion is Reinforced by Progressive De novo DNA Methylation Programming
Authors: Youngblood Ben A , Ghoneim Hazem E. , Abdelsamed Hossam Aly , Carter Robert , Hale Jeffery Scott , Ahn Eunseon , Im Sejin , Ahmed Rafi 
Published: January 2023

Abstract
Antigen-specific CD8 T cells play a critical role in controlling chronic infections and cancer, but progressively lose their effector functions during prolonged antigen exposure. Repression of CD8 T cell effector functions, commonly referred to as T cell exhaustion, limits the ability of the immune system to purge the chronic pathogen from the host. It has recently become recognized that CD8 T cell exhaustion programs can be reinforced and heritably maintained. Therefore in order to develop and/or improve current therapeutic approaches that utilize host antigen-specific T cells to treat chronic infections or cancer a major challenge for the field is to identify mechanisms that stabilize T cell exhaustion. We have recently found that epigenetic modifications acquired in pathogen-specific CD8 T cells during prolonged antigen exposure reinforce T cell exhaustion. Using the LCMV model system of chronic viral infection we investigated the role of Dnmt3a mediated de novo DNA methylation in regulating CD8 T cell exhaustion. Strikingly, conditional deletion of Dnmt3a in activated CD8 T cells blocked the cells from becoming exhausted. Longitudinal analysis of whole-genome methylation programming of WT and Dnmt3a cKO CD8 T cells from chronically infected animals reveals progressive acquisition of Dnmt3a-dependent DNA methylation programs in genes, including interferon gamma, that are coupled to the progressive decline of effector functions. These results have significant implications for therapeutic strategies that utilize reactivation of host pathogen-specific CD8 T cells to control chronic viral infections or cancer and provide a nucleotide-resolution map of epigenetic programs progressively acquired during T cell exhaustion.
Intracellular delivery of anti-pPKCθ (T538) via protein transduction domain mimics for T cell immunomodulation
Authors: Ozay Emrah Ilker 
Published: January 2023

Abstract
Humanized antibodies are increasingly gaining attention for their therapeutic potential in immunotherapy but, to date, the collection of cellular targets has been limited to those that are secreted from cells or are expressed on the cell surface. This approach leaves a wealth of intracellular proteins unexplored as potential targets. We evaluated the potential for delivering a phospho-specific antibody into human peripheral blood mononuclear cells (hPBMCs) as a means to interrogate and modulate their response to activating stimuli. Protein kinase C-theta (PKCθ) is essential to T cell activation, proliferation, and differentiation, and its phosphorylation at specific residues is required for its activity. Here we report on the design, synthesis, and characterization of a protein transduction domain mimic (PTDM) capable of efficiently delivering the antibody against phosphorylated PKCθ (T538) into hPBMCs. We designed an in vitro delivery method for the PTDM:anti-pPKCθ complex then assessed T cell activation and the expression of various downstream indicators of T cell activation and differentiation. In proof-of-principle experiments using a humanized, lymphocyte transfer model of graft-vs-host disease, we evaluated the durability of PTDM:anti-pPKCθ modulation, when delivered into hPBMCs ex vivo. Altogether, these results demonstrate that PTDM:antibody complexes can be readily introduced with high efficacy into traditionally hard-to-transfect human PBMCs to modulate immune function, eliciting a biological response sufficient to alter disease progression. Thus, PTDM:antibody delivery may represent an efficient ex vivo approach to manipulating cellular responses by targeting intracellular proteins.
Amplifying T cell responses to tumor antigens using a synthetic CD8-MyD88 co-receptor
Authors: Kaczanowska Sabina , Davila Eduardo 
Published: January 2023

Abstract
T cell–based immunotherapies are among the most promising approaches for patients with advanced melanoma and other cancers. The goal of this study is to boost T cell anti-tumor responses by harnessing the costimulatory effects of MyD88 signaling in T cells. We previously demonstrated that Toll-like receptor (TLR) engagement on T cells increases their proliferation, effector function, and survival in a MyD88-dependent manner. However, a challenge to stimulating TLRs on T cells in vivo is inefficiencies in delivering TLR ligands to the tumor site or adverse effects of TLR signaling in cancer cells. We hypothesized that coupling TLR and T cell receptor (TCR) signaling would provide antigen-dependent co-stimulation resulting in enhanced anti-tumor function. We developed a novel synthetic co-receptor consisting of a CD8a and MyD88 fusion protein to induce MyD88 signaling in a TCR–dependent but TLR ligand–independent mechanism. We have compelling data indicating that CD8-MyD88 stimulation improves T cell expansion and function in a tumor antigen–dependent manner. Furthermore, CD8-MyD88 T cells exhibit promising anti-tumor responses in mice with established melanoma tumors. Our strategy represents a universal approach since the CD8-MyD88 is not limited to a specific tumor antigen nor MHC haplotype. CD8-MyD88 also improves T cell responses to low concentrations of antigen, providing the opportunity to expand a large repertoire of tumor-reactive T cells. Furthermore, CD8-MyD88 activates MyD88 signaling in the engineered T cells exclusively, thus eliminating undesirable responses of TLR signaling in tumor cells. These studies represent a novel and versatile therapeutic approach for boosting T cell responses to a variety of cancers.
Selected TLR agonists act in synergy to reprogram DC-NK cross-talk and generate effector T cells in nicotinic environment
Authors: Nouri-Shirazi Mahyar A , Tamjidi Saba , Nourishirazi Erika 
Published: January 2023

Abstract

Background
The magnitude of immune responses to vaccination is a critical factor in determining protection from clinical disease. It is recognized that cigarette smoke dampens the immune system and increases the risk of vaccine-preventable diseases. We reported that the nicotine component of cigarette smoke disrupts the differentiation and functional properties of DCs that are pivotal in the initiation of immune response to vaccines.

Objective
In this study, we investigated whether TLR agonist(s) could potentially overcome the degrading effects of nicotine on human DC and DC-NK cross-talk essential for effector T cell generation.

Methods
nicDC, nicDC-NK, and nicDC-NK-T cultures exposed to individual and combined TLR agonists were evaluated for expression of corresponding costimulatory and activation molecules, cytokines, and intracellular cytokine IFN-g using ELISA and flow cytometry.

Results
Our data shows that among the panel of TLR agonists tested, TLR3 and TLR8/7 synergistically optimize nicDC maturation required for IFN-g production by co-cultured NK cells. Importantly, similar to DC-NK, nicDC-NK treated with TLR3 and TLR8/7 and co-cultured with naïve T cells promoted comparable number of effector T cells producing IFN-g.

Conclusion
Our data suggest that the addition of appropriate TLR agonist(s) to vaccine formulation could potentially improve the smokers’ immune response to vaccination. NIH-NIAID 1R03AI103750-01A1
In vivo characterization of the immune response towards the pathogenic Escherichia coli antigen SslE and modulation of the intestinal microbiota
Authors: Naili Ilham , Buonsanti Cecilia , Soriani Marco , Desvaux Mickael , Jubelin Gregory , Vieru Miguela , Baudner Barbara C. , D’Oro Ugo 
Published: January 2023

Abstract
Pathogenic E. coli, both intestinal (InPEC) and extraintestinal (ExPEC), account for a wide range of diseases, which can be fatal, across developing and developed countries. The vast array of virulence factors they can acquire may dramatically increase the severity of the infection. Emergence of resistant strains often renders antibiotics inefficient; in the case of Shiga toxin-producing E. coli, antibiotics can even be detrimental. Hence, a broad spectrum E. coli vaccine could be a promising alternative to prevent the spread of such diseases, while offering the potential for covering against several InPEC and ExPEC at once.
Using the «reverse vaccinology» approach on an ExPEC strain, nine antigens were identified as protective against a mouse sepsis model. With 82% protective efficacy, SslE (Secreted and Surface-associated Lipoprotein of E. coli) was the most potent candidate; additional models demonstrated that SslE was also cross-protective against other ExPEC strains. Functional assays have demonstrated in vitro and in vivo that SslE is a mucinase which plays an important role for colonization and virulence of E. coli.
To better understand the mechanism behind such protection, particularly at the mucosa, we are investigating the intestinal immune response obtained after immunizations with SslE using different routes of injection. In-depth analysis of each regimen will allow us to determine the most efficient method of immunization and adjuvant choice for SslE to be protective. Further, using this model of immunization, we will evaluate the potential perturbation caused by SslE on a human intestinal microbiota. These findings will be important to help us optimize the development of a potential broad spectrum vaccine.
Finding the evolutionary precursor of vertebrate hematopoietic lineage: Functional and molecular characterization of B. schlosseri immune system.
Authors: Rosental Benyamin , Kowarsky Mark A. , Corey Daniel M. , Ishizuka Katherine J. , Palmeri Karla J. , Chen Shih-Yu , Sinha Rahul , Voskoboynik Ayelet , Weissman Irving L 
Published: January 2023

Abstract
We are characterizing the immune system and cell populations of the tunicate model Botryllus schlosseri on functional and molecular levels. The chordate B. schlosseri belongs to a group that is considered the closest living invertebrate relative of vertebrates. Using FACS analysis we can isolate 12 populations of B. schlosseri cells by size, granularity and auto fluorescence. In order to increase the number of isolated cell populations, we used Cytof to scan large numbers of antibodies. Antibodies that differentially bind to B. schlosseri cells were validated by flow cytometry. Serum against the Botryllus Histocompatibility Factor, and lectins and fluorescent reagents activated by enzymatic activity were also used to differentiate live B. schlosseri cell types. Using these markers we isolated 34 cell populations and created RNA libraries for deep sequencing. Additionally, we developed immunological assays for cytotoxicity, phagocytosis and stem cell tracking. Using gene expression analysis we found hematopoietic stem cells and progenitors; in transplantation assays we showed their capability to induce chimerism of pigmentation and differentiation to other cell types, as well as localization to the stem cell niches. We characterized 3 different phagocytic cell types including a previously undescribed myeloid derived cell population. We describe the B. schlosseri cytotoxic cell population that originates as a large granular lymphocyte-like cell type that become morula cells upon allogeneic challenge activation. Altogether, it seems that the common ancestor of tunicates and vertebrates had a true hematopoietic myeloid lineage, while the cytotoxic cells are a convergent evolutionary system.
Polyfunctional cytokine production by central memory T cells from cattle in response to Mycobacterium bovis infection and BCG vaccination
Authors: Maggioli Mayara Fernanda , Palmer Mitchell V. , Thacker Tyler T , Vordermeier Martin , McGill Jodi L , Larsen Michelle H. , Jacobs William R. , Waters W. Ray 
Published: January 2023

Abstract
Polyfunctional T cells simultaneously produce IFN-γ, IL-2 and TNF-α and play relevant roles in several chronic infections, including TB. Mycobacterium bovis infection of cattle elicits ex vivo polyfunctional T cell responses. Vaccine-elicited IFN-γ Tcm (CD4+ CD45RO+ CCR7+) responses correlate to protection in cattle. The objective was to assess polyfunctional Tcm responses to vaccination with BCG or BCG mutants and subsequent M. bovis challenge in cattle. BCG induced polyfunctional Tcm to PPD and Ag85A/TB10.4. After challenge, TNF-α/IFN-γ Tcm responses to PPD, Ag85A/TB10.4 or ESAT-6:CFP10 were greater in non-vaccinates in comparison to vaccinates; while TNF-α/IFN-γ/IL-2 Tcm responses were lower. Both vaccines showed similar protection; however, vaccinated animals that were M. bovis culture negative had lower TNF-α/IFN-γ polyfunctional Tcm responses to ESAT-6:CFP10 3wpi than did culture positive vaccinates. In conclusion, BCG vaccination elicited Tcm polyfunctionality, mainly TNF-α/IFN- γ co-producers. Dampened TNF-α/IFN-γ and greater TNF-α/IFN-γ/IL-2 Tcm responses after challenge may indicate enhanced pathogen control and correlate with vaccine elicited protection.
NK cell activation and metabolic syndrome in a small nonhuman primate model of Hepacivirus infection
Authors: Manickam Cordelia , Reeves Roger Keith 
Published: January 2023

Abstract
Hepatitis C virus (HCV) infects greater than 170 million people worldwide. The disease remains a major source of global morbidity and mortality, but factors leading to resolution or chronicity remain unclear. In this study, we evaluated acute and longitudinal immune responses in an understudied model of HCV infection, GBV-B infection of common marmosets. Plasma viremia was detectable by day 3 post infection (pi) generally peaking by day 14 pi, at which point high virus titers were also found in the liver. Virus clearance was observed in all animals by day 112 pi. Elevated serum levels of ALT, AST and GGT, along with microscopic liver lesions of inflammation and fibrosis were indicative of hepatitis. Increases in serum creatine kinase, triglycerides and glucose, and decreased amylase levels suggested metabolic dysregulation. Interestingly, natural killer cells, which have been linked to bystander damage in the liver, demonstrated increased expression of perforin, Ki-67 and CXCR3 following infection, and were increased up to 5-fold in the liver by day 14 and remained elevated even after viral clearance. Generalized T cell activation was observed by day 28 pi, and GBV-B-specific responses were generally focused on NS3 peptides (IFN-γ ELISPOT) and were up to 10-fold higher in liver compared to peripheral organs. Our results suggest that early NK cell activation could play a major role in limiting hepaciviral infection in marmosets, or alternatively contribute to tissue inflammation and liver damage. Regardless, these data highlight the recapitulation of inflammation and metabolic dysfunction in a tangible small animal model for HCV and suggest innate cell targeting could be an attractive target for future therapeutics.