bluuebunny/crossref_metadata_2025_split huggingface dataset:
Incorporation of a sequence-based HLA typing method into an immunology undergraduate laboratory course. (P4527)
Authors: Garrison Keith , Purtell Sean , Shaw Brian 
Published: January 2023

Abstract
Antibody-based laboratory techniques (e.g. Western blots and ELISA) are relatively easy to implement in an immunology course and widely applicable throughout the subfields of molecular biology. Incorporating lab activities with relevance to T cell immunology is more challenging because they often require proficiency in cell culture and sterile technique. HLA typing facilitates exploration of T cell immunology, including HLA presentation and the development of a T cell response. However, clinical HLA typing kits are costly. Numerous PCR amplifications require high yields of genomic DNA, entailing the challenges of working with blood as a source for DNA in the classroom. Also, typing requires precise gel loading of many samples, which can be challenging. We transformed a sequence-based HLA typing methodology recently developed for research use with plasma samples into a protocol for an immunology course lab. Since the methodology works with the low levels of DNA recoverable from plasma, the technique is well suited for DNA extraction from buccal swabs. The HLA typing process required the following labs to complete: (1) DNA extraction and quantitation (2) primary PCR set-up (3) optional verification of amplification success and secondary PCR set-up (4) verification of nested PCR amplification and sample preparation for sequencing. Additional time was required for editing sequence data and interpreting HLA types using dbMHC.
An immunology course focused on faculty research interest provides key inputs for research productivity at a primarily undergraduate institution (P4529)
Authors: Chatterjea Devavani , Martinov Tijana 
Published: January 2023

Abstract
Rapid turnover of trained undergraduate student researchers and limited time faculty and students can devote to research activities during the academic year are some of the roadblocks to maintaining a productive research program at a primarily undergraduate institution. At Macalester College, a highly selective liberal arts institution, we have addressed these issues, in part, through the design and implementation of a “Research in Immunology” course that is open by application to a group of eight second- or third-year students each spring. The course consists of a journal club with reading, writing and presentation activities that immerse students in the primary literature around the faculty member’s research question, and a laboratory component that emphasizes intensive skill building while fully exposing students to novel, unanswered questions. A subset of these students then enter the lab the following summer with a high level of intellectual and technical preparation and many are highly productive in the laboratory for 2+ years providing continuity of trained personnel, critical peer mentoring and a degree of involvement in and ownership of the research problems that is rare among undergraduates. In the past 6 years, I have trained 40 students in my laboratory most of whom have taken this course. Of these, 30% have completed honors research projects and earned co-authorship on manuscripts, and 6 have gone on to PhD programs in immunology/biomedicine.
Immunology education in an accelerated PharmD program (P4531)
Authors: Mandal Manas , Coffman Renee , Rosenberg Harry 
Published: January 2023

Abstract
Biomedical education and training in professional degree programs in the United States is at a crossroad. Tremendous advancement in biomedical, specifically immunology research has not changed the fundamentals of immunology teaching in recent years at our medical, pharmacy or nursing colleges. Diabetes, obesity and various autoimmune diseases are ever increasing in US population. Majority, if not all of these diseases have their roots in dysregulated inflammatory pathways that strongly argues for improved teaching and understanding of immunology with disease management using new immune-based therapeutics. Here, we describe a unique immunology curricular design and assessment methods in an accelerated PharmD program. Our curricular design is learning objective-based with emphasis on a student-centered mastery of learning educational model where students actively participate in the learning process. Pharmacy-year 1 (P1) students are taught basics of ‘hematology and immunology’, whereas, P2 students are taught ‘clinical immunology’ in a block system. Assessments are administered at the end of the block using a ‘pass’ (90% score) or ‘no pass’ grading system. Cumulative data shows that vast majority of the students pass the assessments with the first attempt and also expressed satisfaction with the curricular design and course contents. Student evaluation of the course contents, faculty teaching and assessment constitutes an integral part of curricular improvement.
High throughput selection of co-stimulation molecules for 3rd generation designer T cells (P4362)
Authors: Guo Xiuyang , Bais Anthony , Gomes-DaGama Erica , Pawar Kartik , Junghans Richard 
Published: January 2023

Abstract
Ample co-stimulatory interactions between antigen-presenting cells and T cells are essential for an effective immune response. Similarly, co-stimulation related immuno-experiences have also been considered critical for the successful tumor eradication by T cells in adoptive immunotherapy cases of: conventional tumor infiltrating T cells for melanoma, chimeric antigen receptor (CAR)-expressing EBV-CTLs for EBV-associated malignancies and the recent CD19 CAR T cells for B-cell neoplasms. However, the specific co-stimulation signals playing the major role in successful immunotherapies are still not identified. Here, a series of constructs has been made to compare the effects of most known co-stimulation molecules, including 4-1BB, CD2, CD27, CD28, CD30, LFA1, GITR, HVEM, ICOS and OX40, in the 1st generation designer T cells bearing anti-CEA scFv-linked CD3ζ signal, and in the 2nd generation designer T cells with anti-CEA scFv linked with CD3ζ plus CD28 signal. Corresponding designer T cells’ function in vitro in proliferation/survival, cytotoxicity and cytokine secretion, and in vivo in animal models, will be tested and compared with the 1st/2nd generation designer T cells. The ones standing out will be delineated for their mechanisms from co-stimulation ligation to specific intracellular signal pathway that lead to the T cell phenotype responsible for final tumor rejection, and will be applied for 3rd generation designer T cell development.
Generation of human T cells expressing only the engineered tumor antigen specific TCR in humanized mice for preclinical research and anticancer therapy (P4364)
Authors: Hu Zheng , Xia Jinxing , Wargo Jennifer , Yang Yong-Guang 
Published: January 2023

Abstract
Clinical application of adoptive transfer of TCR-engineered CTLs, a promising cancer immunotherapy, is limited by difficulties in obtaining sufficient numbers of effective CTLs and by TCR subunit misparing in engineered CTLs. As we reported at the 2012 AAI Meeting, humanized mice, made by transplantation of thymic tissue and CD34+ cells that were transduced with HLA-A2-restricted melanoma antigen (MART-1)-specific TCR, showed efficient development of MART-1-TCR+ human T cells expressing only the engineered TCR. Here we show that although T cells expressing MART-1-TCR were mainly CD8+, CD4 T cells expressing MART-1-TCR were also detected in most humanized mice examined. However, only MART-1-TCR+CD8+ T cells showed specific responses to MART-1 peptide immunization in vivo. In vitro CTL assay revealed that MART-1-TCR+CD8+ cells from humanized mice can mediate robust specific killing of melanoma cells, and fully retain their cytotoxicity after substantial in vitro expansion (up to tens of thousands of times). Adoptive transfer of these expanded CD8 T cells induced potent antitumor responses in both subcutaneous and metastatic tumor models. Furthermore, treatment with IL-15 significantly improved the survival and anti-tumor effects of adoptively transferred MART-1-TCR+CD8+ cells. These findings indicate that humanized mice provide not only an in vivo model for preclinical research, but also a potential means of producing a large quantity of human T cells for cancer immunotherapy.
Polyanhydride particle vaccine platform enhances early antigen-specific cytotoxic T cell responses (P4366)
Authors: Huntimer Lucas , Ross Kathleen , Darling Ross , Ramer-Tait Amanda , Narasimhan Balaji , Wannemuehler Michael 
Published: January 2023

Abstract
Bioerodible nanoparticles have been shown to be useful as vaccine delivery vehicles. Previous work from this laboratory has shown that polyanhydride nanoparticles (NPs) can be used as an effective platform delivery system with the ability to modulate the release kinetics and to physicochemically stabilize protein antigens, activate dendritic cells, and elicit protective immunity to pathogens. The goal of this project was to evaluate the ability of the polyanhydride NPs to enhance cell-mediate immunity. Using a high frequency adoptive transfer model employing 3 x 106 OT I CD8+ T cells, we demonstrated significant expansion of ovalbumin (Ova)-specific CD8+ T cells that expressed an effector memory phenotype (CD44high CD62Llow) following immunization with Ova-loaded NPs. Induction of these effector CD8+ T cells resulted in the development of fewer and smaller tumors after implantation of EG7 tumor cells compared to mice immunized with Alum plus Ova or soluble Ova alone. To better model the naturally occurring low frequency of naïve CD8+ T cells, 3,000 OT I T cells were adoptively transferred prior to immunizing mice with one of three separate polyanhydride NP formulations (20:80 CPH:SA, 20:80 CPTEG:CPH, or 50:50 CPTEG:CPH). The results of these studies demonstrated that immunization with biocompatible and biodegradable 20:80 CPH:SA polyanhydride NPs effectively enhanced the induction of antigen-specific cytotoxic CD8+ T cells.
Inflammatory DCs fail to present antigen but provide a crucial source of IL-12 in priming Th1 responses to CpG based vaccines. (P4514)
Authors: De Koker Stefaan , Willart Monique , Pollard Charlotte , Guilliams Martin , Lambrecht Bart , Grooten Johan 
Published: January 2023

Abstract
DCs govern the induction of antigen specific adaptive immunity and thus constitute the main targets of novel vaccine approaches. Classical steady state DCs consist of multiple subsets, differing in their capacity to respond to TLR stimulation and to present antigen to CD4 and CD8 T cells. Under inflammatory conditions, an additional subset of monocyte derived inflammatory DCs (iDCs) arises. Although the innate role of iDCs in antimicrobial immunity is now clearly established, their role in directing the adaptive response is less evident. Immunization with OVA combined with the TLR9 agonist CpG resulted in the fast recruitment of CCR2 CD64 Ly6Chi CD11bhi monocytes to the draining lymph nodes, where they gradually adopt a CD11cint MHCIIint phenotype. Although a large fraction of these iDCs were OVA+, sorted iDCs failed to present the antigen to OT-I and OT-II T cells. Instead, antigen presentation was limited to migratory DCs. iDCs however proved to be the major source the Th1 polarising cytokine IL12. To investigate the role of iDC-derived IL12 in priming Th1 responses, mixed CCR2-/- /IL12-/- bone marrow chimaeras were immunized with OVA/CpG. In these chimaeras, solely IL12-/- iDCs can reach the lymph node, while classical lymph node DCs are fully competent of producing IL12. When compared to control WT/IL12-/- chimaeras, CCR2 -/-/IL12-/- mice displayed dramatically reduced Th1 responses, establishing the crucial role of iDCs in priming Th1 responses.
Characterization of the immunostimulatory activity and safety of a vaccine adjuvant de-O-acylated lipooligosaccharide (P4520)
Authors: Lee Na Gyong , Han Ji Eun , Cho Yang Je 
Published: January 2023

Abstract
Lipopolysaccharide (LPS), a constituent of the outer membrane of Gram-negative bacteria elicits strong immunopathological reactions observed during bacterial infection, and the lipid A moiety of LPS is responsible for the immunostimulatory activity. Lipid A exerts its biological activity by sending signals via toll-like receptor 4 present on immune cells. Many natural and synthetic analogues of lipid A have been developed as vaccine adjuvant. In this study, we evaluated the immunostimulatory activities of de-O-acylated lipooligosaccharide (deacyl-LOS) on murine and human immune cells in comparison with monophosphoryl lipid A. The results indicated that deacyl-LOS is more potent than MPL in inducing cytokine secretion and activation of monocytes and dendritic cells, which was confirmed to be mediated by TLR4. It also induced allogeneic T cell responses and activated B cell proliferation. The acute toxicity test showed that deacyl-LOS did not cause any pathological effects or death at 0.25, 0.5 or 1 mg per kg body weight in mice. These data suggest a potential of deacyl-LOS as a safe and effective vaccine adjuvant.
The role of the Myc family member, Mnt, in OX40-promoted T cell survival (P4526)
Authors: Lukaesko Lisa , Vasilevsky Nicole , Bambina Shelly , Bahjat Keith , Hurlin Peter , Weinberg Andrew 
Published: January 2023

Abstract
Together with T cell receptor recognition of cognate MHC-peptide complexes, signaling through OX40 promotes late-stage proliferation, survival, and enhanced memory formation of T cells. Despite its known role in co-stimulation, the intracellular signaling pathways regulated by OX40 are not well understood. Recently, we demonstrated that stimulation with an OX40 agonist increases Mnt protein levels by promoting Mnt protein stability. Mnt is thought to play a role in apoptosis, cell survival, and cell proliferation by opposing Myc activity through competition for binding to Max and shared target genes. Using siRNA-directed knockdown of Mnt, we demonstrated that Mnt is necessary for T cell survival ex vivo. In these current studies, we have examined the necessity of Mnt in OX40 signaling and function in the primary T cell response following vaccination. Using OX40-cre/ Mnt-floxed mouse model, we found that Mnt is required for the OX40-mediated enhancement of the primary CD4 T cell response following Listeria monocytogenes vaccination. In addition, we found that OX40 signaling decreased glucose uptake in T cells, a predicted consequence of Mnt upregulation and its antagonism of Myc-mediated glycolytic activity. Hence, future studies will examine if the glycolytic pathway is altered in Mnt knock-out T cells stimulated with OX40 agonists.
Activation of macrophages by natural TLR4 ligand isolated from Paecilomyces cicadae (P4496)
Authors: Kim Hyung Sook , Lee Hong Kyung , Kim Yeon Jin , Ryu Hwa Sun , Kim Ji Sung , Kim Juyoung , Kim Yong Guk , Hong Jin Tae , Kim Youngsoo , Han Sang-Bae 
Published: January 2023

Abstract
Many polysaccharides isolated from mushroom are considered to be biological response modifiers and have been shown to enhance various immune responses. Paecilomyces cicadae have been reported to have immunomodulatory properties. In this study, we investigated the membrane receptor and intracellular signaling responsible for the activation of macrophage by polysaccharide (PCP) isolated from P. cicadae. PCP induced the production of nitric oxide (NO) and the mRNA expression of IL-1β, IL-6, and TNF-α in RAW 264.7 cells. To investigate the membrane receptor involved in the activation of NO production, we examined the effect of PCP on primary macrophages isolated from C3H/HeN mice having normal-TLR4 and C3H/HeJ mice having mutant-TLR4. PCP induced NO production and cytokine gene expression in macrophages from C3H/HeN, but not from tlr4-mutated C3H/HeJ mice, which suggests that TLR4 is the membrane receptor for PCP. As a mechanism of action, PCP induced the phosphorylation of ERK, JNK, and p38, and the nuclear translocation of NF-κB p50/p65, which are the main signaling molecules down-stream from TLR4. In addition, p38 and NF-κB inhibitor attenuated PCP-mediated induction of NO production by macrophages. These results indicate that PCP activates macrophages through the TLR4 signaling pathway
Enhanced expansion of follicular T helper cells following vaccination with gp120-encoding DNA (P4530)
Authors: Dent Alexander , Hollister Kristin , Chen Yuxin , Wu Hao , Lu Shan 
Published: January 2023

Abstract
Follicular T helper (Tfh) cells are essential for germinal center (GC) reactions and somatic hypermutation of antibodies. Vaccination by the DNA prime-protein boost method typically yields antibodies with higher specificity and avidity than conventional approaches, and leads to production of neutralizing antibodies specific for conserved epitopes. Because Tfh cells are crucial for the development of plasma cells from the GC, we hypothesized DNA priming causes more Tfh cell differentiation, producing a more robust GC response. To test this, we primed mice with either gp120-encoding DNA or gp120 protein, three times, at two week intervals. Following a four week rest period, all mice received two gp120 protein boosts, two weeks apart. Priming with gp120-encoding DNA caused increased Tfh cell differentiation, compared to priming with protein alone, both after the priming injections, as well as after the final protein boosts. Additionally, GC B cell numbers were significantly increased with DNA priming. Mice receiving DNA priming had peak Tfh and GC B cell numbers earlier than mice receiving only protein. Furthermore, analysis of memory Th cells shows more Tfh cells being activated out of the memory cell pool when DNA priming is used. Our data suggests a role for Tfh cells in a DNA-prime, protein-boost vaccine strategy, and better expansion of Tfh cells may account for the superior antibody responses seen with this approach.
The development of a novel anti-peptoid antibody for a peptoid-based vaccine platform (P4506)
Authors: Desmond Angela , Case Allison , Yi Kate , Smallshaw Joan , Collins Angela , Dye Kelly , Mapes Kelly , Saherwala Ali , Chen John , Tran Helen , Connolly Michael , Zuckermann Ronald , Chirayil Sara , Luebke Kevin , Astle John , Reddy M. , Kodadek Thomas , Vitetta Ellen 
Published: January 2023

Abstract
Peptoids are synthetic molecules that share structure with peptides, but bear side groups on the backbone nitrogen. They are protease resistant and relatively cheap to synthesize. Since many amines can form peptoid side groups, peptoids are more diverse than peptides and can take on various shapes, making them good B cell epitopes or haptens. We aim to develop peptoid-based vaccines by screening on-bead peptoid libraries we have made with broadly neutralizing monoclonal antibodies against pathogens. Resulting peptoids can be attached to carrier proteins (which provide T cell epitopes) to elicit antibodies that should mimic the screening antibody. To test the immunogenicity of peptoid haptens, we generated the first affinity-purified anti-peptoid antibody by immunizing rabbits with a peptoid linked to a carrier and adsorbed to an adjuvant, alum. Antibodies were produced against the hapten, carrier and linker. After affinity purification, an enzyme-linked immunosorbent assay demonstrated a robust, specific response against the peptoid. This vaccine platform will now be applied using broadly neutralizing monoclonal antibodies against West Nile and hepatitis C viruses, mouse norovirus-1 and HIV. Candidate peptoids will be tested for the ability to induce neutralizing antibodies against the pathogen. If successful, this platform could be applied to any pathogen for which a broadly neutralizing monoclonal antibody is available, without structural knowledge of the native epitope.
Vaccine induced antigen persistence mediated by dendritic cells and lymphatic endothelial cells influences immune memory (P4508)
Authors: Tamburini Beth , Burchill Matthew , Kedl Ross 
Published: January 2023

Abstract
Numerous studies have established that antigen persists for extended periods of time following infection. However, little is known about the functional significance of this persisting antigen. Herein, we demonstrate that antigen can persist in a mouse following infection with both vaccinia virus and listeria monocytogenes far longer than the peak of the immune response and clearance of the infection. Furthermore, using a TLR/CD40 protein combination vaccine as a model we are able to show persistence of antigen for more than 20 days beyond the peak of the primary immune response. Antigen persistence following TLR/CD40 immunization is dependent on the TLR stimulatory component of the vaccine as well as antigen dose. Surprisingly, we find that antigen persists, not on follicular dendritic cells, but on lymphatic endothelial cells, and requires the participation of conventional dendritic cells but not B cells. Expansion of the LECs in the lymph node is required for antigen persistence and this expansion is a direct result of T cell expansion in the lymph node. Taken together, these studies demonstrate that 1) antigen from subunit vaccination can persist for extended periods after immunization, 2) antigen persists on LECs, a cell type not previously known for its capacity to capture and hold antigen for extended periods of time, and requires LEC expansion, and 3) this persistence correlates directly to the induction of protective immunity after vaccination.
Characterization of T cell responses to cryptic epitopes in recipients of a non-codon optimized HIV-1 vaccine (P4495)
Authors: Bet Anne , Sterrett Sarah , Sato Alicia , Bansal Anju , Goepfert Paul 
Published: January 2023

Abstract
Cryptic epitopes (CE) are MHC-I-restricted peptides encoded by any of the 5 alternative reading frames (ARFs) of a gene. CE-specific T cells (Tc) have previously been detected in HIV-1 infected patients but whether responses to CE can be induced by vaccination is currently unknown. Our previous work demonstrated that vaccination with naturally-encoded, non-codon optimized vectors generate CE more frequently than codon-optimized vectors. We therefore analyzed samples from 126 individuals who received either a naturally-encoded MVA/HIV62 double prime-double boost regimen (vaccine) or saline (placebo) during the HVTN205 trial. Peripheral blood mononuclear cells collected at 2 weeks post final vaccination were stimulated in an IFNγ ELISpot assay with overlapping peptide pools (OLPs) for HIV-1 Gag, Pol, and all 5 ARFs of these regions. Vaccinees had significantly more positive responses toward Gag but not Pol than placebo recipients (p=0.020, Mann-Whitney U test). Ex vivo Tc responses to potential CE were low in magnitude and their frequency did not differ significantly between treatment groups. In a cultured assay however, the median magnitude of responses to ARF OLP subpools was significantly greater in vaccinees (p&lt; 0.001, Mann-Whitney U test), indicating CE-specific Tc responses are present but below the IFNγ ELISpot assay’s limit of detection. Thus, we recommend that future vaccines expand the breadth of HIV-1 Tc responses through designs that preserve naturally-encoded CE.