bluuebunny/crossref_metadata_2025 huggingface dataset:
Loss of DRAK2 signaling enhances allogeneic transplant survival by altering effector T cell responses and memory generation (126.24)
Authors: Weist Brian , Hernandez Jeniffer , Walsh Craig
Published: January 2023

Drak2 is a serine threonine kinase that has previously been shown to negatively regulate activation of T cells through the T cell receptor. Here we demonstrate that loss of DRAK2 signaling significantly promotes the acceptance of allogeneic engraftment in two separate transplant models without generalized immunosuppression. Drak2-/- T cells failed to reject allogeneic tumors, and were defective in rejecting Balb/C allogeneic skin grafts on C57BL6/J recipients. A significant fraction of alloreactive Drak2-/- T cells underwent apoptosis following activation, whereas expression of Bcl-xL in Drak2-/- T cells restored rejection. Formation of allogeneic memory was also greatly hampered in T cells lacking the Drak2 gene. Adoptive transfer of memory T cells from Drak2-/- mice failed to promote the rejection of allogeneic tumors, and such cells led to significantly delayed rejection of skin allografts in the Balb/C to B6 model. Costimulatory blockade by in vivo administration of CTLA4-Ig synergized with the DRAK2 deficiency and led to long-term allogeneic skin graft acceptance. Overall, these results demonstrate that DRAK2 plays an important role in primary and memory T cell responsiveness to allografts. Since previous studies have demonstrated that a loss of DRAK2 does not negatively impact antiviral immunity, these studies underscore the potential utility of pharmacological blockade of DRAK2 to achieve transplant maintenance without the imposition of generalized immunosuppression.
Autocrine role for HMGB1 and HSP60 in the growth of murine mammary carcinoma (127.13)
Authors: Kurt Robert , Chalmers Samantha , Eidelman Alec , Ewer Jason , Serrano Antonio , Vail Caroline
Published: January 2023

Previously we reported that Myd88 contributed to growth, metastasis, and expression of CCL2 and CCL5 by mammary carcinoma implying that constitutive signaling through Myd88 contributes to tumor progression. To begin to decipher what may be inducing this signaling we focused on proteins that could function as damage associated molecular patterns (DAMPs) since DAMP expression has been reported in tumors, and certain DAMPs mediate effects through toll-like receptors. A screen of mammary carcinoma for DAMP expression showed HMGB1 and HSP60 were significantly elevated relative to normal mammary epithelium. Although suppression of either HMGB1 or HSP60 using RNAi reduced growth of the tumor cells, a significant reduction in growth as well as CCL2 expression was only found when both DAMPs were targeted. Since, besides their ability to function as DAMPs, HMGB1 and HSP60 have important roles in normal cellular functions we used blocking antibodies to determine whether these DAMPs were mediating effects in an autocrine manner. Indeed, antibodies to these DAMPs inhibited growth of the tumor cells, and analysis using a Myd88 inhibitory peptide suggested that HMGB1 mediated effects in a Myd88 dependent manner, while HSP60 mediated effects in a Myd88 independent manner. Collectively, these data show that mammary carcinoma overexpress HMGB1 and HSP60 relative to normal mammary epithelium, their expression influences tumor growth, and the effects of HMGB1 are dependent upon Myd88.
Autoimmune mechanisms in antitumor immunity: tumor as an immunoprivileged self (127.20)
Authors: Miska Jason , Devarajan Priya , Chen Zhibin
Published: January 2023

Blocking cytotoxic T lymphocyte antigen 4 (CTLA4), a prototypical inhibitory molecule of antitumor immunity, is emerging as a promising immunotherapy against cancers. Its clinical efficacy, however, is intricately associated with autoimmune toxicity. This association presents a challenge to selective immunosurveillance against tumors versus normal tissues, and suggests a paradigm change whereto autoimmunity is exploited as an effector mechanism for the destruction of tumor cells. Aided with well-defined murine models of autoimmunity, this study demonstrated that autoantigen-specific effector T (Teff) cell clones were indeed effective in eradicating tumor cells. However, a combination of the tumor microenvironment and regulatory T (Treg) cells presented a formidable barrier to the autoimmune effectors. CTLA4 critically regulated autoimmunity-mediated tumor destruction, such that a subtle reduction in CTLA4, engineered by shRNA transgenesis to mimic human CTLA4 genetic variations, substantially enhanced antitumor immunity. Surprisingly, this enhancement was not due to an extrinsic mechanism of crippling Treg-cell suppression, but an intrinsic promotion of Teff cell function. Overall, this study suggests that the tumor microenvironment may represent an “immunoprivileged self” status, wherein physiological mechanisms of immunoregulation evolved to protect nonmalignant “self” could be exploited to an increased potency within a solid tumor.
TGF-β-associated miR-27a Inhibits Dendritic Cell-mediated Differentiation of Th1 and Th17 Cells by Targeting TAB3, p38 MAPK, MAP2K4 and MAP2K7 in the Tumor Microenvironment1 (127.22)
Authors: Yang Rongcun , Min Siping , Li Ling , Zhang Miaomiao
Published: January 2023

Abstract The alterations induced in dendritic cells (DCs) in the cancer microenvironment have not been extensively explored. We found that the tumor-associated factor TGF-β may selectively upregulate the expression of miR-27a via the SP1 transcription factor. Importantly, miR-27a altered the activity of NF-κB and the MAPKs p38, JNK and ERK. It influences the production of proinflammatory cytokines by targeting TAB3, p38 MAPK, MAP2K4 and MAP2K7. As a consequence, miR-27a hampered the DC-mediated differentiation of Th1 and Th17 cells in vitro and in vivo, but it promoted the DC-mediated accumulation of Tr1 (CD4+IL-10+) and Treg (CD4+CD25+Foxp3+) cells in vivo. The repeated infusion of miR-27a-engineered DCs into tumor tissues accelerated tumor growth, indicating that miR-27a is a potential target for tumor immunotherapy. (119 words)
Tumor-infiltrating lymphocytes in breast cancer tissue comprise granzyme B-expressing B cells with cytotoxic potential (127.23)
Authors: Hofmann Stefanie , Brüggemann Sabine , Beyer Thamara , Barth Thomas , Deissler Helmut , Lotfi Ramin , Kreienberg Rolf , Möller Peter , Schrezenmeier Hubert , Jahrsdörfer Bernd
Published: January 2023

Certain subtypes of breast cancer are infiltrated by lymphocytes including B cells, correlating them with a favorable prognosis. Recently, we found that B cells can differentiate into granzyme B (GrB)-secreting cells with cytotoxic potential towards certain tumors. After screening paraffin-embedded tissue sections of various tumors, we show here for the first time that breast carcinoma is infiltrated by CD19+ B cells which express significant amounts of GrB. Testing a panel of different cytokines described to be associated with anti-tumor immune responses revealed interleukin (IL-) 21 as a highly potent stimulus to induce GrB in B cells. Induction of GrB by IL-21 was strongly enhanced in the presence of B cell receptor engagement, and was associated with activation of JAK1/3 and STAT1/3, signaling molecules also activated in NK cells and CTL. Given these parallels with other cytotoxic cells, we tested whether or not GrB-secreting B cells are able to directly induce apoptosis in breast cancer cells. Indeed, we found that IL-21-activated, GrB-secreting B cells showed significant cytotoxic activity against breast cancer cells in-vitro, whereas unstimulated B cells had no such effects. The data presented here support the hypothesis that GrB-secreting B cells may be directly involved in anti-cancer immunity. Our findings suggest, that B cell plasticity is higher than previously thought and that B cells may play an immunological role beyond antibody and cytokine production.
Universal tumor-specific T cells: Redirecting T cells to tagged antigens in vivo (127.30)
Authors: Davila Eduardo , Geng Degui , Srivastava Ratika , Bansal Navneeta , Li Chaoyang
Published: January 2023

The development of a universal yet adaptable gene therapy platform that confers immune cells specificity to various cancer types has valuable clinical implications. T cells engineered to express a chimeric antigen receptor (CAR), that bind tumor-associated antigens, show promise in treating patients however several limitations preclude the widespread use of this form of therapy. We improve the existing CAR technology by developing a versatile system which grants T cells specificity to fluorescein isothiocyanate (FITC) molecules. We report that human antiFITC-CAR T cells recognize various cancer types when bound with FITC-labeled antibodies resulting in efficient target lysis, T cell proliferation, and cytokine/chemokine production. Treatment with antiFITC-CAR T cells plus FITC-cetuximab (FITC-Ctx) delayed colon cancer tumor establishment but unexpectedly led to the outgrowth of epidermal growth factor receptor (EGFR)-negative cells. Using pancreatic cancer cells with uniform EGFR expression, antiFITC-CAR T cells plus FITC-Ctx eradicated an established tumor and prevented tumor growth. These studies are the first to describe the generation of a universal and adaptable CAR system which confers T cells specificity to tagged antibodies which when bound to various cancer types mediate tumor destruction. We consider this platform to be an ‘off-the-shelf’ system that considerably advances the existing CAR technology through its potential to target an assortment of tagged proteins.
Meningiomas include an antigen-experienced B cell infiltrate that produces antibodies directed to tumor antigens (127.36)
Authors: OConnor Kevin , Fang Liangjuan , Meizlish Matt , Anderson Richard , Bruce Jeffrey , Huttner Anita , Lovato Laura
Published: January 2023

Meningiomas are among the most common adult neoplasms of the central nervous system. Like several other tumor types, these intracranial tumors often harbor an immune cell infiltrate that can include substantial numbers of B cells. Characteristic details of the B cell infiltrate remain undefined. To gain a deeper understanding of both the B cell repertoire and insight into the role they play in this tumor, we characterized the immune infiltrate of resected meningiomas. Immunohistochemistry revealed a conspicuous B cell infiltrate in the microenvironment of most tumors examined. Flow cytometry of tissue homogenate showed that the infiltrate included naïve and memory B cells and differentiated plasma cells. Molecular characterization of the immunoglobulin variable regions expressed by tumor-infiltrating B cells revealed clear evidence of antigen experience, in that the cardinal features of an antigen-driven B cell response were present: significant somatic mutation, isotype switching and codon insertion/deletion. This characterization also revealed the presence of B cell clonal expansion and intraclonal variation. Testing the specificity of recombinant immunoglobulin derived from single, dissected, intra-tumoral B cells revealed that tumor antigens were recognized. These data indicate that an adaptive and specific humoral immune response exists within meningiomas, further suggesting that tumor antigens may drive B cell maturation within the tumor microenvironment.
Interrogating BDCA3+ dendritic cells in human tumor-draining lymph nodes (127.43)
Authors: van de Ven Rieneke , Haley Daniel , Walker Edwin , Fountain Christopher , Douglas Jeremy , Wood William , Yu Kelvin , Aliabadi Shaghayegh , Fox Bernard , Hu Hong Ming , de Gruijl Tanja , Urba Walter
Published: January 2023

Human blood dendritic cells (DCs) expressing BDCA3 (CD141) have been characterized and described by several groups to represent the human counterpart of the mouse cross-presenting CD8α+ DC. Fully functional cross-presenting DCs are of utmost importance for the induction of tumor-antigen specific CD8 T-cell responses. In the current study, we set out to interrogate BDCA3+ DC within human tumor-draining lymph nodes (LNs) from melanoma and breast cancer patients. For this purpose a 12-color polychromatic flow panel for human DC was developed. CD11c+BDCA3+ cells were found to be of immature phenotype (low in CD80, CD86 and CD83) upon isolation. When comparing a group of 9 tumor-negative LNs with 4 tumor-positive LNs of BrCa patients, the percentage of CD11c+BDCA3+ cells was significantly higher (p<0.03) in the specimen containing tumor metastasis. Moreover, when comparing the activation status of BDCA3+ cells in LNs from advanced melanoma patients that did or did not grossly display evidence of metastatic disease, we observed lower expression of the DC maturation marker CD83 in the grossly positive LNs, suggesting that this population of DC might be affected by active tumor-induced suppression. In ongoing studies we are sorting these BDCA3+ DC to determine their cross-presenting abilities and to determine whether a (tumor-induced) immature phenotype is also reflected in the functional capabilities of these cells.
A critical role of dendritic cells in CD8 T cell IL-10 expression during inflammatory response triggered by CD40-activated B cells (159.16)
Authors: Shimoda Michiko , Bolduc Anna , Powell Domonica , Ametani Yutetsu , Takezaki Mayuko , Nutt Stephen , Kamanaka Masahito , Flavell Richard , Mellor Andrew , Tsubata Takeshi , Koni Pandelakis
Published: January 2023

CD40LBTg mice, expressing a CD40 ligand (CD40L) transgene on B cells, represent a model for human diseases where B cells aberrantly express CD40L or receive excess CD40/CD40L signaling under inflammatory conditions. Here, we show that B cells expressing transgenic CD40L are capable of priming CD8 T cells and generate strong antigen-specific cytotoxicity. Adoptively transferred SIINFEKL peptide-loaded B cells from CD40LBTg but not wild type mice were able to activate self-reactive OT-I CD8 T cells upon immunization with OVA plus alum and trigger diabetes in RIP-OVA mice by enhancing the help of endogenous self-reactive CD4 T cells. CD40L expressing B cells also trigger spontaneous activation of splenic CD8 T cells, which rapidly up-regulate PD-1, Blimp-1 and LAG-3 and lose cytotoxicity along with IL-10 expression via interaction with PDL-1hi CD11c+ dendritic cells in T cell zones. Thus, CD11c+ dendritic cells from CD40LBTg mice exhibit regulatory phenotype, which block granzyme B expression and preferentially induce IL-10 expression in activated CD8 T cells. These results demonstrate that constitutive CD40 signaling on B cells under inflammation increases the risk of breaking peripheral CD8 T cell tolerance. However, the presence of CD40-activated B cells results in PDL-1hi CD11c+ dendritic cells, which exhibit a regulatory role to dampen harmful self-reactive cytotoxicity by promoting IL-10 expression in CD8 T cells.
Diagnostic utility of anti-cyclic citrullinated peptide antibodies in patients with early rheumatoid arthritis in the north of the country in Mexico. (159.18)
Authors: de la Cruz-Galicia Guadalupe , Malacara Mariana , Almanza Rolando , Silva-Belmares Sonia , Rosas-Taraco Adrián
Published: January 2023

Rheumatoid arthritis (RA) is a chronic systemic inflammatory disease which primarily causes a symmetric polyarthritis, which can progress into long-term disability. The diagnosis of RA combines the patient history of joint pain and stiffness, laboratory tests including biomarkers that can provide useful information to confirm the diagnosis of RA. To compare the diagnostic utility of biomarkers to confirm the diagnosis of RA, anticyclic citrullinated peptide (CCP) antibodies, rheumatoid factor (RF), erythrocyte sedimentation rate (ESR), and C-reactive protein (CRP) were tested in patients with early diagnosis RA. We work with 49 patients with RA all fulfilling the revised criteria of the ACR, and healthy volunteers. A commercial ELISA assay kit was used to test for anti-CCP antibodies; RF and CRP were measured by turbidimetric immunoassays; ESR was performed by Wintrobe assay. The levels of anti-CCP antibody titers, and FR were significantly higher in patients with early RA (54 % and 35% respectively) than in healthy controls (p <0.0001), however the 27% of patients were only positive to CCP test. No significant statistical correlation between anti-CCP antibody titers with inflammatory markers such as CRP or ESR was found. A positive predictive value was higher for anti-CCP antibodies in comparison to other markers.
A role for macrophage-derived AIM in humoral immune response and obesity-associated autoimmunity (159.2)
Authors: Arai Satoko , Miyazaki Toru
Published: January 2023

AIM (apoptosis inhibitor of macrophage), a member of scavenger receptor cysteine-rich super family (SRCR-SF), is a secreted protein solely produced by tissue macrophages with a direct regulation by a nuclear receptor liver X receptor/ retinoid X receptor (LXR/RXR) heterodimers. AIM circulates in blood at the concentration of 10-100 μg/mL in human. We have reported that AIM is involved in the metabolic syndromes such as atherosclerosis and insulin resistance, mediated by its functions: inhibition of apoptosis in macrophages and induction of lipolysis in adipocytes, respectively. It has been well-known that AIM binds to IgM in blood. However, its physiological significance has not been yet elucidated. In this study, we found that AIM-IgM complex intensely reinforces the stability of AIM protein in blood, and in the meantime, AIM supports the function of IgM. The AIM-IgM interaction enables the maturation of immunity, transition from innate-like immunity mediated by IgM, to adaptive immunity brought about by IgG. These new observations suggest that the modern life style, such as a continuous high-fat diet, may result in an excessive increase of AIM in blood, and appears to affect not only innate-like but also adaptive immunity by supporting the function of IgM. This may cause various disorders based on chronic inflammation including metabolic and autoimmune diseases.
IgM anti-DNA antibody and C1q Immune complexes regulate the production of Interferon-α in plasmacytoid dendritic cells (159.26)
Authors: Son Myoungsun , Diamond Betty
Published: January 2023

Systemic Lupus Erythematosus (SLE) is an autoimmune disease characterized by the loss of tolerance to autoantigens, and to DNA in particular. Although Immune complexes (ICs) of IgG anti-DNA antibodies are pathogenic, IgM anti-DNA antibodies in SLE are protective. The mechanism responsible for the protective effect of IgM anti-DNA antibody is unclear. We found that a monoclonal IgM anti-DNA antibody cooperated with C1q to suppress interferon (IFN)-α production by plasmacytoid DCs (pDCs). We tested the effects of the antibody on bone marrow-derived pDCs from wild type (WT) and C1q knockout (KO) mice. Interestingly, the absence of C1q significantly enhanced the production of IFN-α induced by IgM anti-DNA antibody. The inhibitory effect of C1q was confirmed using in vivo generated pDCs or in vitro generated pDCs under serum-free conditions. Exogenous C1q suppressed IFN-α transcription in both WT and C1q KO pDCs. Moreover, we identified leukocyte-associated inhibitory receptor (LAIR)-1 as a potential receptor for C1q. Fluorescence labeled C1q strongly bound to pDCs which were highly expressing LAIR-1. LAIR-1 is a collagen receptor containing two ITIMs; its cross-linking with antibody inhibits IFN-a production by pDCs stimulated with DNA containing ICs. In summary, protective IgM anti-DNA antibody/C1q complexes inhibit unwanted IFN-α production through binding to LAIR-1.
The structure of rainbow trout IgM influences antibody-mediated phagocytosis of bacteria (160.16)
Authors: Bromage Erin , Kataria Priti , Ramirez-Gomez Fraancisco
Published: January 2023

The tetrameric IgM molecule is the dominant immunoglobulin found in the serum of fish. In contrast to the fully polymerized mammalian IgM, teleost IgM is variably disulfide cross-linked resulting in considerable structural diversity. Recently, it was revealed that following vaccination, rainbow trout alter the assembly of their antigen-specific IgM. It was hypothesized that these different IgM forms may allow this molecule to switch effector functions. To investigate whether there was a link between structure and function, we used serum with differing IgM structural profiles and evaluated their ability to mediate phagocytosis. We discovered that when IgM was fully disulfide cross-linked it increased phagocytosis of the antibody-opsonized bacteria. Conversely, when a larger percentage of the IgM was held together by electrostatic forces, phagocytosis was diminished. Control experiments proved that differences in antibody coating were not responsible for the differences in phagocytic potential of the antibody pools. Thus, rainbow trout IgM that is more heavily disulfide polymerized facilitates antibody-mediated phagocytosis more effectively. To our knowledge, this is the first time that IgM structural diversity has been demonstrated to affect a biological function critical for the protection against infectious disease.
Differential gene expression changes associated with sub-clinical Mycobacterium avium subsp. paratuberculosis infection in cattle: a transcriptomic study (160.2)
Authors: Purdie Auriol , Begg Douglas , de Silva Kumidika , Plain Karren , Whittington Richard
Published: January 2023

Paratuberculosis or Johne's disease is characterised by a lengthy sub-clinical phase that may persist for years prior to development of clinical disease. Mechanisms leading up to clinical disease are poorly understood although the early sub-clinical phase is reportedly characterised by a pro-inflammatory response largely driven by Th1 cytokines. The objectives of this study were to evaluate early gene expression changes in calves exposed to the causative agent, Mycobacterium avium subsp. paratuberculosis (MAP). Blood samples were drawn at three early time points, from a Johne’s field trial consisting of a mixed population of Holstein and Holstein Red cattle. Four MAP exposed and four unexposed control cattle were sampled at each time point and processed to Affymetrix GeneChip Bovine Arrays. Ontological analysis revealed a number of differences in gene expression between the two populations at the individual time points. In addition a subset of genes associated with the MHC were consistently altered across all MAP-exposed samples in comparison to the unexposed controls, suggesting significant changes to the antigen processing and presentation pathways in MAP-exposed animals. This differential regulation in response to MAP is a significant finding in the understanding of the mechanisms of pathogenesis and may lead to an aid to test for susceptibility.
Induction of pro-inflammatory cytokines by traditional and nano formulations of anti-cancer drug Paclitaxel (161.11)
Authors: Ilinskaya Anna , Dobrovolskaia Marina , McNeil Scott
Published: January 2023

Paclitaxel is a natural product with known anti-cancer activity. Paclitaxel is insoluble in water and requires the use of delivery vehicle. Historically two formulations of this drug were introduced into clinic: traditional, known as Taxol®, and more recently, nanotechnology-based, named Abraxane®. Several studies have reported that in murine macrophages Paclitaxel mimics endotoxin in induction of expression of several proinflammatory genes, and have suggested that Paclitaxel and endotoxin share the same receptor (TLR4) to initiate inflammatory signalling. These earlier studies utilized traditional formulation Taxol®. Nanotechnology offers many benefits to drug delivery such as reduction of drug toxicity, change in its biodistribution and controlled drug release. Clinical use of Abraxane® demonstrated decreased toxicity of this formulation as compared to that of Taxol®, however no study comparing cytokine induction profile of Paclitaxel in nano and traditional formulations has been reported so far. We have revisited this question and conducted an in vitro study evaluating induction of proinflammatory cytokines by traditional and nano formulations of Paclitaxel. Taxol®, but not Abraxane® resulted in induction of proinflammatory cytokines. This data was consistent between three cell culture models - whole blood, PBMC and MonoMac6 cell line. The mechanisms of cytokine induction by Taxol® will be discussed.
AP-1 and NF-κB regulate osteopontin gene expression in anti-CD40 treated B cells (161.16)
Authors: Dsilva Sehba , Hiroaki Kaku , Rothstein Thomas
Published: January 2023

Osteopontin (OPN) is a glycoprotein that is implicated in many physiological and pathological processes. Elevated levels of OPN are linked to autoimmunity and cancer and correlate with disease progression. In the immune system, macrophages, dendritic cells, T and B cells express OPN. The regulatory elements involved in B cell OPN expression are poorly understood. Our objective is to determine the regulatory elements critical for OPN expression in B cells. B cells express OPN when stimulated with anti-Ig post treatment with IL-4 or CD40L. To determine the regulatory elements, we made four overlapping OPN promoter-luciferase constructs extending from -2000bp to +80bp. We then transfected 1C10 (anti-CD40 antibody) treated and anti-Ig stimulated primary murine B cells with the OPN promoter constructs. We saw an increase in luciferase activity in the promoter construct containing binding sites for AP-1, Ets and Runx2. Significantly, mutagenesis of the AP-1 site abrogated this increase in luciferase activity. To investigate the role of NF-κB in OPN expression, we used an NF-κB inhibitor to evaluate OPN expression in B cells stimulated with anti-Ig post treatment with1C10. NF-κB inhibition reduced OPN transcript expression in these cells. In conclusion, preliminary data indicate that AP-1 and NF-κB play a role in OPN expression in anti-CD40 treated B cells.
Under Bcl-2 surveillance, cytokines, autophagy-dependently, induce p53-independent CD54 expression (161.4)
Authors: Wang Chi-Yun , Chiang Tzu-Hui , Chien Shun-Yi , Tseng Po-Chun , Yang Tsan-Tzu , Chuang Ji-Jui , Chen Chia-Ling , Lin Chiou-Feng
Published: January 2023

p53 is required for stress-induced CD54 expression; until now, its role has less investigated for IFN-γ- and TNF-α-induced such effects. However, IFN-γ caused CD54 expression in human non-small cell lung cancer A549 cells even with p53 silence, but controversially had no effects on H1299 and AS2 cells carried p53 mutation. To investigate the unexpected IFN-γ resistance, there was not as much of change on IFN-γ receptor expression among these cells; however, IFN-γ-activated Jak2/STAT1/IFN regulatory factor (IRF) 1 was decreased. Aberrant activation of Src homology domain-containing phosphatase (SHP) 2, but neither suppressor of cytokine signaling (SOCS) 1 nor SOCS3, was essential for IFN-γ resistance to decrease CD54. Notably, autophagy deficiency was shown in resistant cells and determined IFN-γ resistance accompanied by increasing mitochondria and reactive oxygen species. TNF-α also acted accordingly by autophagy to induce IκB degradation, NF-κB activation, and CD54 expression independent of p53. These results demonstrate that under Bcl-2 surveillance, IFN-γ and TNF-α induce p53-independent but autophagy-regulated CD54 expression.
Immunogenic Peptides Derived from the Transcription Factor ERG as a Vaccine for Prostate Cancer (162.17)
Authors: Kissick Haydn , Dunn Laura , Lu Bin , Sanda Martin , Arredouani M. Simo
Published: January 2023

The suitability of immunotherapy as a treatment for prostate cancer (PCa) has recently been demonstrated in a number of clinical trials. While these studies have provided a strong proof of principle, there is considerable room for improvement. One potential strategy is to identify new targets specific to PCa that could be immunized against and with this in mind, we have implemented an in silico-in vitro-in vivo¬ procedure for designing and assessing the suitability of MHC-I-restricted peptides as anti-PCa vaccines. In this study, we investigated peptides designed from ERG, a transcription factor expressed in around 50% of human PCa cases. We used in silico protein cleavage and peptide-binding algorithms to design HLA-A2.1 restricted peptides from the Erg protein. The binding of the in silico designed Erg peptides was then assessed in vitro and shown to bind HLA-A2.1 effectively. Additionally, when peptides were tested in vivo, they generated a strong immune response in humanized HLA-A2.1+ mice and overcame peripheral tolerance in prostate specific Erg expressing TRAMP-HLA-A2.1-ERG+ mice. Finally, it was found that a greater CD8+ T-cell recall response against human ERG+ PCa cell lines occurred in ERG immunized HLA-A2.1+ mice compared to controls. Together these data support the validity of our peptide design method and indicate that peptides derived from the overexpressed gene Erg could be used in an anti-PCa vaccine.
Breaking immune tolerance to prostate-associated tumor antigens through Tim-1 receptor (162.20)
Authors: Arredouani M. Simo , Kissick Haydn , Dunn Laura , Strom Terry , Sanda Martin
Published: January 2023

It has been suggested that one factor limiting cancer immunotherapy in clinical trials are immunological checkpoints that impede vaccine-induced anti-tumor responses. We postulate that manipulating lymphocytes and dendritic cells through the Tim-1 receptor, a membrane protein that regulates the vigor and differentiation of these cells, using an agonist monoclonal antibody could enhance cytotoxic lymphocyte (CTL) responses to prostate tumor-associated antigens. We used an approach that combines concomitant immunization against a model prostate tumor-associated antigen (TAA) SV40 Tag and treatment with an agonist anti-Tim1 antibody and assessed Tag-specific CTL responses two weeks later. Ab treatment of TRAMP mice resulted in elevated Tag-specific CTL in both spleen and prostate-draining lymph nodes, indicating that manipulating Tim1 results in circumventing immune tolerance to Tag antigen. We then tested this strategy for human epitopes derived from the prostate cancer-specific antigen ERG and SIM2 by combining anti-Tim1 antibody with an ERG- and SIM2-derived HLA-A2.1-restricted peptide vaccine in HLA-A2.1 transgenic mice and showed that CTL responses to ERG and SIM2 epitopes are elevated in the presence of anti-Tim1 Ab. Further characterization of lymphocyte differentiation post-immunization/anti-Tim1 treatment revealed that major shifts in T cell differentiation occur. Our findings suggest that targeting Tim1 could be successfully combined with prostate cancer vaccines.
Intraperitoneal immunization prevents autologous and non-autologous tumor growth on the omentum (162.22)
Authors: Sedlacek Abigail , Gerber Scott , Lord Edith
Published: January 2023

Tumor cell metastasis to the peritoneal cavity (PC) is a serious concern for many cancers, including ovarian and colon tumors. Upon introduction to the PC, tumor cells bind to the omentum, a tissue comprised of adipose embedded with organized immune aggregates (IA) thought to have regulatory function. Interestingly, it is to these IA that tumor cells initially bind and thrive. This presents a unique situation in which tumor cells are in close proximity to potential effector cells. However, without a stimulus to override their regulatory function, IA fail to control tumor growth. Thus, we attempted to stimulate a response by immunizing mice with a lethally irradiated colon adenocarcinoma cell line: Colon38 (C38). Our data demonstrates that i.p. immunization with C38 is capable of preventing growth of C38 upon live tumor challenge. Although T cells are necessary to generate this response, when CD8 or CD4 T cells were depleted during the effector phase, tumor growth was still controlled; suggesting T cells are not the effector cells. Interestingly, in mice immunized with C38 and challenged with another tumor cell line, E0771, tumor growth was also prevented. This non-specific response is unique to the PC, as mice that were immunized with C38 and challenged with E0771 by an alternate (i.m.) route developed tumors. Together, these data suggest that i.p. immunization results in T cell induction of an innate/non-specific anti-tumor response.
Therapeutic efficacy of a fully human anti-CD27 monoclonal antibody in a murine colon carcinoma model (162.23)
Authors: Forsberg Eric , He Li-Zhen , Borrelli Kathleen , Boyer James , Gergel Lauren , Pilsmaker Catherine , Round Sarah , Naylor Sarah , Hammond Russell , Marsh, Jr. Henry , Keler Tibor , Thomas Lawrence
Published: January 2023

The costimulatory molecule CD27 is constitutively expressed on the majority of mature T cells, memory B cells, and a portion of NK cells. The interaction of CD27 with its ligand CD70 plays an important role in effector capacity and memory in T cells; in clonal B cell expansion and germinal center formation; and in NK cell cytolytic activity. Agonistic anti-mouse CD27 mAbs have been shown to have potent anti-tumor activity. A panel of fully human antibodies recognizing human CD27 was generated and we have previously shown that our lead clone (1F5) can mediate anti-tumor effects using the BCL1 B-lymphoma line in human CD27-transgenic (hCD27-Tg) mice. In order to further investigate the mechanism of this anti-hCD27 monoclonal and its activity in a therapeutic setting we assessed anti-tumor activity using the CT26 murine colon carcinoma line. CT26 cells were delivered s.c. to hCD27-Tg animals and five doses of 1F5 were delivered to mice i.p. beginning on days 3, 5, or 7. In multiple experiments, tumor growth in mice treated with 1F5 was substantially delayed or prevented compared to that of control-treated mice. When 1F5-treated surviving mice were rechallenged with CT26 cells, without additional anti-CD27 antibody treatment, tumors did not grow in most animals. These data support the continued clinical development of this anti-CD27 mAb to enhance immune responses in cancer therapy. Investigations of immune cell subset depletions and combination therapies are ongoing.
Mantle Cell Lymphoma induced reduction of human NKT cells (162.25)
Authors: Sun Wenji , Kimball Amy , Xu Yan , Brutkiewicz Randy , Strome Scott , Webb Tonya
Published: January 2023

Natural killer T (NKT) cells are important in regulating immune responses against tumors. However, patients with mantle cell lymphoma (MCL), an aggressive subtype of Non-Hodgkins Lymphoma (NHL), have a reduction in both NKT cell number and function. We have found that NKT cells from MCL patients fail to be activated by autologous B cells, even in the presence of α-Galactosylceramide (α-GalCer), a potent activator of NKT cells. NKT cells from MCL patients had a significant reduction in their expansion potential and in cytokine production. Compared to serum from healthy donors, MCL serum induced chronic NKT cell stimulation and a rapid upregulation of PD-1 in cultured primary NKT cells (from healthy donors), resulting in an exhausted or anergic phenotype. Mass spectrometry demonstrated the presence of higher levels of sphingosine 1-phosphate (S1P) in patient sera compared to healthy controls sera. The functional involvement of S1P in NKT activation was supported by restoration of the anti-tumor function of NKT cells against MCL following S1P1 signaling blockade. These data suggest that tumor-related induction of S1P production and/or S1P signaling in MCL patients can inhibit NKT cell function, ultimately resulting in reduced anti-tumor responses.
IL-17D mediated cancer rejection (162.26)
Authors: O'Sullivan Timothy , Saddawi-Konefka Robert , Bui Jack
Published: January 2023

Immune system interaction with cancers can either enhance or prevent tumor formation. Cytokine secretion by immune infiltrates and tumor cells are important in regulating this process. IL-17A, a pro-inflammatory member of the IL-17 family, has been shown to have conflicting results in regards to cancer progression or regression. Other members of the IL-17 family, such as IL-17D, have no known roles in immune responses to cancer, or endogenous function. Our lab possesses matched methylcholanthrene (MCA)-induced tumor cell lines that will continuously grow (Progressors) or reject (Regressors) after transplantation into syngeneic WT mice. The regresssor cell lines can grow in immune-deficient RAG2-/- mice, indicating that their rejection in WT mice requires an intact immune system. Our lab has used these cell lines to identify differential cytokine expression profiles from microarray analysis. We have obtained data that shows IL-17D transcript and protein are significantly overexpressed in regressor cell lines when compared to progressor cell lines. Overexpression of IL-7D in progressor tumor cell lines leads to their rejection in WT mice in a NK cell dependent manner. We hypothesize that IL-17D expression in regressor cell lines leads to immune-mediated tumor rejection by recruiting NK cells that polarize M1 macrophages in the tumor microenvironment.
MyD88 and Stat3 signaling are fundamental to tumor associated macrophage function (162.34)
Authors: Qualls Joseph , Neale Geoff , Haverkamp Jessica , Kratochvill Franz , Smith Amber , Balouzian Liza , Roussel Martine , Lahti Jill , Dyer Michael , Murray Peter
Published: January 2023

Macrophage infiltration of solid tumors correlates with poor prognosis for cancer patients. However, the tumor associated macrophage (TAM) phenotype appears complex and does not emulate the current in vitro paradigms of macrophage activation. To determine TAM phenotypes from a variety of solid tumors from distinct organ types, we analyzed global TAM gene expression profiles. > 90% of all gene expression was similar between TAMs isolated from gliomas, thymomas and spontaneously occurring neuroblastomas in mice, suggesting TAMs have a common phenotype regardless of tumor type or location. Importantly, many of the genes with the highest observed expression are involved in macrophage-mediated regulation of the inflammatory response. We have collected gene expression data from TAMs impaired in IL-1 and TLR signaling (Myd88-/-) and observed a MyD88-depedent regulation of multiple secreted proteins involved in Stat3 signaling (IL-6, IL-10, G-CSF) and T cell functional differentiation (IL-23p19, IL-12p40, IL-12p35). Harvesting macrophages from genetic mouse models, we have developed an in vitro neuroblastoma sphere/TAM tissue culture system to define the autocrine/paracrine mechanisms involved in both MyD88- and Stat3-dependent signaling networks, ultimately leading to defining their involvement in T cell differentiation and/or function. Our data will clarify the tumor/TAM relationship and potentially reveal TAM-specific targets for future therapeutic application.
Regulation of invariant natural killer T cell anti-tumor responses by the adaptor molecule Signaling Lymphocytic Activation Molecule-associated protein (SAP). (162.47)
Authors: Das Rupali , Bassiri Hamid , Banerjee Pinaki , Guan Peng , Wiener Susan , Orange Jordan , Nichols Kim
Published: January 2023

Invariant natural killer T (iNKT) cells comprise a unique sub-lineage of innate-like T lymphocytes that participate in anti-tumor immune surveillance. However, several questions remain regarding the mechanisms by which these cells mediate killing of tumor cells. We observe that primary murine iNKT cell cytotoxicity against tumor cells is T cell receptor (TCR)-dependent and requires target cell presentation of iNKT cell-directed agonistic lipid antigens. Interestingly, expression of the cytoplasmic adaptor molecule SAP in primary murine iNKT cells is required for optimal TCR-mediated iNKT cell cytolytic responses towards B- and T-leukemia cells in vitro as well as iNKT cell-mediated control of leukemia cell growth in mice lacking CD8+ T and NK cells. iNKT cell cytotoxicity relies on expression of the Src family tyrosine kinase Fyn, a known SAP binding protein, and on the engagement of SLAM family receptors on iNKT and target cells. Of note, SAP- or Fyn- silenced human iNKT cells exhibit significantly impaired killing of T-leukemia cells in vitro, suggesting that SAP-associated signals are also critical for human iNKT cell anti-tumor cytotoxicity. These studies provide the first mechanistic insights into the signaling mechanisms underlying TCR-induced iNKT cell killing of tumor cells. Studies are ongoing to further elucidate the specific SLAM receptor(s) and downstream signaling mechanisms by which SAP regulates murine and human iNKT cell anti-tumor responses.
Treg cells modulate chemokines and cytokines in lymph node stromal cells (163.11)
Authors: Broggi Maria , Schmaler Mathias , Rossi Simona
Published: January 2023

The LN is composed of stromal cells that form a conduit system and provide a structural environment for homeostasis and differentiation of lymphocytes. We aim to characterize the effect of stromal cells to attract and instruct DC and T cells to initiate immune responses. In our skin transplantation model, Tregs are able to delay the rejection of the allograft in a cell number dependent manner. We isolated stroma cells from LN of mice rejecting (Teff) or tolerating the graft (Teff:Treg). Only in the Teff group, we found lower numbers of TRCs (gp38+CD31-) and higher numbers of double negative cells (gp38-CD31-). Furthermore, TRC from the Teff group down-regulated the production of the chemoattractants CCL19 and CCL21, and different cytokines including IL-7 and FLT3L. The presence of Tregs inhibited the down-regulation or maintained the expression of analyzed molecules. We found that IL-7 increases the suppressive capacity of Tregs in response to alloantigens in vitro. The induction of NOS3 is enhanced in TRCs during graft rejection, which is probably a response to IFNg release in the Teff but not Teff:Treg group. Taken together our results indicate that Tregs influences the expression pattern of stroma cells by inhibiting effector T cells functions in the LN.
Complementary roles of FADD and RIPK3 in T cell homeostasis and antiviral immunity (163.24)
Authors: Lu Jennifer , Weist Brian , van Raam Bram , Marro Brett , Nguyen Long , Srinivas Prathna , Bell Bryan , Keith Luhrs , Lane Thomas , Salvesen Guy , Walsh Craig
Published: January 2023

T cells lacking caspase 8 develop a hyper-autophagic morphology, and succumb to a programmed necrosis-like death process termed necroptosis. RIP kinases RIPK1 and RIPK3 together facilitate necroptosis in response to TNF-α ligation and mitogenic stimulation. We have previously shown the defective proliferation of T cells lacking caspase 8 activity is rescued by crossing with RIPK3-/- mice, and caspase-mediated cleavage of RIPK1/3 necrosis inducing complexes (necrosomes) is sufficient to prevent necroptosis. Enhanced recovery of these double-mutant T cells following stimulation demonstrates that caspase 8 and RIPK3 are essential for clonal expansion, contraction, and anti-viral responses, though such rescue ultimately leads to lymphadenopathy. Enhanced death in wildtype T cells blocked by RIPK1 inihibitor Nec-1 suggests an important physiological role for necroptotic signaling in normal T cells. The NF-κβ pathway plays a key role in regulating T cell death as well, as blockade of NF-κβ signaling results in increased death that can be rescued with Nec-1. This result implicates the role of NF-κβ in regulating levels of sensitivity to necroptosis in CD4+ vs. CD8+ cells. The mechanism of inhibition by NF-κB signaling as well as the spatial and temporal regulation of necrosomes are critical in regulating downstream activating and inhibitory pathways induced during necroptosis.
Down-regulation of ROS-mediated T cell signaling leading to IL-4 production by SOCS targeting ATFII and STAT6 (163.6)
Authors: Lee Choong-Eun , Kim Seol-Hee
Published: January 2023

Suppressors of cytokine signaling (SOCS) have emerged as multiple role players in the regulation of immune signaling pathways. We have recently reported that SOCS1 suppresses ROS-mediated T cell apoptosis through the induction of thioredoxin which down-regulates intracellular ROS levels and protects protein tyrosine phosphatases (Oncogene 28:3145, 2009). In the present study we have investigated the role of SOCS in ROS-mediated T cell differentiation leading to Th1/Th2 cytokine production. Both in Jurkat T cell lines and mouse splenocytes, mild oxidative stress stimulated TCR-mediated cytokine production toward Th2 differentiation: A prominent induction of IL-4 gene expression was noted without notable effects on IFN-r. Such ROS-stimulated TCR signaling for IL-4 induction was mediated by activation of Jak1/STAT6 and p38/ATFII. Introduction and overexpression of SOCS1 and SOCS3 in Jurkat T cells significantly reduced ROS/TCR-mediated IL-4 gene induction with a concomitant inhibition on ATFII and STAT6 pathways. Under this condition SOCS substantially attenuated TCR/H2O2 -induced intracellular ROS levels, suggesting that ROS-scavenging function of SOCS affects TCR signaling leading to IL-4 gene expression. Since we have previously identified human thioredoxin as a Th1-inducing factor in T cells (BMC Immunol 9:64, 2008), the role of thioredoxin in SOCS-mediated down-regulation of Th2 response in conjunction with the regulation of NADPH-oxidase system is under investigation.
The Role of Regulatory T Cells in Determining Outcomes of Chlamydial Genital Tract Infection (164.15)
Authors: Flitter Rebecca , Mintus Margaret , Darville Toni
Published: January 2023

Females are at risk for severe oviduct disease after genital tract infection with Chlamydia trachomatis. Previous studies in our laboratory demonstrate Toll-like receptor 2 (TLR2) activation plays an important role in the development of chronic oviduct pathology after murine chlamydial genital tract infection. Clearance of chlamydial infection has been shown in human and murine investigations to depend on IFN-γ-producing CD4+ T cells. However, an overly zealous adaptive T cell response may contribute to the development of chronic tissue pathology. Pathogen specific T effector cell responses can be suppressed by CD4+CD25+Foxp3+regulatory T cells (Tregs), whose suppressive activity may be abrogated by TLR2 activation. We hypothesize Chlamydia specific TLR2 activation abrogates Treg function resulting in an exacerbated adaptive immune response, which contributes to tissue pathology. Here, we successfully depleted Tregs in a Foxp3DTR mouse model by administration of diphtheria toxin prior to intravaginal infection with TLR2-stimulating Chlamydia murdarium. Although bacterial burden was unaltered, conditional ablation of Tregs resulted in increased IFN-γ-producing CD4+ and CD8+ T cells. Additional studies will determine if removal of Treg suppression leads to increased immunopathology.
Pathogen-specific Th1 cells are activated by IL-1 family cytokines induced by innate stimulation. (164.2)
Authors: O'Donnell Hope , McSorley Stephen
Published: January 2023

Upon antigen recognition, naïve CD4 T cells can develop into effector Th1 cells capable of trafficking to sites of infection and secreting pro-inflammatory cytokines, such as IFNγ. The mechanism of Th1 activation within tissues is often assumed to require additional TCR ligation. However, we have explored whether Th1 cells can be activated by innate stimulation in Salmonella and Chlamydia-infected mice. Using mixed bone marrow chimeric mice, our data show that bacterial-specific Th1 cells can be activated by PAMPs and that production of IFN-γ is dependent upon intrinsic expression of MyD88, but not intrinsic TLR4. Furthermore, loss of IL-18R and IL-33R reduces the ability of Th1 cells to produce IFN-γ, while IL-1R and IFN-γR are not required. Th1 cells were also activated in vitro by IL-1 family cytokines and IL-12, demonstrating that these are sufficient for reactivation. We are currently determining the minimal requirements for Th1 cell stimulation by IL-1 family cytokines and examining the role of this pathway in infection models. Understanding the mechanism and role of innate Th1 stimulation could have widespread implications for understanding antimicrobial and autoimmune pathways.