bluuebunny/crossref_metadata_2025 huggingface dataset:
Disruption of folliculin interacting protein-1 inhibits Myc-driven glycolysis and sensitizes B cells to apoptosis following activation and chemotherapy. (P1455)
Authors: Ramirez Julita , Tsang Mark , Margineantu Daciana , Hockenbery David , Iritani Brian
Published: January 2023

Folliculin interacting protein-1 (Fnip1) is a cytoplasmic protein originally discovered through its interaction with Folliculin (Flcn), a tumor suppressor mutated in the hamartoma disorder Birt-Hogg Dubé syndrome. Fnip1 and Flcn both interact with the master metabolic regulator AMPK. Our previous studies have shown that Fnip1 is required for the development of B-cells past the late pre-B stage. Fnip1 deficiency also prevented the development of pre-B cell lymphoma in Eµ-Myc transgenic mice. In this study, we examined the potential efficacy of Fnip1 inhibition in cancer and autoimmunity treatments by disrupting Fnip1 expression in B lymphoma cells and activated IgM-expressing B cells. We show that siRNA-mediated depletion of endogenous FNIP1 from a human B cell line expressing a conditional Myc allele increases oxidative phosphorylation in a glutamine-dependent manner and decreases Myc-induced glycolysis, which supports cell division (Warburg Effect). Disruption of Fnip1 also reduces Myc-induced glycolysis and increases apoptosis of murine pre-B Eµ-Myc cells in response to nutrient deprivation and chemotherapeutic drugs ex vivo. Fnip1 deficient B cells expressing an IgMHEL transgene exhibit a “tolerant” phenotype and exhibit increased sensitivity to apoptosis following BCR crosslinking. These results suggest that inhibition of Fnip1 may provide a novel strategy to sensitize lymphoma cells to apoptosis, and/or tolerize or delete activated B cells in autoimmune diseases.
B lymphocyte-induced maturation protein-1 (Blimp-1) maintains plasma cell survival by directly suppressing apoptosis signal-regulating kinase 1 (ASK1) (P1463)
Authors: Lin Kuo-I , Lin Fan-Ru
Published: January 2023

Terminally differentiated, antibody-secreting plasma cells are the end-stage effectors of humoral immune responses. Understanding the regulatory mechanisms that maintain the longevity of plasma cells provides a rational basis for designing strategies to enhance the levels of vaccine-induced antibodies. Blimp-1 orchestrates plasma cell differentiation by silencing the gene expression program of mature B cells. We showed that long-lived plasma cells in the bone marrow require the continuous presence of Blimp-1. Inhibition of Blimp-1 in plasma cells caused apoptosis. We performed microarray study examining the differential gene expression following depletion of Blimp-1 in plasma cells and uncovered that Blimp-1 suppresses apoptosis signal-regulating kinase 1 (ASK1). Blimp-1 directly suppresses ASK1 transcription. Knocking down ASK1 and Blimp-1 together in plasma cells blocked Blimp-1 knockdown-mediated apoptosis. Furthermore, ASK1 plays a negative role in survival of long-lived and short-lived plasma cells. ASK1 activity was induced during differentiation of short-lived plasma cells, and when produced by ASK1-deficient mice these cells survived better than those of control mice. Similarly, antigen-specific long-lived plasma cells generated by immunization accumulated in ASK1-deficient mice. Given that ASK1 induces apoptosis in response to stress, we rationalized that long-lived plasma cells may require sustained suppression of ASK1 by Blimp-1 to maintain survival.
Role of heterotrimeric G-protein exchange factor Ric-8A in B cell development and asymmetric cell division (P1465)
Authors: Boularan Cedric , Kehrl John
Published: January 2023

B lymphocytes likely divide asymmetrically to generate different effector and memory lymphocytes subsets. Studies in C. Elegans have shown that Resistance to inhibitors of cholinesterase (Ric)-8A helps orient the mitotic spindle during asymmetric cell division. However, the role of Ric-8A in lymphocytes development and function during asymmetric cell division is unknown. We have used a mouse strain carrying the Cre recombinase under the control of vav1 promoter or Mb1 promoter to specifically delete a floxed Ric-8a allele in all hematopoietic cells or specifically in B lymphocytes. As Ric-8A is a molecular chaperone for the Gα proteins, we first verified that Ric-8A deletion was correlated with a physical and functional loss of Gαi and Gαq proteins. As only the absolute number of B220+ cells was reduced in lymphoid organs (mostly the marginal zone B cells and T2-B subsets) we focused on the BCR signaling. Ric-8A deletion leads to an altered production of antibodies and defect in calcium response towards IgM stimulation. The simultaneously visualization of the ancestral polarity network member PKCζ with B220 distributions revealed that Ric-8A KO B-cells are less susceptible to asymmetric fate than the WT cells [ImageStream (AMNIS)]. While the molecular mechanisms leading to this phenotype are under investigation, these results indicate that Ric-8A is important for the expansion and/or differentiation of B cells by regulating the asymmetric cell division properties.
CD28 signaling in long lived plasma cells regulates glycolysis for survival and antibody production. (P1469)
Authors: Utley Adam , Carlson Louise , Murray Megan , Lee Kelvin
Published: January 2023

Recent evidence suggests that sustained humoral immunity is dependent upon bone marrow resident long-lived antibody secreting plasma cells (LLPCs). However, the signaling pathways that define LLPC survival and function are not completely understood. We have shown that CD28 signaling in LLPCs is necessary for their survival and sustained antibody production. In T cells, CD28 is known to regulate the induction of glycolysis during activation. What is unclear is how CD28 may regulate metabolism in plasma cells. It is also recognized that many of the molecular pathways governing LLPC cell survival are also observed in multiple myeloma, the transformed counterpart of plasma cells. Here we demonstrate that CD28-mediated signaling induces glut1 expression and that poisoning glycolysis inhibits proliferation and antibody production in both plasma cells and multiple myeloma cells. Interestingly, inhibition of glycolysis inactivates AMPK, a major metabolic sensor responsible for facilitating fatty acid oxidation (FAO). Furthermore, induction of AMPK by AICAR could not rescue the defects induced by glycolytic inhibition, suggesting that LLPCs require CD28-mediated glycolytic end products for both biosynthesis and oxidative energy production. By understanding the metabolic regulation of plasma cell induction, maintenance, and function, we may find insight into how targeting metabolism may alter both long-term humoral immunity and how we can use this knowledge to treat multiple myeloma.
HIV triggers interleukin 21-mediated induction of granzyme B-secreting B cells with T cell regulatory potential. (P1441)
Authors: Jahrsdörfer Bernd , Kaltenmeier Christof , Gawanbacht Ali , Lindner Stefanie , Beyer Thamara , Härter Georg , Grüner Beate , Kern Peter , Kirchhoff Frank , Schrezenmeier Hubert
Published: January 2023

Certain regulatory lymphocyte subpopulations like regulatory T cells can suppress T cell expansion by granzyme B (GrB) secretion. Recently, we found that B cells can also produce GrB and acquire regulatory potential in response to interleukin (IL-)21. Since HIV is associated with elevated serum IL-21 levels, we hypothesized that GrB-expressing B cells may be induced during HIV infection. Here, we show that infection of CD4+ T cells with HIV 1 (NL4-3) induces strong expression of IL-21 without upregulation of CD40 ligand. We demonstrate that such IL-21+CD40Llow T cells secrete IL-21, thereby inducing GrB in cocultured B cells rather than supporting plasma cell differentiation. In line with these results, serum levels of both IL-21 and GrB are significantly higher in HIV-infected patients before HAART as compared to healthy controls. Up to 60% of freshly isolated B cells from HIV-infected patients, but not healthy control B cells, express GrB. Of note, coculture of HIV-infected CD4+ T cells with GrB+ B cells results in GrB transfer, and strongly suppresses both T cell proliferation and viral replication as indicated by significantly reduced p24 levels. The observed effects are enhanced by IL-21, and reduced by GrB inhibition. In summary, GrB induction in HIV-specific B cells may play a role in decelerating expansion of HIV-infected CD4+ T cells, while interfering with their terminal differentiation into plasma cells.
The role of Bcl6 in the regulation of transitional B cells and resting mature B cells (P1449)
Authors: Wu Hao , Hollister Kristin , Dent Alexander
Published: January 2023

As a conserved zinc finger transcription repressor, Bcl6 determines follicular helper T cell differentiation and plays indispensible roles in B cell development and function, especially at the stages of pre-B cell and germinal center B cell. To better understand the role of Bcl6 in B cell lineage, we generate Bcl6 conditional KO mouse by crossing Bcl6 floxed mouse newly made in our lab to CD19cre mouse. Interestingly, B cell development in the bone marrow of conditional KO mice is indistinguishable from control mice. At the transition of T1 B cell to T2 B cell stage, Bcl6 is upregulated and continually maintained in mature B cells. Mainly because of a significant decrease of follicular B cell, conditional KO mice have significantly fewer B220+ cells in spleen compared to control mice. Furthermore, in conditional KO mice, spontaneously-formed GCB cells in Peyer’s patches are decreased dramatically, but there is only slight reduction of GCB cells in spleen after SRBC immunization. The GCB cells in spleen appear to be from incomplete deletion of Bcl6 by CD19cre and by strong proliferation of non-deleted GCB cells. Finally and surprisingly, we detect “truncated Bcl6” in non-GCB cells from Peyer’s patches and spleen in conditional KO mice, which is four times higher than WT Bcl6 in control mice. This indicates an autorepression regulation of Bcl6 in resting mature B cells. Our findings suggest that Bcl6 regulates transitional B cell maturation and maintenance of mature B cell.
IL-22 receptor-expressing stromal cells are located in the germinal center dark zone (P1451)
Authors: Siegers Y. , Reijmers Rogier , Papazian Natalie , Cupedo Tom
Published: January 2023

After antigen encounter and subsequent T cell-mediated B cell activation, germinal centers are formed within B cell follicles of lymph nodes giving rise to high affinity antibody secreting plasma cells and memory cells. Germinal centers are typically divided into a dark zone and light zone in which proliferation, class switching, affinity maturation and selection of B cells takes place. Stromal cells in the light zone are mainly follicular dendritic cells which are important in selection of high affinity B cells through presentation of immune complexes. On the other hand, dark zone stromal cells are still poorly defined. In search for novel markers to identify dark zone stroma we observed expression of IL-22R on stromal cells in the germinal center dark zone 10 days after immunization with TNP-KLH. Flow cytometric analysis confirmed that the IL-22R was expressed by an unidentified CD31-podoplanin- stromal cell subset within lymph nodes and that expression of the IL-22R was only detected after immunization. To understand the signals involved in the induction of IL-22R expression we set up an in vitro culture system of primary human stromal cells. Stimulation of these stromal cells with activated B cell-derived cytokines indicated that IL-22R expression could be controlled by TNF-α. These data suggest that IL-22R may be used as a novel marker for dark zone stromal cells and allow for speculations on a possible role for IL-22R signaling in germinal center reactions.
Blurring of the distinction between hemophagocytic lymphohistiocytosis and macrophage activation syndrome (P3307)
Authors: Cron Randy , Zhang Mingce
Published: January 2023

Hemophagocytic lymphohistiocytosis (HLH) is a fatal disorder of infancy resulting from homozygous mutations in cytolytic pathway proteins [MUNC13-4, RAB27A, PRF1, STX11, STXBP2]. Secondary HLH, or macrophage activation syndrome (MAS), results beyond infancy from rheumatologic, infectious, and oncologic conditions. We screened 21 children with MAS and identified 13 with mutations in 1 of the HLH genes. Three mutations [RAB27A (A87P), PRF1 (A91V), STXBP2 (A433V)] were studied for their effect on NK cell lytic activity. Lentiviral expression vectors were generated with wild-type (WT) or specific gene mutations, sequenced, and introduced into NK-92 cells. Transduction of WT RAB27A into NK-92 had no effect on cytolysis of K562 cells, whereas the mutation decreased cytolytic activity by ~50%. Both the PRF1 and STXBP2 mutants inhibited cytolysis to lesser degrees. The RAB27A mutant, but not the PRF1 or STXBP2 mutants, inhibited degranulation (diminished CD107a upregulation). All 3 mutants had no effect on intracellular perforin or granzyme B levels, or on conjugate formation with target cells. Confocal microscopy revealed the RAB27A mutant disrupted granzyme B polarization to the immunologic synapse. The data suggests single copy HLH gene mutations can partially disrupt cytolytic activity via a dominant negative effect, thus, blurring the genetic distinction between HLH and MAS.
Phenotypic analysis of B cell subsets in HLA*B44 positive identical twins discordant for common variable immunodeficiency and recurrent sino-pulmonary infection (P3331)
Authors: Szymanska Mroczek Ewa , Ippolito Gregory , Zemlin Michael , Hwangpo Tracy , Brand Marsha , Zhuang Yingxin , Crossman David , Osborne John , Schneider David , Liu Cunren , Lefkowitz Elliot , Crowley Michael , Georgiou George , Brown Elizabeth , Schroeder, Jr. Harry
Published: January 2023

Depressed serum immunoglobulin levels (sIgs) and recurrent sinopulmonary infections mark Common Variable Immune Deficiency (CVID). Many family members of CVID patients also suffer recurrent sinopulmonary infection (RESPI) but have normal sIg. We identified HLAB44 positive identical female twins who suffer sinopulmonary infections and are discordant for CVID and RESPI. Flow cytometry subsets showed equivalent numbers of immature B cells (BC) in both twins, but lower numbers of transitional and mature BC in the CVID twin. Deep sequencing of the immunoglobulin (Ig) repertoires expressed by the transitional and mature BC showed a significant divergence in the utilization of VH1 and VH4 family gene segments, with CVID favoring VH4 and RESPI VH1. RESPI twin used JH6 more frequently, whereas CVID twin used JH3. The amino acid composition of CDR-H3 repertoire was compared with a control; the twin and control tyrosine usage in transitional BC was similar (~15%) but greatly diverged in mature BC (control 15%, RESPI 25%, CVID < 10%). Whole genome sequencing revealed homozygosity for a rare CD21 S639N polymorphism and heterozygosity for CD19 L174V. These findings suggest that in addition to an acquired block in BC development at the transitional stage, the CVID twin produces an Ig repertoire that is markedly depleted of tyrosine. This may explain why the function of the Ig repertoire in CVID is more impaired than what might be expected by sIgs levels.
Regulation of CD19 surface expression in human B cells by CD81 (P3323)
Authors: Levy Shoshana , Kuo Chiung-Chi , Sagi Yael , Chen Homer , Kela-Madar Neta , van Zelm Menno , van Dongen Jacques
Published: January 2023

A girl was diagnosed with primary antibody deficiency. Her B cells lacked surface CD19, although both alleles were normal. The cause was a homozygous exon splice site mutation in CD81(1). This human deficiency differs from CD81 knockout mice, in which surface CD19 is lower but not completely absent. The patient’s EBV-transformed B cells were used to determine how CD81 controls the trafficking of CD19. We show that the CD81 mutant (CD81mut) protein is expressed intracellularly in the patient’s B cells. Using a proximity ligation assay we also demonstrate that the truncated CD81mut protein interacts intracellularly with CD19. This interaction with the CD81mut protein abrogates carbohydrate maturation and trafficking of CD19 to the cell surface. However, expression of CD81mut did not exert a dominant negative effect on CD19 surface expression in B cells that express or lack CD81. Finally, using a reconstitution system we identify specific CD81 domains that restore carbohydrate maturation and cell surface expression of CD19 in the patient’s B cells. This specific case of antibody deficiency was manifested because of lack of surface expression of CD19, an important B cell signaling molecule. However, the maturation of CD19 and its trafficking to the cell surface require the presence of specific domains of the tetraspanin CD81 molecule. 1.van Zelm et al (2010) CD81 human gene defect disrupts CD19 complex formation and leads to antibody deficiency. J Clin Invest. 120: 1265.
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

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

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

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

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

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

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

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

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

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

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

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

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

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

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< 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.
Stabilized lipid nanocapsules eliciting potent mucosal immune response (P4509)
Authors: Li Adrienne , Moon James , Irvine Darrell
Published: January 2023

We investigated the use of a novel lipid nanocapsule, Interbilayer-Crosslinked Multilamellar Vesicles (ICMV), for mucosal vaccine delivery. ICMV co-encapsulated with MPLA and/or polyI:C as adjuvants, was used to vaccinate mice using model antigen and SIV-gag epitopes. When delivered via the lungs, we found that ~60-fold more antigen transported to the draining lymph nodes than equivalent subcutaneous vaccination, mediated by the vast number of antigen presenting cells in the lungs. Potent CD8 T-cell response was elicited by ICMV pulmonary vaccine and established a substantially greater memory population than a soluble vaccine, with 5.2-fold and 4.8-fold higher frequencies of antigen-specific cells in lungs and spleen, at 11 weeks post-priming. T-cells primed by ICMV pulmonary vaccination were also found to be strongly biased toward an effector memory phenotype, and a 5-7-fold higher number of effector memory cells were found in both the local and distal mucosa compared to soluble vaccines. Protection against a viral challenge was observed in animals given ICMV pulmonary vaccine while animals that received subcutaneous ICMV or pulmonary soluble vaccines showed steady weight loss that led to 100% mortality by day 5 post challenge. These particles can be prepared using aqueous solution processing techniques currently used for production of commercial liposomal products. They can also be freeze-dried for long term storage and have shown to retain immunogenicity after 1 month.
Humoral immune responses elicited by the polyanhydride particle vaccine platform are facilitated by enhanced T follicular helper cell phenotype (P4511)
Authors: Huntimer Lucas , Ross Kathleen , Ramer-Tait Amanda , Narasimhan Balaji , Wannemuehler Michael
Published: January 2023

Previous studies have shown that the use of the polyanhydride nanoparticle (NP) vaccine platform provides innate immunomodulatory activity while also controlling antigen release kinetics that enhance antibody-mediated immunity. The goal of this project was to demonstrate that the increased antibody responses detected by utilizing NP-based vaccines induced of robust T helper cell responses to promote B cell activation and differentiation into antibody secreting cells. Using the ovalbumin (Ova) specific T cell (OT II) model, we evaluated the mechanisms associated with the expansion and activation of Ova-specific CD4+ T after immunization using three different polyanhydride NP formulations that contained encapsulated Ova. Significant expansion of CD4+ T cells occurred following immunization with Ova-loaded 20:80 CPH:SA or 20:80 CPTEG:CPH PNPs. Similar to Alum and MPLA adjuvanted responses, these NP regimen induced T follicular helper cells (CXCR5high PD-1high) that are important for initiating and maintaining germinal centers. Following a single administration, PNP vaccines induced robust Ova-specific IgG1 antibody titers. Upon secondary immunization, the NP vaccinated mice effectively responded with robust Ova-specific IgG1 and IgG2c antibody responses. The NP vaccinated mice presented with more antigen-specific Tfh (vα2+, vβ5+) CD4 T cells and more numerous germinal centers cells (B220+ CD23high PNA+) that supported more avid Ova-specific IgG1.
Analysis of the humoral immune response to HSV-2 glycoproteins in ACAM529-vaccinated mice. (P4519)
Authors: Hernandez Hector , Hamberger John , Lai Charles , Kleanthous Harold , Parrington Mark , Delagrave Simon
Published: January 2023

HSV-2 infection is a global, serious public health problem affecting over 530 million people. ACAM529, a vaccine candidate targeting genital herpes infections, is an HSV-2 virus rendered incapable of replication, through deletion of the genes UL5 and UL29. Blockage of viral DNA replication is expected to prevent true late gene expression, yet this vaccine successfully protected animals against infection and lethal challenge in preclinical studies (Delagrave et al., 2012, PLoS One, 7(10): e46714). Here we present the IgG responses against viral glycoproteins elicited by ACAM529 vaccination. We compare the ability of ACAM529 to generate IgG responses to gG and gD, two leaky late (γ1) HSV-2 antigens, and gC a true late antigen (γ2).
The role of antibody and CD4 follicular helper cells during malaria liver stage infection (P4485)
Authors: Keitany Gladys , Jang Ihn Kyung , Finney Olivia , Smithers Hannah , Wang Ruobing
Published: January 2023

The liver stage malaria provides an excellent target for malaria vaccine: eliminating the parasite at this stage using immunization with attenuated parasites that arrest in the liver results in long-term sterile protection. This immune response has been associated with CD8 T cells, as well as antibodiesdirected against the sporozoite. In this study, we hypothesized that genetically attenuated pasite (GAP) immunization induces T follicular helper cells (Tfh), which in turn induce memory B cells responses, leading to long-lived and high avidity functional antibodies to pre-erythrocytic malaria. Using immunization with GAP that arrest during late liver-stage development, we showed that immunization induces high titer antibodies, and that these antibodies can inhibit parasite growth in liver cells in vitro by 99%. To understand the induction of the antibody response, we characterized both the B cell response and the Tfh response in the liver, the spleen and the lymph nodes. Furthermore, in order to determine the localization of the priming of the response, immunization via mosquito bite and intravenous injection were compared. Understanding the role of Tfh in liver stage malaria and how they are induced will be crucial in designing an effective antibody based malaria vaccine.
AERAS-402/Ad35 vaccine induces CD4+ and CD8+ responses in HIV-infected, BCG-vaccinated adults (P4487)
Authors: Hokey David , Pandian Ramanan , Graves Andrew , Churchyard Gavin , Snowden Peggy , Douoguih Macaya , Landry Bernard
Published: January 2023

One-third of the world's population is estimated to be infected with TB. The WHO no longer recommends that the currently available TB vaccine, BCG, be given to infants born to HIV+ mothers due to safety concerns. An improved vaccine that is effective and safe for HIV+ infants is needed to protect this vulnerable population. AERAS-402 is a novel replication deficient Ad35 TB vaccine candidate that expresses a fusion protein of the Mtb antigens 85A, 85B, and TB10.4 and has been shown to safely induce CD8+ T cell responses in healthy adults. This study examined the safety and immunogenicity of a single IM dose of AERAS-402 in HIV+ adults. No vaccine-related serious adverse events were reported, suggesting the vaccine is safe in this population. CD4+ and CD8+ T cell responses were evaluated using ICS. Prior studies in healthy adults revealed primarily CD8+ responses to vaccination with AERAS-402. Vaccination of HIV+ adults resulted in the elicitation of both CD4+ and CD8+ responses of ~0.1% to 0.2% of the parent population largely directed to 85A and 85B. CD4+ responses were primarily polyfunctional (IFN-γ, IL-2, and TNF) and bifunctional (IL-2 and TNF) while CD8+ responses were largely bifunctional (IFN-γ and TNF) and monofunctional (IFN-γ) with lower levels of polyfunctional cells observed. Together the data suggest that vaccination with AERAS-402 in HIV+ adults is safe and immunogenic, resulting in a mixed CD4+ and CD8+ multifunctional T cell response to encoded antigens.