bluuebunny/crossref_metadata_2025 huggingface dataset:
Fullerene C60 derivatives suppress the acquired immune responses (THER5P.832)
Authors: Yoshioka Yasuo , Udaka Asako , Hirai Toshiro , Aoshima Hisae , Kokubo Ken , Oshima Takumi , Ohe Tomoyuki , Mashino Tadahiko , Tsunoda Shin-ichi , Higashisaka Kazuma , Tsutsumi Yasuo
Published: January 2023

Fullerene C60 derivatives are the most promising nanomaterials because of its unique ability to scavenge large numbers of free radicals. Therefore fullerene C60 derivatives with antioxidant effects have been expected to apply to the medicine for inflammatory disease such as inflammatory bowel disease. We have previously shown that fullerene C60 derivatives have the anti-inflammatory effects independent of an antioxidant effects in vitro. However, little is known about the effect of fullerene C60 derivatives on acquired immune system. Here, we examined the effects of fullerene C60 derivatives on the acquired immune responses. At first, we examined the effects of fullerene C60 derivatives on ovalbumin (OVA)-induced immune responses in vivo. Treatment of OVA plus fullerene C60 derivatives induced a lower level of OVA-specific IgE and IgG than did treatment of OVA alone, suggesting that fullerene C60 derivatives suppressed acquired immunity. Next, we investigated the influence of C60 fullerene derivatives on T cell proliferation in a mouse mixed lymphocyte reaction. The fullerene C60 derivatives suppressed the production of IL-2 from responder splenocytes. Furthermore, fullerene C60 derivatives also suppressed the IL-2 production from CD4+ T cell stimulated by anti-CD3 and anti-CD28 antibodies, although NAC (anti-oxidant medicines) didn’t show such effect. These results show that the fullerene C60 derivatives have the effect to suppress the acquired immune system.
Human gingiva-derived mesenchymal stromal cells inhibit graft-versus-host disease through CD39 and IDO (THER5P.844)
Authors: Zheng Song Guo , Chen Maogen , Wang Julie , Olsen Nancy
Published: January 2023

Mesenchymal stem cells have the capacity to maintain immune homeostasis and prevent autoimmunity. We recently reported that human-derived gingival mesenchymal stem cells (GMSCs) have strong capacity to suppress immune responses and T cell-mediated collagen-induced arthritis in animals. However, it is unclear whether these cells can suppress human T cell-mediated diseases. Here, we used a xeno-GVHD model to investigate the interventional role of human GMSCs in NOD/SCID mouse, which is a best animal model to study human cell therapy prior to a clinical trial. We found that GMSCs potently suppressed the proliferation of PBMC and T cells in vitro. Co-transfer of GMSC and human PBMC significantly prolonged the mouse survival. Moreover, we demonstrated that GMSCs inhibited human PBMCs-initiated xenogenic responses via CD39 and IDO signals. Thus, GMSCs can suppress human immune responses and immune system-mediated diseases, offering a potential clinical option to be used for modulating GVHD and other autoimmune diseases.
Development of human B-lymphocyte targeted bi-specific DART® molecules for the treatment of autoimmune disorders (THER5P.830)
Authors: Chen Wei , Nordstrom Jeffery , Burke Steve , Shah Kaplana , Ciccarone Valentina , Li Haiquan , Hotaling Tim , Li Jonathan , Gorlatov Sergey , Li Hua , Alderson Ralph , Koenig Scott , Bonvini Ezio , Johnson Syd , Moore Paul
Published: January 2023

B-cell targeted immunotherapy for autoimmune disorders is often limited by general immune suppression caused by significantly altered B-cell homeostasis. To overcome this, we developed MGD010, a bi-specific Dual-Affinity Re-Targeting (DART) molecule that coligates the inhibitory Fcγ receptor IIb (CD32B) and the B-cell receptor component, CD79B, on B cells. Previous work has shown that CD32BxCD79B DARTs engage CD32B in a BCR-dependent manner and inhibit B-cell activation without B-cell depletion. MGD010, a CD32BxCD79B DART bearing an Fc domain to enhance pharmacokinetic (PK) properties, was generated to support potential clinical development. Favorable PK properties and lack of safety signals, including no cytokine release nor B-cell depletion, were observed in non-human primate dose-escalated with MGD010 to up to 10 mg/kg. Pharmacodynamic analysis demonstrated dose-correlated target binding to B cells and reduced B-cell calcium flux induced by ex vivo BCR-crosslinking. In addition, MGD010 blocked B-cell function ex vivo in samples from autoimmune patients. Repeat administration of MGD010 also inhibited both humoral immune responses and the development of chronic graft-versus-host disease in a humanized mouse model. Thus, MGD010, an Fc-bearing CD32BxCD79B DART with extended PK, may represent a promising B-cell intervention strategy for the treatment of autoimmune diseases, with limited impact on B-cell homeostasis.
Targeting natural killer cell reactivity by employing antibody to NKp46: implications for type 1 diabetes (THER6P.857)
Authors: Yossef Rami , Hadad Uzi , Jonjić Stipan , Mandelboim Ofer , Porgador Angel
Published: January 2023

Natural killer (NK) cells belong to the innate lymphoid cells. NK cells are able to eliminate transformed cells and virus-infected cells without the need of prior antigen stimulation. Their cytotoxic activity is regulated through the delicate balance between activating and inhibitory signals. NKp46 is a member of the primary activating receptors of NK cells; and it is reported to interact with the hemagglutinin (HA) of the influenza virus, heparan sulfate and vimentin. Moreover, our group and others have reported that NKp46 is involved in the development of type 1 diabetes (T1D) autoimmune disease. Subsequently, we hypothesized that blocking of the NKp46 could prevent T1D development or prolong its honeymoon period. To address this goal we characterized monoclonal antibodies for the murine NKp46. One mAb, F15, was able to down-regulate the surface expression of NKp46 on primary murine NK cell following antibody injection. Additionally, F15 treatments were able to down-regulate cytotoxic activity mediated by NKp46, but not by other NK receptors. To test our primary assumption, we examined T1D development in two models, NOD mice and low-dose streptozotocin. The results showed significantly lower incidence of diabetic mice in the F15-treated group comparing to control groups. This study directly demonstrates the involvement of NKp46 in T1D development and suggests possible treatment for early insulitis
Identification of swine leukocyte antigen-derived peptides inducing epitope-specific CTL responses in HLA-A2 transgenic mice (TRAN3P.882)
Authors: Lee Chong-Kil , Im Sun-A , Park Chan-Su , Kim Kyungjae
Published: January 2023

Xenotransplantation of porcine organ is considered a potential solution to the problem of allograft shortage. A xenograft is rejected by cross-primed CD8 cytotoxic T cells as these cells show cytotoxicity against xenografts by recognition of swine leukocyte antigen (SLA). However, the precise swine leukocyte antigen (SLA)-derived immunogenic peptides that are presented in the context of human HLA molecules remain to be elucidated. In the present study, the SLA-derived peptides that bind to HLA-A*0201, a representative of the A2 supertype, were predicted using a computer-assisted algorithm. The candidate peptides were synthesized, and then the stability of peptide/HLA-A*0201 complexes was compared by MHC stabilization assays using HLA-A*0201-expressing Tap knock out T2 cells. Of 15 candidate peptides, two peptides, peptide-35 and peptide-43, were selected to have high affinity and stability with HLA-A*0201. Immunization of HLA- A*0201-transgenic mice with peptide-35 elicited potent CD8 CTL cell responses, while immunization with peptide-43 did not induce noticeable CTL responses. Induction of peptide-35-specific CD8 T cells was also confirmed by intracellular IFN-g staining of CD8 T cells from peptide-35 primed mice. This study has important implications not only for the identification of an immunogenic HLA-A*0201-restricted indirect epitope derived from SLA, but also for the development of epitope-specific immunoregulation strategies.
Different competency of CMV-pp65 or Flu-M1 specific CD8+ T cells in naïve and central memory population in healthy human adults (TRAN3P.885)
Authors: Chen Guobing , Solokina Arina , Li Huifen , Truong Thai , Oelke Mathias , Wersto Robert , Schneck Jonathan , Leng Sean , Weng Nan-ping
Published: January 2023

The competency of antigen specific (Ag+) lymphocytes plays a critical role in the outcome of an adaptive immune response during an infection. Although the precursor frequency of Ag+ T cells often serves as an indicator, the evaluation of the proliferative capacity of Ag+ T cells of healthy humans to known viral antigens is limited. Here, we compared CD8 T cell responses to the antigen from cytomegalovirus (CMV-pp65) or influenza virus (Flu-M1) using an artificial antigen presenting system in vitro. We found that CD8 T cell responses (proliferation and Granzyme B production) to these two antigens varied in 23 healthy adults analyzed. The antigen specific CD8 T cell expansion did not correlate with the precursor frequency of total Ag+ CD8 T cells. Further comparing the response of isolated naïve (Tn) and central memory (Tcm) CD8 T cells showed that Tcm expanded more than Tn did to both antigens. Finally, we found that longer telomere lengths in CD8 T cells were positively correlated with the better expansion of these Ag+ CD8 T cells. Together, our study shows that antigen specific CD8 T cell response to CMV-pp65 and Flu-M1 is varied in healthy adults and the capacity of proliferation of these Ag+ CD8 T cells depends partly on the telomere length in these cells.
Trafficking of intra-graft T cells to recipients’ lymphoid organs after skin transplantation (TRAN3P.916)
Authors: Marino Jose , Tocco Georges , Mordecai Scott , Benichou Gilles
Published: January 2023

The skin contains T lymphocytes including significant numbers of FoxP3+ regulatory T cells (Tregs). However, their role in skin transplantation is still unknown. In this study, we investigated the trafficking of intra-graft donor T cells to the recipient’s lymphoid organs after skin transplantation in mice. To test this C57BL/6 (B6) mice received a skin transplant from a double transgenic B6 donor whose T cells expressed DSred while FoxP3 expression was associated with GFP (Tregs being double positive). At different time points after transplantation, cell suspensions of recipients’ lymph nodes, spleen and thymus were prepared. The presence and characterization of fluorescent donor cells was investigated using imaging flow cytometry analysis. First, the presence of T cells (DSred+ FoxP3-) was detected only at d4-d7 post-transplantation i.e. after skin graft revascularization and essentially in the host’s thymus. On the other hand, double positive (DSred+ FoxP3+) Tregs were found in all recipient’s lymphoid organs. Surprisingly, high frequencies of single positive GFP+ cells were found in both ipsilateral lymph nodes and thymus. The nature of these cells is being investigated. In summary, our results show that most T cells leaving the graft are Tregs spreading through all recipient’s lymphoid organs while other intra-graft T cells migrate preferentially to the host’s thymus.
Control of B lymphoma through regulation of TRAF protein degradation during nutrient stress (TUM7P.932)
Authors: Abdul Sater Ali , McPherson Ann , Laister Rob , Minden Mark , Watts Tania
Published: January 2023

The TNFR family of receptors includes members, such as CD40, that mediate important immune functions. TNFRs play critical roles in regulating cellular survival, where they utilize TNFR associated factors (TRAFs), including TRAF1 and TRAF2, as adaptors to signal through NF-κB and MAPK pathways. TRAF1 is linked with increased lymphoma cell survival and is overexpressed in over 50% of human lymphomas, with highest expression in most refractory B-CLL. Thus there is interest in blocking TRAF1 induced survival signaling in lymphoma. Here we show that during nutrient deprivation, induced by low glutamine in the media or by treatment with the licensed drug Erwinaze, TRAF1 is essential for maintaining protein levels of another pro-survival signaling adaptor, TRAF2, in RAJI cells (Burkitt's lymphoma). The PKC, PKN1, has been shown to phosphorylate TRAF1. Utilizing shRNA knockdown, we show that loss of PKN1 reduces TRAF1 expression, suggesting that PKN1 phosphorylation stabilizes TRAF1. Importantly, we demonstrated that Erwinaze treatment causes cell death in TRAF1 deficient RAJI cells but not in wild type cells. Moreover, PKN1 deficient RAJI cells, which exhibit lower TRAF1 expression, were also susceptible to treatment with Erwinaze. Together, these findings suggest that a combination therapy that targets TRAF1 for degradation (i.e. PKN1 inhibitor) along with Erwinaze to cause nutrient stress mediated loss of TRAF2 constitutes an effective and aggressive therapy for B cell lymphoma
Hematopoietic expression of CXCR3 is required for melanoma engraftment of lung (TUM7P.936)
Authors: Clancy-Thompson Eleanor , Mullins David
Published: January 2023

Chemokine receptors have an established role in immune cell trafficking, and a less well-defined role in metastasis. Recent literature has demonstrated a role for tumor-expressed CXC chemokine receptor 3 (CXCR3) in metastasis to lymph nodes, but no study has evaluated the role of host CXCR3 expression in melanoma metastasis. To assess this possibility, we injected B16 melanoma intravenously into CXCR3wt or CXCR3-/- mice and assayed for melanoma-specific gene expression (tyrosinase) 24 hours later. Gene expression was significantly (9.5-fold) higher in CXCR3wt mice. At 24h, this is likely due to differential engraftment of the melanoma in the lung, as opposed to different growth. To determine if the CXCR3-expressing host contribution is an immune component, we phenotyped cells in the lungs 24 hours after melanoma injection. Neutrophils, CD8+ T cells, and B cells were significantly increased in CXCR3-/- mice versus CXCR3wt mice after melanoma injection, whereas macrophages/DCs, CD4+ T cells, and NK cells were not significantly different. Using bone marrow chimeras, we have shown that melanoma engraftment in the lungs requires a host CXCR3-expressing cell of the hematopoietic cell lineage. A role for host CXCR3-expressing cells to mediate melanoma engraftment in the lungs has not been previously shown, and these studies may predict future targets for the reduction or prevention of melanoma metastases in patients.
Development of protease activated interleukin-12 cytokine fusion proteins for tumor immunotherapy (TUM7P.946)
Authors: Skrombolas Denise , Maharaj Shivana , Wylie Isabel , Sullivan Mark , Shenoy Gautam , Bankert Richard , Frelinger John
Published: January 2023

Local expression of IL-12 in the tumor microenvironment results in greatly enhanced anti-tumor responses. However, achieving local expression of cytokines at disseminated sites is challenging and systemic delivery has significant toxicity. We describe a novel approach employing an activatable fusion protein (FP) to reduce the negative side-effects of systemic delivery of the cytokine but which can preferentially affect all tumor sites. This FP consists of a cytokine linked to an inhibitory single chain antibody fragment (scFv) separated by a specific protease cleavage sequence. Importantly, the scFv does not bind to the tumor but rather binds to the cytokine keeping it inactive. However, at the tumor site, the FP can be cleaved by proteases such as Matrix metalloproteases (MMPs) that are over-expressed at tumor sites. This cleavage allows the release of the cytokine from the scFv and its binding to higher affinity receptors on immune cells. We will present data on the construction, expression, and characterization of a panel of murine and human IL-12 FPs. Using phage display, we have identified a panel of 15 scFv that bind to epitopes of murine IL-12 on either the P35 or P40 subunit as well as scFv that bind to human IL-12. Functional and biochemical data on baculovirus expressed IL-12 FPs will be presented. This strategy has the potential to reduce the negative side effects associated with systemic delivery of constitutively active cytokines yet retain their efficacy.
Serum prevalence of B cell stimulatory TLR ligands, elevated B cell TLR2 expression and regulatory B cell frequency precede the genesis of HIV-non-Hodgkin’s lymphoma (NHL) in ART treated HIV+ subjects (TUM7P.951)
Authors: Siewe Basile , Pham Joey , Cohen Mardge , Hessol Nancy , Levine Alexandra , Martinez-Maza Otoniel , Landay Alan
Published: January 2023

TLR2-signaling may contribute to B-cell activation during HIV infection leading to HIV-NHL genesis; a common malignancy in anti-retroviral therapy (ART) treated patients. TLR-signaling and anti-tumor immunity inhibitory regulatory B cells (Bregs) contribute to lymphomagenesis but their roles in HIV-NHL genesis remain unexplored. We investigated, if in HIV- donors, TLR-activated B cells mimic an activated/germinal center (GC)-like phenotype aberrantly expressed in B-cell lymphomas. Further, in HIV+ART subjects: (cases = <1 year pre-NHL, controls = HIV+ART but don’t develop NHL), we assessed: 1) B-cell TLR2 and TLR2 modulatory miRNA expression 2) Breg frequency and 3) serum levels of TLR-ligands. In B cells from HIV- donors only TLR2- (unlike TLR4 nor 9) stimulation (p=0.03), leads to an aberrant phenotype (CD10+CD71+CD86+AID+) identified in lymphomas. Compared to controls, cases exhibit significantly elevated: 1) frequency of CD10+CD71+CD86+AID+ B cells (p=0.01) and Bregs (p=0.0071), 2) serum levels of TLR2 and TLR9 ligands (lipoteichoic acid, p=0.03 and 16s DNA, p=0.03 respectively), 3) B-cell TLR2 expression (p=0.0024). Finally, in cases, the activated/GC-like cells exhibit dysregulation of miRNA regulating TLR2-expression including miR-9, miR-19a and miR146a. Our data suggest that HIV-NHL genesis is associated with aberrant GC-like B cells, elevated Bregs and a possible role for TLR2-stimulation.
CD28 mediated survival in plasma cells involves redox regulator thioredoxin (TUM7P.954)
Authors: Nair Jayakumar , Koorella Chandana , Murray Megan , Carlson Louise , Boise Lawrence , Lee Kelvin
Published: January 2023

Very little is known about the molecules that mediate essential prosurvival interactions between plasma cells (PC) and bone marrow stroma. We have shown that both normal and transformed plasma cells (myeloma, MM) express CD28 (prototypic T cell costimulatory molecule) that correlates with increased survival in MM. We had previously shown that CD28 activation directly with agonistic antibodies or indirectly with CD80/CD86+ (CD28 ligands) dendritic cells protect MM cells against cell death. MM-CD28 also interacts with other CD86+ MM cells to maintain enhanced survival that can be reversed with CD86 blockers like CTLA4Ig. Additionally, we also show that CD28 activation is essential to help plasma cells overcome intrinsic redox stress, a result of increased protein synthesis and metabolic activity (Bertolotti et al., 2010) which if not regulated can lead to cell death. For the first time, we show that CD28 activation not only drives survival via increased NFκB activity, but it also decreases ROS levels via the redox regulator thioredoxin (TRX-1), inhibition of which increased ROS, decreased TRX-1 expression and NFκB activity and reversed CD28 mediated survival in MM. Also, TRX-1 inhibition enhanced CTLA4Ig’s anti-survival effect on MM. Interestingly, gene expression datasets of long lived plasma cells from patients correlate with TRX-1 expression with increased survival, highlighting the vital role this redox regulator plays in PC survival.
Increased lung/liver metastasis of human breast cancer cells in NOD.SCIDIL2rγ-/- mice after treatment with soluble CD200Fc (TUM7P.922)
Authors: Gorczynski Reg , Chen Zhiqi , Khatri Ismat , Podnos Anna , Kos Olha
Published: January 2023

CD200 overexpression increases metastasis of tumor cells to draining lymph nodes. Increased levels of a soluble form of CD200, sCD200, occur in serum of ~50% breast cancer patients, and an immunoadhesin, linking CD200 to an immunoglobulin Fc region (CD200Fc) increases metastasis in experimental models. We investigated whether CD200 levels were increased in human breast cancer cells in mice, and if overexpression of CD200 increased metastasis. NOD.SCIDIL2rγ-/- mice received 5x105 cells of different human breast cancer cell lines (MCF7; HTB19; MDA-MB-231) injected into the mammary fat pad. Groups of mice received ongoing infusion of human CD200Fc (20μg/mouse at 72hr intervals for 4 weeks). Tumors were resected at ~0.7cm3, and snap frozen for PCR analysis. Mice were monitored post resection, and sacrificed 2-3 months later. Lung and liver metastatic nodules were enumerated, and tissue snap frozen for PCR analysis. All tumors growing in NOD.SCIDIL2rγ-/- mice expressed CD200, though parent (in vitro) cell lines were uniformly negative. CD200Fc increased lung and liver metastasis of all breast cancer lines tested. Tumor metastasis was associated with changes in gene expression in tumor tissue, with significant alterations in genes encoding matrix metalloproteinases, and transcription factors modulating inflammation. Further changes in gene expression were attributed to the effect of CD200Fc infusion. Our data suggests increased soluble CD200 promotes human breast cancer metastasis.
TLR7/8 agonists enhance FcγR in CLL patient monocytes (TUM7P.958)
Authors: Elavazhagan Saranya , Shah Prexy , Fatehchand Kavin , Patel Hemal , Cheney Carolyn , Muthusamy Natarajan , Vasilakos John , Matthews Maura-Ann , Dietsch Gregory , Hershberg Robert , Byrd John , Tridandapani Susheela , Butchar Jonathan
Published: January 2023

Monoclonal antibody therapy has been used to treat cancer since 1997, but there is a continued need to improve its effectiveness. We have previously shown that the TLR7/8 dual agonist R-848 could modulate monocyte FcγR expression and function, and that it synergized with antibody to reduce tumor growth rate in vivo. Hence, the purpose of this study was to test whether these agonists could enhance antibody-mediated effects in patient samples and to determine whether it was TLR7, TLR8 or both that drove these FcγR responses. Using peripheral blood samples from Chronic Lymphocytic Leukemia (CLL) patients we found that treatment with the TLR7/8 dual agonist R-848 significantly enhanced monocyte FcγR-mediated TNFα production. In patient Peripheral Blood Mononuclear Cells (PBMC) treated with the anti-CD20 antibody Rituximab, R-848 increased both cytokine production and CLL cell clearance. We next compared the effects of TLR7- versus TLR8-selective agonists on monocytes and found that TLR8 agonists led to greater FcγR-mediated cytokine production, higher levels of miR-155 and lower levels of SHIP-1. Unexpectedly, while all agonists upregulated the activating FcγR, only TLR8 agonists downregulated the inhibitory FcγRIIb. These results suggest that TLR8 agonists are superior to those for TLR7 with regard to enhancing monocyte FcγR expression and function, and may be better adjuvants for antibody therapy.
Direct T cell activation via CD40 ligand generates high avidity CD8+ T cells capable of breaking immunological tolerance for the control of tumors (VAC10P.965)
Authors: Soong Rueyshyang , Wu Tc , Hung Chien-Fu
Published: January 2023

CD40 and CD40 ligand (CD40L) are costimulatory molecules that play a pivotal role in the proinflammatory immune response. Here, two tumor-associated antigen (TAA) animal models, a p53-based and a GP100-based model, were utilized to examine the ability of CD40-CD40L to improve antigen-specific CTL-mediated antitumor immune responses. Although p53 and GP100 are self-antigens that generate low affinity antigen-specific CD8+ T cells, studies have shown that their functional avidity can be improved with CD40L-expressing APCs. Therefore, in the current study, we immunized mice with a DNA construct encoding a TAA in conjunction with another encoding CD40L via intramuscular injection followed by electroporation. We observed a significant increase in the antigen-specific CTL-mediated immune responses and potent antitumor effects in both models. Antibody depletion experiments demonstrated that CD8+ T cells play a crucial role in eliciting antitumor effects in vaccinated mice. Furthermore, we showed that in vitro stimulation with irradiated tumor cells expressing both TAA and CD40L improved the avidity of antigen-specific CD8+ T cells. Thus, our data show that vaccination with a TAA/CD40L DNA can induce potent antitumor effects against TAA-expressing tumors through the generation of high avidity antigen-specific CD8+ T cells. Our study serves as an important foundation for future clinical translation.
Tumor-specific CD8+ Tc9 cells are superior effector than Tc1 cells for adoptive immunotherapy of cancers (VAC11P.1000)
Authors: Lu Yong , Hong Bangxing , Li Haiyan , Zheng Yuhuan , Zhang Mingjun , Qian Jianfei , Yi Qing
Published: January 2023

Because cytokine-priming signals direct CD8+ T cells to acquire unique profiles that affect their ability to mediate specific immune responses, here we generated a novel IL-9-skewed (Tc9) CD8+ T cells by priming with Th9-polarized condition. Compared with Tc1 cells, Tc9 secreted different cytokines and were less cytolytic in vitro but surprisingly elicited greater antitumor responses against advanced tumors in OT-I/B16-OVA and Pmel-1/B16 melanoma models. After adoptive transfer, Tc9 cells persisted longer and differentiated into IFN-g- and granzyme-B (GrzB)-producing cytolytic Tc1-like effector cells. Phenotypic analysis revealed that adoptively transferred Tc9 cells secreted IL-2 and were KLRG-1low and IL-7Rahigh, suggesting that they acquired a signature of “younger” phenotype or became long-term lived cells with capacity of self-renewal. Our results also revealed that Tc9-mediated therapeutic effect was critically dependent on IL-9 production in vivo. These findings have clinical implications for the improvement of CD8+ T cell-based adoptive immunotherapy of cancers.
α-Galactosylceramide stimulates splenic lymphocyte proliferation in vitro and increases antibody production in vivo in the late neonatal-age mouse. (VAC12P.1028)
Authors: Chen Qiuyan , Ross A.
Published: January 2023

The neonatal stage is characterized by weak responses to various infections and vaccines; thus the development of efficient formulas to improve vaccine effectiveness is of high priority. The glycolipid alpha galactosylceramide (αGalCer) is known as a potent immune modulator mainly due to NKT cell activation. Using a mouse tetanus toxoid (TT) immunization model, we observed that neonatal mice given αGalCer at the time of primary immunization on day (d) 17 had a significant higher TT-specific IgM response (ELISA), while given αGalCer at d7 during immunization only resulted in a marginal boosting. Consistently, immunostaining of the spleen sections from αGalCer-treated d17 immunized neonates showed higher number of Ki67+ cells in the splenic germinal center area, suggesting stronger response after immunization. In vitro kinetic studies revealed that spleen cells from newborn to d7 neonates did not respond to αGalCer stimulation, whereas cell proliferation was markedly increased by αGalCer after d7, and became dramatic around neonatal d17-18, which was accompanied by increased B and T lymphocyte populations in the spleen. In addition, in d17 spleen cells, αGalCer significantly stimulated the proliferation of CD23+ B cells, a subset of B cells enriched in germinal centers. These data suggest that αGalCer is an effective stimulator of the immune system in late neonatal stage and thus may be useful as a potential adjuvant to achieve an efficient immunization.
In vivo immunogenicity of Tax 11-19 epitope in HLA-A2/DTR transgenic mice: implication for dendritic cell-based anti-HTLV-1 vaccine (VAC12P.1020)
Authors: Sagar Divya , Masih Shet , Schell Todd , Jacobson Steven , Wigdahl Brian , Khan Zafar , Jain Pooja
Published: January 2023

Viral oncoprotein Tax plays key roles in transformation of human T cell leukemia virus (HTLV-1)-infected T cells leading to adult T cell leukemia (ATL), and is the key antigen recognized during HTLV-associated myelopathy (HAM). In HLA-A2+ asymptomatic carriers as well as ATL and HAM patients, Tax(11-19) epitope exhibits immunodominance. Here, we evaluate immunotherapeutic potential of this epitope in HLA-A2 transgenic mice in the presence and absence of dendritic cells (DCs) given the recent encouraging observations made with Phase 1 DC-based vaccine trial for ATL. To facilitate these studies, we first generated an HLA-A2/DTR hybrid mouse strain carrying the HLA-A2.1 and CD11c-DTR genes. We then studied CD8 T cell immune response against Tax(11-19) epitope delivered in the absence or presence of Freund’s adjuvant and/or DCs. Overall results demonstrate that naturally presented Tax epitope could initiate an antigen-specific CD8 T cell response in vivo but failed to do so upon DC depletion. Presence of adjuvant potentiated Tax(11-19)-specific response. Elevated serum IL-6 levels coincided with depletion of DCs whereas decreased TGF-β was associated with adjuvant use. Thus, Tax(11-19) epitope is a potential candidate for the DC-based anti-HTLV-1 vaccine and the newly hybrid mouse strain could be used for investigating DC involvement in human class-I-restricted immune responses.
Mitochondrial DAMPs stimulate TNF-alpha production by canine splenocytes in an in vitro model of SIRS inflammation (VET2P.1045)
Authors: Strange Heather , Friedenberg Steven , VanGundy Zachary , Guillaumin Julien , Crouser Elliot , Papenfuss Tracey
Published: January 2023

The systemic inflammatory response syndrome (SIRS) is a life-threatening condition that arises secondary to massive immune system activation or dysregulation. Damage associated molecular patterns (DAMPs) are endogenous danger signals released after trauma or sepsis that trigger these potent immune responses. DAMPS derived from mitochondrial fractions have been shown to be pro-inflammatory in mouse models and play a role in human disease. However, the role of mitochondrial DAMPs as a trigger of SIRS in dogs has not been explored. In this study we characterized the response of canine splenocytes to mitochondrial DAMPs and developed an in vitro model system to study SIRS-induced immune dysregulation in dogs. We demonstrated that splenocytes respond to crude fractionated mitochondrial and soluble protein fractions by upregulating TNF-alpha, a described biomarker for SIRS. We further studied the effects of these DAMPs on splenocyte myeloid cell subpopulations (i.e. dendritic cell and macrophage) and found similar upregulation of TNF-alpha. Our findings mimic studies in mouse splenocyte models where mitochondrial DAMPs were shown to promote TNF-alpha release, suggesting that the in vitro model employed here is useful to study the impact of mitochondrial DAMPs in dogs. Given that dogs are increasingly being used to model and optimize therapy for human disease, these results have direct applications in understanding the role of mitochondrial DAMPs in both canine and human SIRS.
Evaluation of a Pmp18D vibro cholerae ghost vaccine against chlamydia abortus in mice (VET2P.1046)
Authors: Pan Qing , Pais Roshan , Ohandjo Adaugo , He Cheng , Eko Francis
Published: January 2023

Chlamydia abortus (C. abortus) is an obligate intracellular pathogen that causes abortion in pigs and poses a zoonotic risk in pregnant women. Currently available attenuated and inactivated vaccines show marginal protective efficacy underlining the need for the development of effective vaccines. Recombinant proteins expressed on gram negative bacterial envelopes are known to induce protective immunity (Th1) without the need of any additional adjuvant. Pmp18D is a member of polymorphic outer membrane protein family of C.abortus. The PmpD protein of C. abortus is expressed throughout the chlamydial developmental cycle. Pmp protein family members are known to exhibit higher immunogenicity. In this study an evaluation of immune responses to Vibrio cholerae ghosts expressing Pmp18D(rVCG) was done in comparison to rPmp18D codelivered with either Flt3L or CpG. Mice were immunized 3 times intranasally 2 weeks apart .By day 14 post last immunization, the rVCG vaccine elicited significantly higher levels of antigen-specific IgG2a and IgA antibodies both in serum and in vaginal washes. Proliferative responses of mucosal (genital tract) as well as splenic (systemic) T cells were significantly higher in mice receiving rVCG vaccine.These results indicate that the rVCG-Pmp18D vaccine is capable of inducing antigen-specific cellular and humoral immune responses that may provide protective immunity against a live challenge infection with C.abortus.
Suppression of T and B cell responses by myeloid derived suppressor cells after natural CD4 T regulatory cell depletion and LP-BM5 retrovirus infection (VIR7P.1057)
Authors: O'Connor Megan , Green William
Published: January 2023

Immunomodulatory cellular subsets, including myeloid derived suppressor cells (MDSCs) and T regulatory cells (Tregs), contribute to the immunosuppressive tumor microenvironment and have been targets of immunotherapy, but their role in retrovirus-associated immunosuppression is less understood. Tregs are associated with HIV-infected patients, however it remains unclear whether Tregs suppress the anti-viral immune response or dampen HIV viral spread; and also unclear is the stage(s) of infection of Treg involvement. MDSCs have only recently been identified in HIV-infected patients, and thus the phenotype and mechanism(s) of suppression are still being defined. Due to known crosstalk between Tregs and MDSCs in the tumor microenvironment, it is of interest to look at the interplay between these cellular types during LP-BM5-induced murine AIDS (MAIDS). Following in vivo nTreg depletion, using adoptive transfer approaches, and subsequent LP-BM5 retroviral infection, enriched MDSCs from infected nTreg-depleted mice exhibit increased suppression of T cell, but not B cell, responses, as compared to MDSCs derived from non-depleted LP-BM5 infected controls. In addition, MDSCs from LP-BM5-infected nTreg-depleted mice have altered phenotypic subsets, which may correlate with functional differences. These collective data may provide insight into the possible immunotherapeutic targeting of Tregs and MDSCs during retroviral infection. (NIH AI007363, CA50157)
Synergistic effects of the inhibitory Fc receptor FcγRIIB deficiency and lupus-associated SLAM family genes on breaking germinal center tolerance checkpoint (BA2P.126)
Authors: Soni Chetna , Wong Eric , Khan Tahsin , Wakeland Edward , Rahman Ziaur
Published: January 2023

The inhibitory Fc receptor FcγRIIB deficiency and lupus-associated signaling lymphocyte activation molecules (SLAMs) are suggested to contribute to autoimmunity in FcγRIIB deficient mice generated using 129Sv/J ES cells and backcrossed to B6 mice (B6-129.RIIBKO). To study the individual contribution of FcγRIIB deficiency and 129-derived SLAM genes to loss of germinal center (GC) tolerance we compared B6 mice deficient in FcγRIIB (B6.RIIBKO) and B6 mice congenic for 129-derived SLAM locus (B6.129-SLAM) with B6-129.RIIBKO and B6 controls. B6-129.RIIBKO mice developed numerous spontaneous germinal centers (Spt-GCs) with increased size producing high titers of autoantibodies against nuclear (ANA) and other self-antigens, alongside a hyperactive T cell phenotype. We also observed elevated Spt-GC and T-cell responses in B6.RIIBKO and B6.129-SLAM mice than B6 mice although much lower when compared to B6-129.RIIBKO mice. B6.RIIBKO and B6.129-SLAM mice also failed to produce high titers of ANAs in comparison to B6-129.RIIBKO. Ex vivo assays revealed that differentially expressed 129-SLAM genes promoted higher expression of activation molecules on B cells, T cells and DCs while FcγRIIB deficiency was sufficient to drive proliferation of B cells and T cells. Our results suggest that the co-operative effects of 129-derived SLAMs on activation and FcγRIIB deficiency on the proliferation of B cells and T cells are essential for breaking the GC tolerance checkpoint in B6-129.RIIBKO mice.
p85a recruitment by the CD300f phosphatidylserine receptor mediates apoptotic cell clearance required for autoimmunity suppression (BA4P.222)
Authors: Tian Linjie , Choi Seung-Chul , Murakami Yousuke , Allen Joselyn , Morse Herbert , Qi Chen-Feng , Krzewski Konrad , Coligan John
Published: January 2023

The rapid and efficient removal of apoptotic cells (AC) prevents the release of the potentially toxic or immunogenic components from dying cells, thereby reducing inflammatory and autoimmune responses. Phagocytosis is the major means of clearing AC, which is dependent on the interaction between “eat-me” signals on AC and receptors on phagocytes. The externalized phosphatidylserine (PS) on AC is the best-known “eat-me” signal. We demonstrate that mouse CD300f (CLM-1) recognizes outer membrane-exposed PS and regulates AC phagocytosis. CD300f accumulates in phagocytic cups at AC contact sites. Phosphorylation within CD300f cytoplasmic tail tyrosine-based motifs initiates signals that positively or negatively regulate AC phagocytosis. Y276 phosphorylation is necessary for enhanced CD300f-mediated phagocytosis through the recruitment of the p85a regulatory subunit of phosphatidylinositol-3-kinase (PI3K). CD300f-PI3K association leads to activation of downstream Rac/Cdc42 GTPase and mediates changes of F-actin that drive AC engulfment. Importantly, primary macrophages from CD300f-deficient mice have impaired phagocytosis of ACs. The biological consequence of CD300f deficiency is predisposition to autoimmune disease development, as FcgRIIB-deficient mice develop a systemic lupus erythematosus-like disease at a markedly accelerated rate if CD300f is absent. This is the first report identifying the mechanism and role of CD300f in AC phagocytosis and maintenance of immune homeostasis.
Ultraviolet B irradiation accelerates the development of lupus-like autoimmunity in wild-type mice induced by topical treatment with TLR7 agonist (BA4P.212)
Authors: Sano Shigetoshi , Kataoka Sayo , Kamijima Reiko , Fujimoto Chisa , Ohko Kentaro , Takaishi Mikiro , Nakajima Kimiko
Published: January 2023

SLE is an autoimmune disease characterized by the production of autoantibodies and inflammatory pathologies such as glomerulonephritis. A number of studies have demonstrated that altered Toll-like receptor 7 (TLR7) signaling contributes to the initiation of SLE in humans and in murine models. Indeed, introduction of Tlr7 null mutation in lupus-prone mice resulted in amelioration of lupus nephritis and reduction of autoantibodies, clearly indicating the contribution of TLR7 to lupus phenotype. We have recently demonstrated that epicutaneous application of TLR7 agonists, imiquimod (IMQ) or R848, leads to systemic autoimmunity in wild-type mice. They developed lupus-like systemic autoimmunity from 4 weeks of topical treatment, but not by peritoneal injection of IMQ, showing elevated autoantibodies to double-stranded DNA and glomerulonephritis, hepatitis, carditis and photosensitivity, indicating that the skin as the primary organ that allows TLR agonists to initiate lupus. Since ultraviolet (UV) irradiation is a worsening factor in SLE, we examined the effect of UVB on the initiation of TLR agonist-induced lupus. Strikingly, UVB irradiation of Balb/c mice facilitated the emergence and increase in the titers of autoantibodies including anti-double stranded DNA induced by topical treatment with R848 from 2 weeks, which was much earlier than R848 alone, strongly suggesting that UV accelerated the TLR7-mediated systemic autoimmunity.
Anti-scavenger receptor immune complexes disrupted marginal zone macrophage integrity via bruton's tyrosine kinase (BA4P.213)
Authors: Li Hao , Wu Qi , Li Jun , Yang PingAr , Luo Bao , Edberg Jeffrey , Hsu Hui-Chen , Mountz John
Published: January 2023

Macrophage receptor with collagenous structure (MARCO) and scavenger receptor A (SR-A) are marginal zone macrophage (MZM) restricted scavenger receptors that play important roles in apoptotic cell (AC) clearance in the spleens. We identified that in SLE patients, higher serologic levels of anti-scavenger antibodies at early stage were associated with the late development of end-stage-renal disease. In lupus prone BXD2 mice, elevated anti-MARCO/anti-SR-A antibodies in the sera correlated with the loss of MZMs in the spleens. To determine the molecular connection between anti-MARCO/anti-SR-A and MZM loss, immune complexes (ICs) generated from a mixture of AC-Ags and autoantibodies (autoAbs) produced from BXD2 mouse were administrated to B6 mice. Interestingly, disruption of MZMs was observed in mice administered ICs generated from AC-Ags plus polyreactive autoAbs that exhibit high reactivity to both MARCO and SR-A. Confocal imaging analysis showed that, in these mice, there were high levels of bruton's tyrosine kinase (Btk) phosphorylation (pBtk) in the MZMs. Increased pBtk was also observed in MZMs of BXD2. Delivery of Ibrutinib, a Btk inhibitor. systemically or Ibrutinib-liposome as MZM targeted, prevented MZM loss and attenuated autoantibodies and IC-mediated glomerulonephritis in BXD2 mice. As we also identified the loss of MARCO+ MZMs in SLE patient spleens, the present study suggests the potential in development of anti-Btk in MZMs as a novel therapy for lupus.
Aryl hydrocarbon receptor attenuation of sub-chronic cigarette smoke-induced pulmonary neutrophilia is associated with retention of nuclear RelB and suppression of intercellular adhesion molecule-1 (CAM1P.233)
Authors: Rico de Souza Angela , Zago Michela , Eidelman David , Hamid Qutayba , Baglole Carolyn
Published: January 2023

Cigarette smoke exposure is associated with chronic and enhanced pulmonary inflammation characterized by increased cytokine production and leukocyte recruitment to the lung. Although the aryl hydrocarbon receptor (AhR) is well-known to mediate toxic effects of environmental contaminants, the AhR has emerged as a suppressor of acute cigarette smoke-induced neutrophilia. As there is currently no information on the AhR prevention of lung inflammation due to varied and prolonged exposure regimes, we exposed control and AhR-/- mice to cigarette smoke for 2 weeks (sub-chronic exposure) utilizing low and high exposure protocols and evaluated pulmonary inflammation. Sub-chronic cigarette smoke exposure increased pulmonary neutrophilia dose-dependently in AhR-/- mice. There was no difference between smoke-exposed AhR+/- and AhR-/- mice in the expression of cytokines associated with neutrophil recruitment. However, expression of pulmonary intercellular adhesion molecule-1 (ICAM-1), an adhesion molecule involved in neutrophil migration and retention, was higher in AhR-/- mice. Nuclear RelB, an anti-inflammatory NFkB transcription factor, was significantly lower in sub-chronically exposed AhR-/- mice. These data support that absence of the AhR contributes to heightened pulmonary neutrophilia in response to on-going cigarette smoke exposure. Inter-individual variations in AhR expression may enhance the susceptibility to cigarette smoke induced diseases.
Aryl hydrocarbon receptor negatively regulates type I interferon production and the development of murine lupus (CCR1P.245)
Authors: Lee Soyoung , Ripley Barry , Chinen Ichino , Millrine David , Kishimoto Tadamitsu
Published: January 2023

Systemic lupus erythematosus is a highly heterogenous multi-organ autoimmune disease characterized by the production of diverse autoantibodies and a broad spectrum of clinical manifestations. The etiology and pathogenesis of lupus remain unclear. Our current understanding highlights an important role for autoantibodies and numerous studies in both animals and humans now suggest an important role for type I interferon (IFN) in the pathogenesis of lupus. Type I IFN signaling and subsequent feedback induction of type I IFN production, are critically dependent on the STAT1 transcription factor. How type I IFN activity is regulated through inhibition of STAT1 function is poorly understood. Here, we show that type I IFN production and signaling both in cultured plasmatoid dendritic cells, and in vivo, is significantly higher in Aryl hydrocarbon receptor (AHR) knockout mice compared to wild-type mice, following stimulation with TLR7/9 agonists. We demonstrate that through TLR7/9 signaling or by IFN-α, expression of AHR is induced, which in-turn forms an inhibitory interaction with STAT1. This interaction attenuates both type I IFN signaling and in-turn, positive-feedback induction of type I IFN. Furthermore, in pristane-induced murine lupus, we found that production of type I IFN and expression of IFN-stimulated genes in AHR knockout mice is higher than in wild-type mice. In conclusion, AHR negatively regulates type I IFN signaling and production and the development of murine lupus.
Design of a bioinformatics practicum aimed at integrating concepts in immunology, virology, and pharmaceutical sciences in pharmacy curricula (EDU1P.253)
Authors: O'Donnell Lauren , Andrick Benjamin , Meng Wilson
Published: January 2023

The purpose of this educational design is to develop hands-on experiences to equip pharmacy students with bioinformatics skills through analysis of immunological problems. The instructional template uses bioinformatics tools available in the public domain to predict potential adverse drug events through identification of shared T-cell epitopes between commonly prescribed protein drugs and viral antigens. We developed a module using the web-based tools Immune Epitope and Database and Analysis Resource (http://www.iedb.org) to predict histocompatibility complex (MHC) molecules ligands and the NCBI protein database to select viral and peptide drug sequences for comparison. IEDB contains scanning tools by which sequences retieved from NCBI-PB can be analyzed for shared epitopes. Initially, students analyzed tumor necrosis factor-alpha inhibitors, a class of protein drugs that are often prescribed for chronic inflammatory diseases. Students were tasked to construct matrices representing overlapping MHC-II ligands for each drug-antigen pair. A preliminary analysis identified in the drug infliximab (heavy-chain 52-61: IRSKSINSAT) a molecular mimicry of a known T cell epitope in influenza A hemagglutinin (322-334: PRYVKQNTLKLAT)). The ultimate goal is to develop transportable exercises that can be implemented throughout the curriculum and to provide experience to students in analyzing major classes of biologic drugs and infectious pathogens.
Smurf2 regulates hematopoietic stem cell self-renewal and aging (HEM2P.256)
Authors: Trabucco Sally , Ramkumar Charusheila , Kong Yahui , Zhang Hong , Gerstein Rachel
Published: January 2023

The age-dependent decline in the self-renewal capacity of stem cells plays a critical role in aging, but the precise mechanisms underlying this decline are not well understood. By limiting proliferative capacity, senescence is thought to play an important role in age-dependent decline of stem cell self-renewal, although direct evidence supporting this hypothesis is largely lacking. We have previously identified the E3 ubiquitin ligase Smurf2 as a critical regulator of senescence. In this study, we found that mice deficient in Smurf2 had an expanded hematopoietic stem cell (HSC) compartment in bone marrow under normal homeostatic conditions, and this expansion was associated with enhanced proliferation and reduced quiescence of HSCs. Surprisingly, increased cycling and reduced quiescence of HSCs in Smurf2-deficient mice did not lead to premature exhaustion of stem cells. Instead, HSCs in aged Smurf2-deficient mice had a significantly better repopulating capacity than aged wild-type HSCs, suggesting that decline in HSC function with age is Smurf2-dependent. Furthermore, Smurf2-deficient HSCs exhibited elevated long-term self-renewal capacity and diminished exhaustion in serial transplantation. As we found that the expression of Smurf2 was increased with age and in response to regenerative stress during serial transplantation, our findings suggest that Smurf2 plays an important role in regulating HSC self-renewal and aging.