bluuebunny/crossref_metadata_2025_split huggingface dataset:
Characterization of the Periplaneta americana immune response using image-based flow cytometry (P6068)
Authors: Herlands Robin , Qadir Anam 
Published: January 2023

Abstract
Several reports have emerged demonstrating that many insect species have unanticipated “trained” immune responses, where immunization with a pathogen has a significant and lasting protective effect. This is particularly interesting considering that many of the well-characterized vertebrate adaptive cell subsets and humoral tools associated with immunological memory are absent in invertebrates. Here we report our investigations of immunity in Periplaneta americana, the cockroach, and the use of imaging flow cytometry to investigate cellular changes in response to pathogen exposure. Staining for cellular features such as nuclear and cell membrane morphology, lysosomes, and other subcellular structures were used to categorize cells and monitor their changes over the course of an immune response. Stains are also used to reveal proliferation of certain cell subsets. As cockroaches are worldwide pests in both homes and hospitals, how they manage infection will be of broad interest. Additionally, elucidating rudimentary systems of adaptive immunity in invertebrates may facilitate our development of therapeutics for immunocompromised vertebrates whose well-understood mechanisms of defense are dysfunctional.
DC-sign as a marker for inflammatory skin dendritic cells in the pig (P6074)
Authors: Ricklin Meret , Summerfield Artur 
Published: January 2023

Abstract
The objective of this study was to phenotypically characterize dendritic cells (DC) associated with inflammatory skin diseases in the pig. To further study dermal DC, the expression profile of DC-sign, a C-type lectin and human DC-marker was characterized on various populations of DC. To this end, we isolated peripheral blood mononuclear cells (PBMCs), analysed blood monocytes, conventional blood DCs (cDC) and plasmacytoid DCs (pDC). Furthermore monocytes were differentiated to monocyte-derived DC (MoDCs) or monocyte-derived macrophages (MMθ). In addition, DC were isolated from separated epidermis and dermis of lesional and non-lesional skin. In the blood unstimulated pDCs, cDC and monocytes were negative for DC-sign expression. After LPS stimulation a slight upregulation could be observed. MoDC expressed higher levels of DC-sign than MMθ. Langerhans cells as well as dermal DC from normal skin were DC-sign-. However, after induction of an inflammation the newly immigrated DC were DCsign+. In contrast to Mθ these were CD14-CD163-. These results indicate that in the pig DC sign represents a marker for monocyte-derived inflammatory DC of use to study the pathogenesis of inflammatory skin diseases.
Analysis of the expressed porcine antibody repertoire in porcine and reproductive and respiratory syndrome virus infected pigs (P6077)
Authors: Schwartz John , Murtaugh Michael 
Published: January 2023

Abstract
Adaptive humoral immunity is mediated by three separate loci (one heavy chain and two chains) that each encode multiple gene segments which recombine to generate a vastly diverse antigen-binding repertoire. We have characterized the diversity of the porcine heavy and light chain repertoires in pigs that were either infected or uninfected with porcine reproductive and respiratory syndrome virus (PRRSV) using amplicon-based 454 high-throughput sequencing. Bioinformatic analysis revealed that a small number of variable gene segments dominate the expression of the immunoglobulin repertoire. Among both heavy and light chain transcripts, several exceptionally common immunoglobulin sequences were identified in PRRSV-infected pigs which were rare in uninfected pigs, suggesting that we detected clonally expanded PRRSV-specific B-cells. The total richness was estimated to be approximately 3.5x10^5 for heavy chain, 1.5x10^5 for kappa light chain, and 2.3x10^5 for lambda light chain. Our calculated richness is similar to that reported for humans, suggesting that the swine antigen-binding repertoire is similarly complex, despite lack of diversity in the porcine IGHV framework regions. Genetic heterogeneity was detected among pigs in their lambda light chain loci, including the identification of a novel IGLV3 gene segment that is highly expressed in pigs possessing it. Our analysis suggests that this heterogeneity influences gene segment usage and thus, may affect humoral immunity.
Infection with foot-and-mouth disease virus induces a natural killer cell response in cattle (P6080)
Authors: Patch Jared , Dar Pervaiz , Waters Ryan , Toka Felix , Golde William 
Published: January 2023

Abstract
Natural killer (NK) cells play an important role in innate immune defense against viral diseases. Although they develop from the same lymphoid precursor cells as B and T lymphocytes, NK cells lack an antigen specific receptor, and are regulated in an antigen-independent manner yet have effector functions similar to T cells. The activation state of NK cells is determined by the balance of activating and inhibitory signals transmitted through surface receptors. Target cell expression of MHC class I serves as a powerful inhibitory signal for NK cells. Foot-and-mouth disease (FMD) is a viral disease of cloven-hoofed animals that is of particular concern for livestock species including cattle, swine, sheep, and goats. Clinical disease is characterized by fever, viremia, and the presence of vesicles in the mouth and on the feet and teats. FMD virus (FMDV) has evolved several immune-evading characteristics, including inhibition of MHC class I surface expression. Although this loss of class I MHC expression would be expected to result in NK cell activation during FMDV infection, swine with FMD exhibit a loss of NK cell function that cannot be overcome by ex vivo cytokine stimulation. Here we show that, in contrast to swine, FMDV-infected cattle exhibit an increase in NK cell cytolytic activity that is accompanied by an early and brief increase in NK-like cytolysis by γδ T cells. These data represent a difference in disease pathogenesis between these two susceptible species.
Differential immune regulation of mir-155 in tumor growth (P2113)
Authors: Zhang Bin , Chen Siqi , Fan Jie , Dominguez Donye , Ye Cong 
Published: January 2023

Abstract
MiR-155 is upregulated in both myeloid and lymphoid cells following their activation. To address the role of host miR-155 in tumor growth, we examined the contribution of miR-155 to antitumor immunity using multiple models of syngeneic solid tumor growth in mice. Host miR-155 deficiency inhibited growth of transplanted LLC1 lung tumor cells but not B16 melanoma cells. Interestingly, there was a specific contribution of both T-cell-intrinsic and dendritic cell-intrinsic function of miR-155 positively regulating antitumor T cell immunity. However, host miR-155 deficiency did not lead to overt immune suppression, and instead stimulated robust T-cell- mediated antitumor immunity. On the other hand, the accumulation and function of myeloid-derived suppression cells (MDSC) were impaired in tumor- bearing mice lacking miR-155 and miR155-deficient MDSCs failed to induce T cell antigen unresponsiveness. Moreover, in vivo tumor-inhibitory effect of MDSC depletion by 5-FU was completely abrogated in miR155-deficient mice. Thus, the net antitumor effects of host miR-155 deficiency indicate that the impairment of immunosuppressive MDSCs possibly outweighs the potential suppressive effects of miR-155 deficiency in T cell and dendritic cell compartments. Our study identifies the differential immune regulation for miR-155 during tumor growth, and provides valuable insights for therapeutically intervention in cancer or autoimmune diseases using highly specific methods of modulating miRNAs.
Activating the TNFR super family member 4-1BB pathway for the expansion of tumor infiltrating lymphocytes for adoptive T cell therapy for metastatic melanoma patients. (P2119)
Authors: Chacon Jessica , Kale Charuta , Hwu Patrick , Radvanyi Laszlo 
Published: January 2023

Abstract
Adoptive T cell therapy using tumor infiltrating lymphocytes ‘TIL’ is the most effective therapy for metastatic melanoma patients that have failed first and second line therapies. The expansion of TIL from melanoma tumors occurs in two phases. The first phase expands the TIL from the excised melanoma tumor fragments using IL-2. The second phase is the Rapid Expansion Protocol, in which the TIL are activated with anti-CD3 antibody, IL-2 and feeder cells and expanded for 2 weeks. We believe there are shortcomings of Adoptive T cell therapy, such as the inability to grow TIL from all melanoma patients, the amount of time it takes to grow out the TIL and the lack of adequate co-stimulation for the TIL during expansion. 4-1BB is a co-stimulatory molecule expressed on CD8+ T cells after TCR stimulation. We found that 4-1BB is expressed on freshly isolated melanoma CD8+ TIL, suggesting recent tumor antigen exposure. Our hypothesis is that providing co-stimulation through the 4-1BB signaling pathway during the initial TIL outgrowth from tumor fragments accelerates TIL expansion, improves the output of CD8+ T cells, their effector-memory phenotype and anti-tumor specificity. We found that 4-1BB co-stimulation increased CD8+ TIL expansion, maintained CD28 expression and increased Granzyme B expression. Our findings suggest that augmenting the TNFR pathway through 4-1BB co-stimulation during initial melanoma TIL expansion improves proliferation and effector phenotype of the TIL.
Turning T cells on: epigenetically enhanced effector T cell activity following tumor cellirradiation (P2091)
Authors: Kumari Anita , Cacan Ercan , Garnett-Benson Charlie , Greer Susanna 
Published: January 2023

Abstract
While sub-lethal tumor irradiation enhances killing of colorectal carcinoma cells by tumor-specific CD8+ CTLs, the mechanisms responsible for increased activity of CTLs to irradiated tumor cells remain unknown. Two members of the tumor necrosis family super family of receptors, OX40L/TNFSF4 and 41BBL/TNFSF9, enhance CTL activation and survival. Our results indicate sub-lethal doses of radiation increase tumor cell expression of 41BBL and OX40L via epigenetic mechanisms as inhibition of HDACs (with TSA) and of DNMTs (with 5-AZA) results in significantly increased OX40L and 41BBL mRNA and protein. The combination of radiation and epigenetic modifiers results in greater expression of both receptors, suggesting overlap in the mechanisms by which radiation, 5-AZA, and TSA regulate OX40L and 41BBL. Finally, chromatin immunoprecipitation experiments reveal significantly increased H3 acetylation of 41BBL promoters following irradiation. Our results show non-lethal doses of radiation make human tumors amenable to immune system attack through epigenetic modulation of genes stimulatory to CTLs. The use of ionizing radiation to specifically enhance cancer immunotherapy strategies, through epigenetic modulation of genes stimulatory to CTLs, will have a broad impact on cancer therapy strategies and will extend the use of radiation in new directions.
Non-redundant requirement for CXCR3 chemokine receptor in T cell trafficking across tumor vascular gateways (P2137)
Authors: Mikucki Maryann , Skitzki Joseph , Fisher Daniel , Curry Kathryn , Frelinger John , Luster Andrew , Evans Sharon 
Published: January 2023

Abstract
Recent advances in the treatment of metastatic melanoma are designed to promote the cytotoxic CD8 T cell response (e.g., CTLA-4 blockade and adoptive cell transfer), but it remains poorly understood how T cells enter tumor tissues to contact tumor targets. Here, we investigated the hierarchy of chemokine receptor requirements during the trafficking of blood-borne T cells to melanoma tumors by studying three molecules associated with infiltration, i.e., CCR2, CCR5, and CXCR3. These studies used B16-F10 murine melanoma to discriminate the contributions of individual chemokine receptors to T cell trafficking as opposed to T cell retention, proliferation, and survival in situ. Tumor-reactive cytotoxic T cells expressed functional CCR2, CCR5, and CXCR3 in vitro, and their cognate chemokines (CCL2, CCL5, and CXCL10, respectively) were also detected in the tumor microenvironment. Unexpectedly, loss of CCR2 and CCR5 did not impact T cell delivery to melanoma tissues; only CXCR3 was required for extravasation of adoptively transferred T cells in tumors. Intravital microscopy further revealed that T cells require CXCR3 to transition from transient rolling to firm arrest during the stepwise migration cascade at the tumor vascular interface. Taken together, these studies establish the obligate role for CXCR3 in the migration of effector T cells into tumor tissue and support the development of chemokine-targeted strategies to improve T cell entry during cancer immunotherapy.
Pam3Cys4 internalization by naïve CD4+T cells accelerates t-bet promoter epigenetic modification and restrains TGF−β1 expressing tumor growth (P2153)
Authors: Ibrahim Mohsen , Toth Kelsey , Ponti Donatella , Calogero Antonella , Andreetti Claudio , Huang Howard , Kreisel Daniel , Kreisel Sasha , Rendina Erino , Gelman Andrew 
Published: January 2023

Abstract
To promote Th1 responses TLR2 engagement is conducted on ex vivo-activated T cells but this hinders Ag-specific clonal expansion in vivo. We asked if naive CD4+T cells could internalize the TLR2 agonist Pam3Cys4 to promote Th1 responses in vivo. Methods: WT, TLR2-/- or OT2 CD69-CD25-CD44-CD62L+CD4+T cells were incubated with rhodamine-labeled or unlabeled Pam3Cys4 for 3 hrs and extensively washed to remove all soluble agonist. CD4+T(iPam) activation ex vivo was conducted under Th1 conditions +/- TGF-β1 and bisulfite conversion t-bet promoter sequence analysis was performed. 4x10(5) OT2 or OT2(iPam) cells were adoptively transferred into B6 mice with established EG-7 OVA tumors. Results: Intracellular FACS revealed WT or TLR2-/- CD4+T cells readily internalize Pam3Cys4. As compared to untreated CD4+T cells CD4+T(iPam) displayed &gt; than 50% t-bet promoter CpG hypomethylation at -134 and -75 at 36 hrs post-activation leading to sharply higher t-bet and IFN-γ expression in a TLR2 dependent manner. OT2 (iPam) halted EG-7 OVA growth with high numbers of OVA-specific CD4+ and CD8+ T cells in the DLN and tumor. In contrast, untreated OT2 induced little Th1 accumulation and accelerated tumor growth when compared to mice that did not receive T cells but this rapid growth pattern could be reversed with TGF-β1 Abs. Also, unlike with untreated OT2 cells TGF-β1 could not block naïve OT2(iPam) differentiation into Th1 cells. These data suggest a novel approach to tumor immunotherapy.
Role of Th17/IL-17 inflammatory axis in the progression of malignant gliomas (P2095)
Authors: Parajuli Prahlad , Mandalaparty Mouli , Brodie Chaya , Lum Lawrence , Thakur Archana , Mittal Sandeep 
Published: January 2023

Abstract
Recent studies on extra-cranial tumors have demonstrated a strong relationship between tumor progression and inflammation. The Th17 cells, an inflammatory T cell subtype, have been implicated with either pro- or anti-tumor activity, depending on the tumor type. Th17 cells or the cytokine IL-17 remain to be studied in malignant gliomas. The objective of our study is to explore the axis of inflammatory interactions in glioma progression by focusing on the functional significance of IL-17 and IL17 receptor (IL17R) in the tumor. Our experimental strategies include in vitro 3D culture of IL17R+ glioma cells for proliferation/differentiation and cell signaling assays and ex vivo analysis of clinical glioma tissues via multi-color flow cytometry and immunohistochemistry. We report here that: 1) There is considerable prevalence of IL-17-secreting and IL17-inducing (Th17 and CD11b+ myeloid) cells among glioma-infiltrating immune cells; 2) Th17 cells induced by glioma-derived factors, in the presence of TGF-β1, shows potential immune-suppressive phenotype; 3) IL17R is preferentially expressed in glioma stem-like cells (GSCs); 4) IL17 enhances the proliferation of GSCs in matrigel cultures; 5) IL17 induces phosphorylation of STAT3, NF-κB, and GSK-3β, while also enhancing β-catenin activity in GSCs. This study provides novel insight into inflammatory axis in glioma progression, which may have significant impact on clinical interventions in patients with malignant gliomas.
IL-21 and CD40L signals from autologous T cells can induce antigen-independent proliferation of chronic lymphocytic leukemia cells (P2167)
Authors: Pascutti Maria Fernanda , Jak Margot , Tromp Jacqueline , van Bochove Gregor , Derks Ingrid , Luijks Dieuwertje , Pals Steven , van Lier Rene , Kater Arnon , van Oers Marinus , Eldering Eric 
Published: January 2023

Abstract
Chronic Lymphocytic Leukemia (CLL) is a malignancy of mature B cells that depend on host factors in the tissue microenvironment for survival and proliferation. While it is well established that monoclonal malignant B cells multiply in secondary lymphoid tissue, the mechanisms leading to their proliferation are still uncertain. In addition to BCR-triggered signals, other microenvironmental factors might well be involved. In proliferation centres, leukemic B cells are in close contact with CD4+CD40L+ T cells. Therefore, we here dissected the signals provided by autologous activated T cells (Tact) to CLL cells. Although the gene expression profile induced by Tact was highly similar to that induced by sole CD40 signaling, an obvious difference was that Tact induced proliferation of CLL cells. We next determined that stimulation with only CD40L+IL-21 was sufficient to induce robust proliferation in CLL cells. Then, we defined an IL-21-induced gene signature in CLL, containing components of JAK/STAT and apoptosis pathways in addition to the previously described Granzyme B (GZMB) expression. This signature could subsequently be detected in LN samples from CLL patients. Finally, we demonstrated presence of IL-21 RNA and protein in LN samples from patients. These results indicate that, in addition to BCR signaling, activated T cells might contribute to CLL cell proliferation via CD40L and IL-21. Targeting this interaction might offer new venues to the treatment of CLL.
Targeted disruption of lectin-glycan interactions promotes remodeling of tumor vascular networks and restores immune function. (P2173)
Authors: Croci Diego , Cerliani Juan , D Alotto Tomás , Mascanfroni Ivan , Salatino Mariana , Mendez-Huergo Santiago , Rabinovich Gabriel 
Published: January 2023

Abstract
The mechanisms linking tumor neovascularization and immunity are poorly understood. We previously demonstrated an essential role of galectin-1 (Gal-1) in tumor-immune escape and angiogenesis. The present study was conducted to elucidate whether Gal1-glycan lattices can link tumor angiogenesis to immunity. We examined the glycophenotype of endothelial cells (ECs) in resting, proliferative, tolerogenic or inflammatory conditions. ECs exposed to tolerogenic or proliferative microenvironments exhibited a substantial up-regulation of cell surface glycans critical for Gal-1 binding (p&lt;0.01). In vivo disruption of Gal1-glycan lattices in B16 melanomas attenuated hypoxia-driven angiogenesis, while promoting vascular remodeling (p&lt;0.01) in tumors treated with anti-Gal1 blocking mAb. Moreover, anti-Gal1 treated tumors showed a significant reduction in tumor growth (p&lt;0.01) and evoked a T-cell specific immune responses, as shown by increased infiltration of CD8+ T-cell (p&lt;0.05), increased proliferation of tumor-specific CD4+ T-cells (p&lt;0.01) and augmented IFN-γ and IL-17 (p&lt;0.05) production. Moreover, tumor-draining LN of mice receiving anti-Gal-1 mAb showed lower frequency of FoxP3+ Treg cells (p&lt;0.05) and lower IL-10 secretion (p&lt;0.05). Hence, disruption of lectin-glycan lattices, not only evokes an unleashed anti tumor immune response, but also favors remodeling of tumor vascular networks, highlighting the versatility of endogenous lectins during cancer progression.
Comparison of infiltrating lymphocytes in colon tumor versus adjacent normal mucosal tissue (P2179)
Authors: Sherwood Anna , Emerson Ryan , Scherer Dominique , Habermann Nina , Buck Katharina , Staffa Jürgen , Desmarais Cindy , Ulrich Cornelia , Robins Harlan 
Published: January 2023

Abstract
Tumor immunogenicity is emerging as an important but incompletely understood contributor to cancer progression. Tumors from colorectal cancer (CRC) are generally immunogenic and commonly infiltrated with T lymphocytes. However, the details of the adaptive immune reaction to these tumors are poorly understood. We have accrued both colon tumor samples and adjacent healthy mucosal samples from 15 CRC patients to study lymphocytes infiltrating these tissues. We apply a method for detailed sequencing of T-cell receptor (TCR) sequences from tumor infiltrating lymphocytes (TILs) in CRC tumors at high throughput to probe T-cell clones in comparison to the TCRs from adjacent healthy mucosal tissue. The variation in diversity of the TIL repertoire was far wider than the variation of T-cell clones in the healthy mucosa, and the oligoclonality was higher on average in the tumors. However, the diversity of the T-cell repertoire in both CRC tumors and healthy mucosa was on average 100-fold lower than in peripheral blood. Using the TCR sequences to identify and track clones between mucosal and tumor samples, we determined that the immune response in the tumor is different than in the adjacent tissue, and the number of shared clones are not dependent on distance between the tissues. Together, these data imply that CRC tumors induce a specific adaptive immune response, but that this response differs widely in strength and breadth between patients.
Effector memory CD8 T cells and central memory CD4 T cells dominate a proliferation deficient T cell population in the primary tumor of breast cancer patients (P2207)
Authors: Egelston Colt , Simons Diana , Miyahira Andrea , Lee Peter 
Published: January 2023

Abstract
Much of our knowledge on breast cancer derives from research on animal models of the disease. Studies on human breast cancer patient samples offers potential data that is more readily translatable to clinical therapeutics. We found a significant reduction in the proliferative capability of ex vivo T cells from primary tumor tissue compared to those from paired sentinel lymph nodes (SLNs). Central memory T cells have previously been shown to have a higher proliferative capacity than effector memory T cells. We found higher proportions of effector memory cells among tumor infiltrating CD8 T cells as compared to SLN CD8 T cells. Conversely, the CD4 compartment in both tumors and SLNs was dominated by central memory CD4 T cells. We therefore considered that the diminished T cell proliferative capacity may also be explained by a CD4 compartment comprised of anergic or exhausted cells. Further examination of the tumor and SLN CD4 T cells showed only a small population producing IFNγ or IL-17 in response to TCR stimulation, which may support this possibility. We hypothesize that these observations are symptoms of an inefficient T cell response to tumor cells in breast cancer patients. Diminished T cell proliferative capacity in the tumor may be explained by the predominant effector memory status of TIL CD8s and reinforced by the presence of anergic CD4 T cells. Future characterization of these T cells will be useful to design treatments that enhance their anti-tumor ability.