bluuebunny/crossref_metadata_2025 huggingface dataset:
Differential RNA expression of TLR pathway signaling pathways in macrophages from chickens stimulated with Interferon γ in disease resistant B2 haplotypes versus susceptible B19 haplotypes. (P1216)
Authors: Drechsler Yvonne , Keeler Calvin , Carr Thomas , Griggs Lisa , Collisson Ellen
Published: January 2023

MHC has been associated with immunity in several species and studies have demonstrated association of MHC B haplotype in chickens and resistance to a variety of viral pathogens including Marek’s disease virus, avian leucosis virus, Newcastle disease virus and Rous sarcoma virus. Previous studies in our laboratory have shown that chicks with the homozygous B2 MHC haplotypes are more resistant to respiratory illness following avian coronavirus infection, than homozygous B19 chicks. Further studies demonstrated more vigorous responses of B2 macrophages to Poly I:C and IFNγ stimulation than responses of B19 derived macrophages. Macrophages play a critical role in the early innate response and mobilization of the adaptive immune responses. Next Generation Sequencing of IFNγ stimulated macrophages show differential expression of genes of the toll-like receptor signaling pathway. Macrophages were stimulated for 0, 1, 2, 4, 8, 16, 24 and 48 hours. At most time points more genes were stimulated in macrophages of the disease resistant B2 haplotype and quantification of RNA expression (fold-increase) indicates earlier and more robust stimulation of several genes.
Fli-1 transcription factor is involved in cytokine production in spleen cells upon Toll-like receptor stimulation (P1218)
Authors: Brandon Danielle , Williams Sarah , Zhang Xian
Published: January 2023

Friend leukemia integration-1, a member of the Ets family of transcription factors, is highly expressed in splenocytes. Dysregulation of Fli-1 occurs in Systemic Lupus Erythematosus patients and lupus prone mice. Systemic Lupus Erythematosus is an autoimmune disease utilizing the innate and adaptive immune response. Reduction of Fli-1 expression in NZM 2410 and MRL/lpr lupus prone murine models resulted in decreased severity of disease. Studies of Toll-like receptor 7 and 8 reveal various implications in autoimmunity. Inflammatory cytokine IL-6 plays a pivotal role in the pathogenesis of SLE. The role of Fli-1 in Toll-like receptor signaling pathways in splenocytes is unknown. In this study, we generated Fli-1 heterozygous knockout (Fli-1+/-) B6 mice that expressed decreased levels of Fli-1 and investigated the impact of Fli-1 on splenocyte development and cytokine production. Spleen cells were isolated from Fli-1+/- B6 mice and wild-type controls. Examination of cell populations in the spleen revealed unaffected populations of B, T, Natural Killers and Dendritic cells between Fli1+/- mice and wild-type littermates. Spleen cells isolated from Fli-1+/- mice produced increased levels of IL-6 compared to wild-type mice upon stimulation with Toll-like receptor 7 ligand R848. Our results suggest that Fli-1 is involved in IL-6 cytokine production in spleen cells.
An alternative role for FoxP3 as an effector T cell regulator controlled through CD40 (P1027)
Authors: Wagner David , Carter Jessica
Published: January 2023

The BDC2.5 T cell clone is highly diabetogenic, but the transgenic mouse generated from that clone is surprisingly not. CD4+ cells expressing the CD40 receptor defined as Th40 cells are highly diabetogenic in NOD mice and BDC2.5.TCR.Tg mice have a high number of these effector T cells. Given the importance of CD40 for pathogenic T cells, BDC2.5.CD40-/- mice were created where Tregs, CD4+CD25hiFoxP3+, develop normally and pathogenic effector cells are severely ablated. Tregs in both wild type, BDC2.5.wt and CD40 knockout , BDC2.5.CD40-/-, mice express membrane TGFbeta and are fully functional; however Th40 cells are less responsive to Treg control than CD4+CD40- cells. Blocking membrane TGFbeta significantly ablates suppression of CD40- effector T cells with little impact on Th40 cells. Th40 cells from diabetic BDC2.5.wt mice rapidly induces diabetes in NOD.scid recipients but cells from pre-diabetic mice transfer diabetes very slowly. Demonstrating an important paradigm shift, those effector T cells are FoxP3+; but at diabetes onset Th40 cells become FoxP3-. CD28 engagement induces FoxP3 but CD40 induces a smaller FoxP3 not yet reported in murine T cells. This poses a new consideration for the function of FoxP3, not as a molecule defining Tregs, but rather a molecule controlling effector T cell function. The smaller form of FoxP3 therefore is associated with effector cell pathogenicity.
CD4+ T regulatory cells mitogenic signaling is activated by IL-1 and IL-6 produced by serum amyloid A-stimulated monocytes (P1037)
Authors: Macaubas Claudia , Nguyen Khoa , Truong Phi , Wang Nan , Hou Tieying , Yoon Taejin , Mellins Elizabeth
Published: January 2023

We have previously identified serum amyloid A (SAA) as a selective mitogen for regulatory T cells (Treg). However, the precise molecular mechanism by which SAA induces Treg proliferation has been unknown. Here we provide evidence that IL-1 and IL-6 are directly involved in the SAA-mediated reversal of Treg anergy. By engaging its several cognate receptors, SAA induces IL-1 and IL-6 secretion by monocytes. This monocyte-derived inflammatory milieu is required for Treg expansion as pharmacologic inhibition and genetic deletion of IL-1 and IL-6 abrogate the ability of SAA to reverse Treg anergy. Furthermore, while both IL-1 and IL-6 are required for ERK1/2 activation, only the former is necessary for AKT signaling in proliferating Treg. Collectively, these results point to a novel mechanism, by which SAA initiates a monocyte-dependent production of inflammatory cytokines to drive mitogenic signaling in Treg.
MHC composition and variation in turkeys of the Americas (P6066)
Authors: Reed Kent , Monson Melissa , Settlage Robert , Mendoza Kristelle , Dalloul Rami
Published: January 2023

Variation in the MHC is associated with disease susceptibility/resistance in avian species of agricultural importance. Variation includes individual sequence differences, but also structural differences (gene rearrangement) and copy number variation. The directionality of structural changes can be determined through comparison of closely related species in a phylogenetic context. The MHC has now been described for multiple galliform species including the chicken and the domestic turkey (Meleagris gallopavo). Analysis of the turkey MHC is limited primarily to broad taxonomic contrasts with chicken. This study applies high-throughput sequencing approaches to investigate MHC structure of turkeys of the Americas. Sequence assemblies of the Eastern wild turkey (M. g. silvestris) and the Ocellated turkey (M. ocellata; native to the Yucatán Peninsula of Mexico, Guatemala and Belize) were constructed from whole genome sequencing efforts. Assemblies were compared with the MHC of the domestic turkey. In addition, an MHC-specific SureSelect capture array was developed for sequencing multiple birds from different genetic backgrounds. Sequence libraries were created for 14 turkeys (domestic, heritage and wild birds), a second Ocellated turkey, and a chicken. Libraries were hybridized to over 12K baits (120 bp) and the resulting pools were sequenced producing an average of 439K paired reads per library. Results of the sequence analysis and variant discovery are presented.
Cranberry products alter immunity after a Salmonella challenge in pigs (P6084)
Authors: Eicher Susan , Stanley Megan , Richert Brian , Rostagno Marcos
Published: January 2023

Cranberry products decrease bacterial adhesion and alter some immune functions. Our objective was to determine if feeding 3 different cranberry products to pigs would alter mesenteric lymph node (MLN) or spleen cell (SPL) immune functions, with or without a Salmonella challenge. Pigs were fed 1) whole cranberry powder (1-2 g/d) in dry feed plus free choice water, 2) cranberry fiber (4%) in dry feed with free choice water, 3) control dry feed with cranberry juice (10%) as the only liquid for 10 d post-weaning and 2 d pre-challenge, or 4) control diet with free choice water. Pigs were sampled (MLN and SPL) at weaning (19 days), 10, 17, and 24 days post-weaning. On d 28 post-weaning, an oral 10^5 CFU Salmonella enterica serovar Typhimurium dose was given, and 24 h and 7 days post-challenge MLN and SPL tissues were collected. CD14 and CD18 expression and ability to phagocytize fluorescently labeled bioparticles was determined by flow cytometry. SPL did not differ before the challenge; phagocytosis by SPL was increased at 24 hours post-challenge by the powder supplement. However, juice decreased phagocytosis by SPL (% and mean fluorescence) and CD14 expression at 14 days post-weaning. In contrast, on d 14 post-challenge, MLN of the powder supplemented pigs expressed more CD14 and CD18. Phagocytosis was not different for any cells on d 14 post-challenge. Cranberry products affected SPL and MLN differentially, but primarily enhanced MLN activation after the challenge.
Characterization of the Periplaneta americana immune response using image-based flow cytometry (P6068)
Authors: Herlands Robin , Qadir Anam
Published: January 2023

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

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

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

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

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

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

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

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

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 > 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

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

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

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<0.01). In vivo disruption of Gal1-glycan lattices in B16 melanomas attenuated hypoxia-driven angiogenesis, while promoting vascular remodeling (p<0.01) in tumors treated with anti-Gal1 blocking mAb. Moreover, anti-Gal1 treated tumors showed a significant reduction in tumor growth (p<0.01) and evoked a T-cell specific immune responses, as shown by increased infiltration of CD8+ T-cell (p<0.05), increased proliferation of tumor-specific CD4+ T-cells (p<0.01) and augmented IFN-γ and IL-17 (p<0.05) production. Moreover, tumor-draining LN of mice receiving anti-Gal-1 mAb showed lower frequency of FoxP3+ Treg cells (p<0.05) and lower IL-10 secretion (p<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

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

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.
Immunosurveillance of cancer stem cells (P2217)
Authors: Gross Emilie , Peinado Carlos , Han Semi , Washington Allen , Bui Jack
Published: January 2023

The cancer stem cell (CSC) hypothesis stipulates that tumors follow a hierarchical organization where a subpopulation of cells endowed with stem cell characteristics initiates and maintains the malignant progression. Using the murine MCA sarcoma model system, a widely recognized model for studying cancer immunosurveillance and rejection mechanisms, we sought to explore how hierarchical tumor progression occurs during cancer immunosurveillance. The first step was to define CSC in the MCA sarcoma model. We identified an interesting heterogeneity of the tumor population based on Sca1 and CD90 expression. Purification of the Sca1+ and Sca1+/CD90+ in various cell lines and subsequent in vitro culture demonstrated that the Sca1+/CD90-population is able to regenerate the initial tumor heterogeneity. Transplantation of those two populations in RAGxγc-/- hosts demonstrated either a more rapid onset of sarcoma in mice injected with Sca1+ cells. Interestingly, phenotypic analysis of escaping tumors revealed an increase in Sca1+ cells. Finally, in vitro treatment with IFNg led to a substantial increase in Sca1 expression that correlated with an increase in cells' stemness properties as suggested by the elevated sphere forming capacity of IFNg treated cells. This data suggest that cancer stemness may be modulated by anti-cancer immune responses and may represent an additional mechanism of tumor immune escape.
The role of macrophages and T regulatory cells in capsaicin mediated regression of colon cancer (P3217)
Authors: Kadaveru Krishna , Hodio Jennifer , Basu Sreyashi
Published: January 2023

Colorectal cancer (CRC) is the third most commonly diagnosed cancer in both men and women. In humans genetic changes in intestinal epithelia commencing with inactivation of the APC tumor suppressor gene along with mucosal inflammation, facilitate the development of intestinal adenomas. The role of host immune system to detect and eliminate these altered cells is debatable based on its failure to prevent tumor growth. A physiological approach to prevent CRC formation is to use dietary compounds, which can inhibit inflammation and induce apoptosis to transformed cells. Capsaicin (CP), the principal pungent ingredient of red chili peppers is highly consumed spice throughout the world. It exhibits a wide range of immunological and pharmacological properties. We have previously reported that orally administered CP enhances a discreet population of CD11b+/F4/80+ macrophages and a CD4+ T cells population in the gut associated lymphoid tissue (GALT) expressing anti-inflammatory factors such as IL-10 and PD-L1. Here we demonstrate that orally administered CP can suppress spontaneous intestinal polyposis in the Apc(Min/+) mouse model, a widely used spontaneous colorectal cancer model for human familial adenomatous polyposis (FAP) in a time and dose dependent manner. We further demonstrate the role of different immune cells (myeloid and lymphoid) in CP mediated prevention of intestinal tumorigenesis. The use of dietary CP will be a novel approach for chemoprevention of human CRC.
The meninges: A staging site for immune cell interactions in early experimental autoimmune encephalomyelitis (P4166)
Authors: Hatfield Julianne , Brown Melissa
Published: January 2023

Multiple sclerosis (MS) is a demyelinating disease affecting the central nervous system in which immune mediate damage to white matter myelin is well characterized. However, improved imaging has revealed that inflammation in the meninges, tissues that are proximal to the CNS, also occurs and corresponds to cortical grey matter lesion formation in early disease. Together with evidence of activated T cells and tertiary lymphoid follicle formation within these tissues, these data suggest that the meninges are critical sites of immune cell activity. Yet little is known about the basal and disease-associated immune status of these tissues. Using a murine model of MS, we evaluated the immune cell makeup of the meninges in naïve mice and asked how this changes during the course of disease. CD4+ and CD8+ T cells, macrophages, dendritic cells, mast cells and neutrophils are detected in naïve mice and increased numbers of neutrophils and macrophages are observed in preclinical disease. A population of innate lymphoid cells (ILCs: CD3-, CD4+/-, RORγt+, IL-7Rα+) also normally resides in the meninges and this population is reduced in mast cell deficient mice. These data confirm that the meninges are an active site of immune response in early disease. We speculate that meningeal ILCs may provide a mechanism for the development of tertiary lymphoid follicle formation as well as contribute to Th17 cell activation and thereby amplify meningeal inflammation.
HMGB1-LPS complex promotes the transformation of SFs to a RASF-like phenotype (P4168)
Authors: Qin Yanghua , Chen Yan , Shen Qian
Published: January 2023

The specific pathogenesis of rheumatoid arthritis (RA) remains unclear. Current views hold that some inflammatory antigens recognize the Toll-like receptors on the synovial fibroblasts (SFs) and then activate downstream signals, leading to the formation of RASFs and the induction of RA. Our previous studies showed that intra-articular injection of the complex of HMGB1 and lipopolysaccharide (LPS) directly induced experimental arthritis in DBA/1 mice; histopathological analysis further revealed synovial thickening with excessive proliferation of SFs. In the present study, osteoarthritis synovial fibroblasts (OASFs) were co-cultured with HMGB1-LPS complex in vitro for 5-8 generations to induce the transformation of human SFs to RA-like SFs (tOASFs). Then the pathogenicity of tOASFs was evaluated, both in vitro and in vivo. Our results showed that tOASFs attached to, invaded and degraded co-implanted cartilage in a SCID mouse model. In addition, histochemistry showed excessive proliferation of tOASFs and the expression of MMPs. In vitro, cell cycle analysis showed more tOASFs passing through the G1/S checkpoint and moving to S or G2 phase. Flow cytometry and confocal microscopy showed significantly reduced apoptosis and enhanced autophagy in tOASFs compared to OASFs, respectively. Finally, we showed upregulated expression of certain receptors and adhesion molecules in tOASFs. This accumulated evidence led to the conclusion that HMGB1-LPS complex promoted the formation of RASFs.
Cell intrinsic role for MAVS in regulating CD8 T cell fitness during acute virus infection (P6113)
Authors: Thomas Sunil , Huang Albert , Loo Yueh-Ming , Ramos Hilario , McMillan Aimee
Published: January 2023

Mitochondrial antiviral Signaling molecule (MAVS) has been described in it’s role as an adaptor molecule that links viral RNA sensing by RIG-I-like receptors (RLR)s to type I IFN and inflammatory cytokine production. However it’s role in adaptive immune responses to viruses is not well defined. Using an adoptive cell transfer system we examined the requirement for MAVS directly in CD8 T cells during adaptive responses following acute Lymphocytic choriomeningitis virus (LCMV) infection. Here, we found that MAVS plays an RLR-independent role in determining CD8 effector T cell fitness. MAVS-deficient cells were deficient in virus-induced expansion and cytokine production. This deficiency correlated with altered cell size and granularity, differential mitochondrial membrane potential and reduced levels of steady-state intracellular calcium. We found cytokine production deficiencies were acute at earlier stages of CD8 cell expansion, suggesting that the requirement for MAVS is most critical when cellular need for mitochondrial metabolism is maximal. These results suggest that MAVS is required for metabolic support of CD8 effector cell expansion during acute virus infection. Our study therefore assigns a novel metabolic role to MAVS in adaptive CD8 T cell energetics. Defining the function of MAVS in enhancing T cell metabolism therefore has implications for vaccine strategies that aim to increase the quality and quality of the CD8 T cell response to virus infection.
An in vitro culture system for studying the human B cell repertoire (P1459)
Authors: Su Kuei-Ying , Nojima Takuya , Kuraoka Masayuki , Moody M. Anthony , Kelsoe Garnett
Published: January 2023

Defining the human B-cell repertoire is important for understanding human B-cell development and autoimmunity. Currently, re-expression of immunoglobulins from single B cells is the standard method to study human B-cell repertoire; however, this method is labor-intensive and costly as it requires large numbers of recombinant antibodies to be generated by single-cell PCR and expression of recombinant heavy and light chain pairs. We have developed an alternative method to characterize the B-cell repertoire by culture that supports the proliferation and differentiation of human pre-B, immature/transitional, and mature B cells recovered from bone marrow, fetal liver, or peripheral blood. Briefly, single B cells are cultured in IL-2, IL-4, IL-21, and BAFF on CD154+ stromal cells. This culture supports extensive cell proliferation and differentiation into IgG-secreting plasmacytes. The monoclonal IgG antibodies in each cell culture can be readily screened by ELISA, Luminex, or immunofluorescence against endogenous and exogenous antigens. We find that autoantibodies from pre-B cell cultures have higher avidities for self-antigens than do those from immature/transitional and mature B cell cultures, suggesting the loss of high avidity cells at the first tolerance checkpoint. V(D)J rearrangements are easily recovered from selected clones for sequencing and expression. This culture method offers a pathway to determine B-cell repertoires with high throughput and reduced cost.
CD28 promotes long lived plasma cell survival, sustained antibody responses and upregulation of BLIMP-1 expression through its distal proline motif. (P1475)
Authors: Lee Kelvin , Utley Adam , Murray Megan , Carlson Louise , Farren Matthew , Russell Lisa , Nair Jayakumar , Garrett-Sinha Lee Ann , Green Jonathan , Boise Lawrence , Rozanski Cheryl
Published: January 2023

Durable humoral immunity is dependent upon the long term maintenance of serum antibody titers. Evidence now suggests that antibody levels are maintained by a distinct subset of bone marrow resident long lived plasma cells (LLPC). However, the molecular basis for the longevity of LLPC is largely uncharacterized. We have recently shown that CD28 signaling supports the survival and function of LLPC but not short lived splenic PC (SLPC). We have now extended these studies by characterizing the signaling pathways and molecular targets downstream of CD28 in PCs using CD28 knock-in mice in which the CD28 cytoplasmic tail is mutated at either the Y170MNM (CD28-Y170F) or P187YAP190 (CD28-AYAA) regions, resulting in loss of downstream PI3K or Grb2-Vav signaling, respectively. Interestingly, only the CD28-AYAA animals had significantly diminished LLPC numbers with no effect on SLPC numbers in vivo, consistent with the inability of CD28 activation to rescue CD28-AYAA LLPC from serum starvation-induced death in vitro. Further characterization of the CD28 (PYAP)-Vav-NFκB pathway in PC uncovered a previously undescribed regulation of the central transcription regulator BLIMP-1 through a CD28 responsive element in the prdm1 promoter. These results demonstrate that CD28 (PYAP)-Vav-NFκB activation in LLPC but not SLPC induces BLIMP-1 expression, which then can regulate the broader transcriptional reprogramming allowing for LLPC function and survival.
B cell development and IL-10 secretion (P1476)
Authors: Basu Uttiya , Sun Jianbo , Wang Jiguang
Published: January 2023

Accumulating evidence confirms the existence of regulatory B cells (Bregs). Bregs are hypothesized to negatively regulate immune responses in autoimmunity and inflammation through the production of regulatory cytokines such as IL-10 and TGF-β. The precise cellular origin and function of Bregs remains unknown. IL-10 is known to exert broad anti-inflammatory effects which may negatively influence the activation of T cells, macrophages, and dendritic cells (DCs) and thereby influence both cellular and humoral immunity. Many questions exist regarding the function and biogenesis of Bregs. Do IL-10-generating Bregs only transiently express IL-10 during particular developmental stages? And what is the functional consequence of B cell mediated IL-10 secretion in vivo? We observe that in a population of naïve splenic B cells, a significant fraction is competent to secrete IL10. Even in B cells stimulated for class switch recombination (CSR), the unswitched population shows a high proportion of IL-10 secreting Bregs. Finally, we discuss the expansion of IL-10 secreting Bregs and their ex vivo matured progenitors in activation-induced cytidine deaminase (AID) deficient mice which are incompetent to undergo CSR and somatic hypermutation, two processes that generate antibody diversity. Put together, these observations point to a correlation between Breg biogenesis and antibody diversification processes.
Endothelin-1 but not endothelial nitric oxide synthase polymorphisms are implicated in sickle cell disease (P3311)
Authors: Thomas Bolaji , Thakur Tanya , Guindo Aldiouma , Ott Jurg , Diallo Dapa
Published: January 2023

Sickle cell disease (SCD) shows marked variability in severity among individuals; this variability probably linked to differential expression of adhesion molecules. Polymorphisms of the endothelial nitric oxide synthase (eNOS) have been implicated in disease severity, potentially delineating vascular complications. There is paucity of information on endothelin-1 (ET-1) diversity and disease severity or ethnic stratification. We examined genomic diversity and estimated haplotype frequency of eNOS and ET-1 polymorphisms in children with SCD. Thirteen SCD children (HbSS) and 176 adult and pediatric controls (HbAA) from Mali were recruited. Genotypic analysis of eNOS variants (T786C, Glu298Asp and intron 4) and ET-1 (G5665T) was carried out with a PCR-RFLP assay. Our results show the frequency distribution and allelic diversity of eNOS and ET-1 variants. Endothelin-1 (G5665T) mutant form was the most frequent, and probably most significant among children with SCD, though the distribution is out of Hardy-Weinberg equilibrium (P=0.0008). eNOS variants showed no difference between SCD and controls. We show that eNOS variants are less frequent, and may have no functional significance in SCD. Additionally, eNOS allele and genotype frequencies are not significantly different in cases and controls. Association between ET-1 and eNOS variants in African versus African American SCD children will be explored and the reason(s) for the HWE deviation will be investigated.