bluuebunny/crossref_metadata_2025_split huggingface dataset:
Protease activated interleukin-2 cytokine fusion proteins for tumor immunotherapy (46.17)
Authors: Skrombolas Denise , Puskas John , Sedlacek Abigail , Narrow Wade , Mastrangelo Michael , Bowers William , Lord Edith , Frelinger John 
Published: January 2023

Abstract
Cytokines are being used for tumor immunotherapy, but when delivered systemically, negative side effects limit their utility. To mitigate these side effects but maintain their anti-tumor effects, we have constructed a panel of fusion proteins (FPs) with IL-2 joined to a specific inhibitor, separated by specific protease cleavage sequences. The rationale is that at the tumor site, where the concentration of a protease is high, the FP will be cleaved, allowing the IL-2 to dissociate from the inhibitor, increasing the biological activity of the cytokine and enhancing anti-tumor effectors. We have expressed these FPs and showed that cleavage in vitro results in increased biological activity of IL-2. In the Colon 38 peritoneal tumor model, delivery of a FP results in increased frequency of T cells (p&lt; 0.0001) and NK cells (p&lt; 0.01) compared to vehicle control treatment, and a corresponding decrease in tumor growth. To test for systemic side effects and evaluate the effects of FP delivery, we used an adeno-associated virus (AAV) delivery system to achieve sustained, high systemic levels of the FPs in mice. AAV-expressed FPs persist at high levels (~500 pg/ml) for at least 9 weeks and do not appear immunogenic. Importantly, unlike injection of free IL-2, expression of the FPs did not generate an inflammatory cytokine response as determined by a 22-analyte Luminex assay. Thus, protease activated cytokine fusion proteins may be a valuable means to enhance tumor immunotherapy.
Development of protease activated cytokines that can be used to alter cytokine tumor microenvironments (46.20)
Authors: Frelinger John , Skrombolas Denise , Puskas John , Sullivan Mark 
Published: January 2023

Abstract
We are developing an immunotherapy approach in which cytokines can be preferentially activated by proteases at the site of growing tumors, thus altering the tumor microenvironment. This approach employs a novel fusion protein (FP) in which a cytokine is joined to a specific binding moiety, such as a single-chain antibody (scFv), separated by a protease cleavage site. The rationale is that before cleavage, the cytokine is largely inactive, but that after cleavage by a protease preferentially expressed at the tumor site, the cytokine becomes biologically available as the inhibitory scFv dissociates from the cytokine. To test this approach, we have isolated scFvs using phage display that bind to human IL-2, including one that can be inhibited in its binding to IL-2 by the neutralizing anti-human IL-2 antibody MQ1-17H12. This scFv has been used to construct and express a FP containing a protease cleavage site. Immunoblot analyses revealed that the FP can be cleaved in vitro and that cleavage results in increased functional activity of the IL-2. To investigate how the increase in functional activity of the IL-2 depends upon the binding characteristics of the scFv, we have created mutations that alter its affinity for IL-2, which are being evaluated for how they affect the characteristics of the FP. Since scFvs that bind to essentially any molecule can be readily isolated, this should be a general approach for increasing the biologic activity of immuomodulators at tumor sites.
The immune system is pivotal in mediating the anti-tumor effects of radiation therapy (46.3)
Authors: Gerber Scott , Sedlacek Abigail , Lord Edith 
Published: January 2023

Abstract
A new paradigm has emerged demonstrating that the immune system mediates many of the anti-tumor effects of radiation therapy (RT). RT continues to be a cornerstone in the treatment for many cancers, unfortunately not all individuals respond effectively to RT resulting clinically into two groups consisting of non-responders (progressive disease) and partial/complete responders (tumor control/cure). We have designed a unique model that mirrors this clinically relevant phenomenon in which mice bearing colon38, a colon adenocarcinoma, were treated locally with 15Gy RT resulting in both non-responders and responders. Interestingly, our data uncovered a window of interest four days following RT in which critical events occurred that ultimately determined the effectiveness of therapy. Intratumoral IFNγ was increased in responsive tumors and licensed CD8 T cells to exhibit potent lytic activity against tumor cells; a response that was greatly diminished in tumors refractory to RT. We explored whether enhancing IFNγ and CD8 T cells via immunotherapy would increase the responder to non-responder ratio. Indeed, multi-modality therapy comprised of IL-12 and RT in mice bearing colon38 tumors resulted in complete remission of cancer in 100% of cases compared to a cure rate of only 40% with RT alone. Our data suggests that the efficacy of RT may depend on the immune response generated shortly after treatment and targeting this component may greatly enhance the effectiveness of RT.
A novel anti-B7-H4 human recombinant antibody overcomes TAM-mediated T-cell inhibition (46.43)
Authors: Dangaj Denarda , Zhao Aizhi , Lanitis Evripidis , Joshi Shree , Sandaltzopoulos Raphael , Powell Jr Daniel J , Scholler Nathalie 
Published: January 2023

Abstract
Tumor polarization of macrophages represents an essential immune escape mechanism thus targeting tumor-associated macrophages (TAMs) could boost anti-tumor immune response. To model tumor-induced macrophage polarization, we developed an in vitro system of transwell co-culture with human macrophages, T cells and ovarian cancer cells (OVCAR3) where in vitro-matured macrophages and CD3/CD28 subactivated T cells were separated from tumor cells; in vitro-generated TAMs overexpressed the immunosuppressive molecule B7-H4 and inhibited T cell functions. We then hypothesized that targeting B7-H4 could block T cell inhibition mediated by B7-H4+ TAMs. We isolated anti-human B7-H4 scFvs from a novel yeast-display scFv library derived from B cells of ovarian cancer patients’ ascites. T cell proliferation and activation were restored in presence of anti-B7-H4 scFv #56 during transwell co-culture, suggesting that scFv #56 could block B7-H4 inhibitory signals mediated by TAMs. To further address whether scFv #56 activity was TCR-dependent, we set up experiments with antigen-specific T cells (MART1) and wild type or B7-H4-transduced artificial APCs (T2) loaded with MART1 peptide. T cell cytokine response was downregulated by B7-H4 expression on T2 and partially restored in presence of scFv#56. Our results indicate that competing with B7-H4 inhibitory signals on TAMs can promote T cell activity. We propose that in vivo use of anti-B7-H4 scFv could increase anti-tumor T-cell immunity.
Overcoming obstacles to naïve T lymphocyte trafficking in tumor-draining lymph nodes (46.47)
Authors: Muhitch Jason , Ito Fumito , Fisher Daniel , Diehl Michael , Appenheimer Michelle , Evans Sharon 
Published: January 2023

Abstract
Effective tumor immune responses rely on trafficking of rare tumor-specific T cells to tumor-draining lymph nodes (TdLN) where they undergo activation and expansion, and subsequently to tumor tissues to gain access to neoplastic targets. Here, we evaluated the trafficking of naïve CD8 T cells to TdLN. Lymphocyte entry into TdLN was diminished by ~50% in mice bearing B16 melanoma, suggesting a localized mechanism of immune escape. Intravital microscopy revealed that while normal proportions of lymphocytes initiated tethering and rolling in high endothelial venules (HEV) of TdLN (34%), the fraction of lymphocytes that transitioned from rolling to firm arrest was reduced compared to unchallenged lymph nodes (8 and 20%, respectively). Diminished trafficking in TdLN was attributed to suboptimal presentation of the CCL21 chemokine in HEV. We further determined that inflammatory cues triggered by systemic thermal therapy (STT, 39.5 ± 0.5°C, 6 h) could restore normal T cell entry in TdLN, paralleling our previous findings in non-tumor-challenged mice. Improved homing was due to a reduction in rolling velocity and an increase in proportion of T cells undergoing firm arrest. STT was further shown to enhance the display of trafficking molecules required for firm arrest, i.e., CCL21 and ICAM-1 in HEV. These findings suggest that therapeutic targeting of HEVs for improved entry of naïve T cells in TdLN may be a novel strategy to overcome immune evasion.
CD28-mediated pro-survival signaling in multiple myeloma (46.6)
Authors: Murray Megan , Nair Jayakumar , Lee Kelvin 
Published: January 2023

Abstract
Multiple myeloma (MM) is a plasma cell neoplasm which requires molecular bridges to the bone marrow microenvironment, one of which may be CD28. It has been observed that CD28 is expressed on MM cells and is correlated with progressive disease and worse outcomes. We have previously shown that activation of CD28 protects MM cells from chemotherapy. We have also observed that co-culture of MM cells with bone marrow stromal cells such as dendritic cells (which express the CD28 ligands CD80 and CD86) protect MM cells from chemotherapy or serum starvation. This protection is entirely abrogated by either a CD28 blocking antibody (CD28.6) or by CTLA4-Ig, which binds to the ligands CD80 and CD86. Since CD28 in T cells signals via a PI3K-Akt axis, we interrogated whether or not this pathway is crucial for CD28-mediated MM cell survival. We observed that blockade of PI3K signaling using chemical inhibitors abrogates CD28-mediated survival. Since the pro-apoptotic molecule Bim is both critically important in MM biology and downstream of PI3K-Akt, we next examined whether or not it is regulated by CD28. When CD28 is activated, Bim mRNA and protein levels are downregulated, and when CD28 signaling is blocked, Bim protein is upregulated. Moreover, when Bim is knocked down with siRNA, MM cells are no longer sensitive to CD28 blockade. Taken together, these data show that CD28-mediated survival is regulated (at least in part) by downregulating Bim and thus preventing apoptosis.
DC enzymes as early biomarkers of an effective vaccine (46.7)
Authors: Marvel Douglas , Finn Olivera 
Published: January 2023

Abstract
Previous work by our lab and others has shown that human MUC1 transgenic mice (MUC1.tg) are hyporesponsive to vaccination with some forms of MUC1 and fail to mount a spontaneous immune response against MUC1 positive tumors. An interesting correlate of hyporesponsiveness to MUC1 vaccination is that as early as 24 hours post vaccination, splenic dendritic cells (DC) in wild type (WT) mice upregulate expression of a group of proteolytic enzymes previously characterized as functioning primarily in pancreatic catabolism (e.g. Trypsin-1, Trypsin-4, Carboxypeptidase B1). This upregulation does not occur in the MUC1.tg animals. Using an in vitro co-culture system and in vivo antibody depletion, we show that regulation of enzyme expression in DC is dependent on their interaction with CD4 T cells such that CD4 effector T cells (Teff) up-regulate these enzymes, whereas the presence of activated CD4 regulatory T cells (Treg) actively suppress this activity in an IL-10 dependent manner. TLR agonists do not upregulate enzyme expression, suggesting differences are due to alternative DC activation pathways following their interaction with T cells. We propose that one or more of these enzymes, which appear to be co-regulated, could serve as early markers of, and most likely also as contributors to, DC activation by helper T cells. In contrast, lack of their expression marks insufficient activation or active suppression by Tregs.
Effects of aging on the human adaptive immune system revealed by high-throughput DNA sequencing of T cell receptors (47.16)
Authors: Robins Harlan , Desmarais Cindy 
Published: January 2023

Abstract
As we age, our adaptive immune system undergoes changes that affect vaccine efficacy as well as susceptibility to disease and infection. Identifying the factors that determine immune repertoire diversity and studying the impact of changes in diversity through the life span is therefore an important public health concern. The objective of our study was to characterize alterations that occur in the T cell receptor repertoire as we age. To address these questions we utilized a high-throughput sequencing (HTS) approach developed by Adaptive Biotechnologies. This technology (immunoSEQ) amplifies rearranged T-cell receptor CDR3 chains and uses HTS to capture millions of CDR3 sequences. CD4+ Memory, CD4+ Naïve, CD8+ Memory and CD8+ Naïve T cell populations from peripheral blood were isolated from a total of 15 healthy volunteers, ranging in age from 25 to 80. The CDR3 region of each TCRB chain was then sequenced, generating a comprehensive profile of the T cell receptor repertoire for each cell population. We observed a reduction in the relative contribution of CD8+ naïve T cells with advancing age. This effect was exacerbated by CMV infection. These results provide a basis to further characterize the effects of immunosenescence on the adaptive immune system.
The forkhead transcription factor, Foxo1, coordinates an adaptive, pro-survival response to calorie restriction in T cells (47.3)
Authors: Gubbels Bupp Melanie , Edwards R. 
Published: January 2023

Abstract
In addition to longevity enhancement, calorie restriction has been reported to slow the appearance of aging-related T cell abnormalities. However, the molecular mechanism underlying this phenomenon in T cells is ill-defined. We report that short-term calorie restriction in wild type mice inhibited the spontaneous apoptosis of naïve CD8+ T cells by approximately three fold, as compared to cells isolated from mice fed ad libitum. An ortholog of the forkhead transcription factor, Foxo1, is required for the longevity benefits of calorie restriction in C. elegans. Therefore, mice with a conditional, T cell-specific deficiency of Foxo1 (Foxo1 cKO) were also subjected to calorie restriction. Indeed, the pathway through which calorie restriction results in reduced naïve CD8+ T cell apoptosis appears to involve Foxo1, as calorie restriction in the absence of Foxo1 did not result in reduced naïve CD8+ T cell apoptosis. Instead, naïve CD8+ T cell apoptosis was similarly high for cells isolated from ad libitum wild type, ad libitum Foxo1 cKO, and calorie restricted Foxo1 cKO mice. Thus, Foxo1 coordinates an adaptive, pro-survival transcriptional response to calorie restriction. To our knowledge, Foxo1 is the first transcription factor identified to contribute to this process in mice.
IL-15 inhibits caspase-3-mediated T cell apoptosis (47.9)
Authors: Thai Phan , Fortner Karen , Budd Ralph 
Published: January 2023

Abstract
Active caspase-3 is an effector cysteine protease crucial for apoptosis. T lymphocyte survival impinges on the ability of the cell to keep the levels of active caspase-3 in check. Previous studies have shown that despite IL-2 and IL-15 sharing γc and IL-2Rβ (CD122) receptor subunits and being capable of stimulating proliferation and activation of T cells, these cytokines differentially regulate the survival of T cells. Although IL-2 plays a crucial role in activation-induced cell death (AICD) of peripheral T cells whereas IL-15 extends survival of T cells by inhibiting AICD, the mechanism behind this difference in susceptibility to AICD is not well understood. In this study, we report that IL-2-cultured primary murine T cells manifest considerably higher levels of active caspase-3 compared to IL-15-cultured T cells. This was independent of initiator caspases, such as caspase-8 and -9. Furthermore, there was a very robust increase in caspase activity as well as cell death following α-CD3 restimulation of IL-2-cultured T cells, whereas the IL-15-cultured T cells manifested almost no increase in caspase activity or cell death. Taken together these data support the hypothesis that IL-15 can inhibit T cell apoptosis and therefore prolong T cell survival by maintaining low levels of active caspase-3.
Monocytic lineage myeloid-derived suppressor cells are the principal suppressor population (48.11)
Authors: Haverkamp Jessica , Smith Amber , Qualls Joseph , Balouzian Liza , Bronte Vincenzo , Opferman Joseph , Murray Peter 
Published: January 2023

Abstract
Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of immunosuppressive cells and are identified in mice as Gr-1+ (Ly6G/C) CD11b+ cells with the ability to inhibit T cell proliferation. However, the key functional immunosuppressive MDSC population remains controversial. Functional dissection of MDSCs is limited by population heterogeneity because Ly6C is expressed on mature and immature monocytic and granulocytic cells, each of which could potentially mediate immunosuppression. Here we describe genetic and chemical strategies allowing us to ablate the entire population of granulocytic or monocytic MDSCs specifically. Our data define the monocytic-macrophage MDSC population as the predominant suppressor population. Furthermore, our data have implications for the engineering of MDSCs as active suppressors for autoimmune diseases and targeted therapies aimed at selective ablation of monocytic MDSC in the treatment of cancer.
Tolerogenic DC differentiation and development of immunosuppressive DC precursors (48.13)
Authors: VanGundy Zachary , Taylor Cassandra , White Angela , Baker Julie , Muth Dillon , Papenfuss Tracey 
Published: January 2023

Abstract
Tolerogenic dendritic cells (tDCs) are important myeloid cells which potently regulate innate and adaptive immune responses. We have previously found that the steroid hormone estriol (E3) generates tDCs in vivo which protect mice from developing autoimmune disease. To better understand tDC generation, we developed an in vitro tDC differentiation model. Myeloid progenitors were differentiated with GM-CSF in the presence of steroid hormones E3 and retinoic acid (RA) and both differentiated tDCs and their precursors (pre-tDCs) were phenotypically and functionally evaluated. We found that both E3 and RA tDCs had increased expression of stimulatory and inhibitory costimulatory markers and suppressed proliferation of responder immune cells in a dose-dependent manner, similar to in vivo tDCs. Interestingly, we also found that pre-tDC had enhanced immunosuppressive abilities over differentiated tDCs without marked changes in phenotypic cell composition. Although CD11b+Gr1+ populations were unchanged, increases in FoxP3+ expression were seen in responder splenocyte populations when co-cultured with pre-tDCs. These results suggest that pre-tDCs may be acting as myeloid derived suppressor cells (MDSCs) and are a potently immunosuppressive immature myeloid population that gives rise to tDCs. A better understanding of how tDCs are generated has potential therapeutic relevance for the treatment of numerous inflammatory diseases.
Role of C-type lectin receptor CLEC12A in human basophil activation (48.6)
Authors: Divekar Anagha , Conklin John , Yang Xifeng , Ransom John 
Published: January 2023

Abstract
CLEC12A is a member of the C-type lectin receptor family with a cytoplasmic immunoreceptor tyrosine-based inhibitory motif. Murine CLEC12A is expressed on variety of cell types including dendritic cells, monocytes/macrophages, B cells, neutrophils and basophils but not NK cells. In humans, CLEC12A is reported to be expressed on dendritic cells (DC, both myeloid and plasmacytoid), monocytes, B cells (normal and leukemic) and NK cells, but not on T cells or basophils. Performing flow cytometric analysis of human peripheral blood leukocytes using an anti-human CLEC12A antibody (clone 50C1), we report CLEC12A expression on a subset of CD3, CD19 and CD56 negative cells. Further analysis revealed these cells to be CCR3+ basophils. Antibody mediated ligation of CLEC12A on DCs results in cytokine production and cellular maturation and can inhibit NK cell mediated cytotoxicity. In response to IgE and IL-3, basophils can induce Th2 differentiation and amphiregulin production, respectively. We hypothesize that ligation with CLEC12A will alter IL-3 and/or IgE mediated basophil activation, which will be studied in our ongoing work.
IL-27 and B7-H1 mediate liver plasmacytoid DC ability to suppress delayed-type hypersensitivity responses (48.9)
Authors: Matta Benjamin , Raimondi Giorgio , Rosborough Brian , Thomson Angus 
Published: January 2023

Abstract
IL-27 and B7-H1 are regulatory molecules that modulate immune responses. Plasmacytoid (p) dendritic cells (DC) can promote tolerance. We investigated the contribution of IL-27 and B7-H1 to the ability of murine pDC to regulate T cell responses and to suppress delayed-type hypersensitivity (DTH) reactions to ovalbumin (OVA). PDCA-1+ pDC were purified from livers and spleens of Flt3L-mobilized, C57BL/6J wild type (WT), Ebi3 knockout (KO), or B7-H1 KO mice. pDC phenotype was evaluated by flow cytometry. IL-27p28 was quantified by ELISA and flow cytometry. Ebi3 expression was evaluated by Western blot. T cell allostimulatory capacity of pDC was determined in CFSE MLR. For DTH, mice were immunized with OVA-pulsed bone marrow-derived conventional DC with or without pDC from mice fed OVA. Mice were challenged with heat-aggregated OVA and footpad swelling and T cell responses quantified. Liver pDC express high levels of IL-27p28 and Ebi3 (the components of IL-27) but low IL-12p35 (that forms IL-35 with Ebi3). They express high levels of B7-H1 compared to spleen pDC. Ebi3 KO and B7-H1 KO pDC show enhanced T cell allostimulatory capacity compared to WT pDC. Liver but not spleen pDC suppress DTH responses to OVA, a phenomenon dependent on IL-27 and B7-H1. In conclusion, liver pDC exhibit high expression of IL-27 and B7-H1 that mediate their regulatory ability in vitro and in vivo. Liver pDC, IL-27, and B7-H1 may be important therapeutic targets to promote tolerance or enhance immunity.
The role of a cx3cr1+/egfp mucosal resident dendritic cell network in mucosal homeostasis (49.10)
Authors: Velazquez Peter , Moriel Gabriela , Xu Cassie 
Published: January 2023

Abstract
Cxcr1 is expressed in all CD11c+ mucosal dendritic cells (DC). Mucosal DCs are perivascular and are interconnected to form a network. However, the importance of the network in maintaining mucosal homeostasis is poorly understood. We first aimed to gain insight on the structure of the DC network. Using multi-photon intravital microscopy, we demonstrate that the cx3cr1+/egfp DC network interconnects DCs between villi and throughout small and large bowel. The network overlaps a radiation resistant non-hematopoietic cell network. We next aimed to examine the role of DCs in vascular permeability. cx3cr1+/egfp DCs were selectively depleted by targeting depletion with CD11c-diptheria toxin receptor (DTR) transgenic intercross. Preliminary data suggests that DCs are not necessary to maintain vascular integrity, as measured by permeability of 70kDa dextran fluor. Critical for maintaining adaptive immunity is spatial temporal regulation of T cell migration as T cells survey antigen present cells (APC). Since DCs are a critical APC, T cells patrolling was examined following depletion of the mucosal DC network. Adoptive transfer studies demonstrate that CD4 and CD8 T cell migration remains intact despite selective depletion of DCs. In summary, the mucosal DC network represents a higher order of organization of DCs than previously appreciated. Resident DCs may play a role in maintaining vascular permeability but surprisingly do not play a role in regulating mucosal T cell migration.