bluuebunny/crossref_metadata_2025 huggingface dataset:
Inhibition of glycogen synthase kinase 3 beta promotes long term cytokine-free survival of chimeric antigen receptor-expressing T cells (VAC11P.1008)
Authors: Sengupta Sadhak , Sampath Prakash
Published: January 2023

Application of chimeric antigen receptors (CAR), where an antibody or ligand-binding domain is fused with the zeta signaling chain of the T cell receptor (TCR), is a novel and alternative strategy to bypass the failure of cytotoxic immune response induction by most tumor cells. The resulting CAR+ T cells are redirected by the neo-specificity to attack tumors expressing the surface antigen or receptors recog¬nized by the gene-modified TCRs. Although CARs have shown impressive preclinical efficacy, translational success is restricted due to the limited viability of the T cells in absence of IL2 consequent to preconditioning chemotherapy. Although multiple doses of T cell transfer and systemic administration of IL2 are effective for short time periods, unregulated overdose of T cells and IL2 result in immune toxicity. In this study we have identified that inhibition of glycogen synthase kinase 3 beta (GSK3β) maintained increased survival of the CAR+ T cells in the absence of IL2, upon activation with target-specific tumor cells. Increased CAR+ T cell proliferation was observed upon GSK3β-inhibition in comparison to non-treated CAR+ T cells. GSK3β-inhibited CAR+ T cells also showed increased CD127 (IL7R) expression compared to the bystanders as well as non-treated CAR+ T cells indicating a possible development of memory population. This is a fundamental progress towards the development of a cancer immunotherapy strategy that will avoid the complications arising from IL2-toxicity.
Autologous tumor vaccine modified with recombinant new castle disease virus expressing IL-7 promotes anti-tumor immune response (VAC12P.1022)
Authors: Liu Haiyan , Zhao Lixiang , Mei Yu , Sun Qing , Guo Linghua , Liu Xiufan
Published: January 2023

Autologous tumor cell modified with non-lytic Newcastle Disease Virus (ATV/NDV) is a promising vaccine for cancer immunotherapy. IL-7 plays a critical role in lymphocyte development and homeostasis. In order to improve the efficacy of ATV/NDV, we inserted IL-7 gene into the genome of non-lytic NDV strain LX using reverse genetic system. The insertion of the IL-7 gene neither affected the main features of NDV replication nor its tumor selectivity. The gene product was biologically active and stable. Then we tested the anti-tumor effects of the autologous tumor vaccine modified with LX/(IL-7) in the melanoma model. We showed that B16-F10 cells modified with LX/IL-7 induced a strong anti-tumor activity against melanoma both in prophylaxis and therapeutic models. The IFN-γ production and the cytotoxicity of tumor-specific CD8+ T cells were significantly enhanced after immunization with B16-F10 cells modified with LX/(IL-7) in both models. Although the tumor infiltrating CD4+ T cells and CD8+ T cells were both increased and their IFN-γ productions were also up-regulated, the anti-tumor activity of the tumor vaccine modified with LX/(IL-7) was dependent on CD8+ T cells. Our results demonstrated that the autologous tumor vaccine modified with NDV/(IL-7) could promote the anti-tumor immune responses mediated by CD8+ T cells and significantly improve the efficacy of the ATV/NDV.
Adenoviral-vectored tuberculosis vaccine AERAS-402 enhances T cell cytokine response in TB+ patients. (VAC12P.1026)
Authors: Graves Andrew , van Zyl-Smit Richard , Esmail Ali , Bateman Mary , Dawson Rodney , Goldin Jonathan , van Rikxoort Eva , Douoguih Macaya , Pau Maria , Sadoff Jerald , Bennett Sean , Snowden Margaret , Gearhart Jacqueline , Ishmukhamedov Sadritdin , Schrager Lewis , McClain J. , Bateman Eric , Hokey David
Published: January 2023

Development of a vaccine to prevent pulmonary tuberculosis remains a public health priority. AERAS-402 is a live, replication-deficient adenovirus 35-vectored TB vaccine expressing the mycobacterial antigens 85A, 85B, and TB10.4. While this vaccine has been shown to be safe in TB uninfected patients, little is known about the performance of this vaccine in TB-experienced populations. The safety and immunogenicity of AERAS-402 was evaluated in a Phase 2a randomized, placebo-controlled, double-blinded dose-escalation study in HIV-negative South African adults with either active or recent pulmonary TB infection. Peripheral blood mononuclear cells isolated from trial patients were assessed via intracellular cytokine staining (ICS) examining T cells for interferon-γ (IFN-γ), TNF, and IL-2 production. While modest CD4+ T cell responses were observed, the ICS data indicate the induction of a robust, dominant CD8+ T cell response not observed in placebo patients, including instances of total cytokine responsive cells approaching 6% of CD8+ T cells in some subjects. Polyfunctional analysis revealed that vaccine-specific T cells were primarily secreting IFN-γ either alone or in combination with TNF following stimulation. These data suggest that AERAS-402 is able to boost T cell responses in patients with a history of TB infection.
Gp96-Ig DNA transfer into muscle by electroporation induces antigen specific cellular immune responses (VAC12P.1027)
Authors: Strbo Natasa , Gonzalez Louis , Podack Eckhard
Published: January 2023

The secreted gp96 vaccine approach is based on the use of cellular vaccines (293 cells) that continuously secrete gp96-Ig associated with antigenic peptides that are cross presented to CD8 T cells in vivo. Cellular gp96 vaccines are highly active in generation CTL for cancer therapy and SIV prophylaxis. Here, we investigate the use of gp96-Ig cDNA as vaccine platform. An optimized electroporation approach was used to deliver the B45 plasmid encoding secreted gp96-Ig and HIV-gag-gfp or ovalbumin genes. The B45 plasmid is a bovine papilloma virus derived vector from which the potentially transforming genes E5, E6, and E7 have been removed as well as the L1 and L2 viral capsid genes. The plasmid (50ug) was injected into the muscle (musculus tibialis anterior) of C57/Bl6 mice followed by electric field pulse that induced temporary and reversible pores in the muscle cell membrane. 48h after i.m. injection and EP-delivery, we found gfp (HIV-gag-gfp) expression in the muscle cells. To determine gp96-Ig secretion and cross presentation to CD8 cells we used cDNA for ovalbumin as antigen in conjunction with gp96-Ig and expansion of OT-I TCR-transgenic cells as readout. Vaccination by electroporation increased the frequency of OT-I in the spleen and in the mucosal compartment (lamina propria, small intestine).This simple, efficient and reproducible method of delivery may have great potential for further development of DNA gp96-Ig vaccination.
Dasatinib promotes an expanded and therapeutically-superior T cell repertoire in response to vaccination against melanoma (VAC12P.1014)
Authors: Storkus Walter , Taylor Jennifer , Lowe Devin , Bose Anamika , Lin Yan , Kirkwood John , Tawbi Hussein
Published: January 2023

Dasatinib (DAS) is a potent inhibitor of the BCR-ABL, SRC, c-KIT, PDGFR and ephrin tyrosine kinases that has demonstrated only modest clinical efficacy in patients with melanoma. Given reports suggesting that DAS enhances T cell infiltration into the tumor microenvironment (TME), we analyzed whether therapy employing the combination of DAS plus specific active vaccination (VAC) would promote superior anti-melanoma efficacy in mice bearing s.c. M05 (B16.OVA) melanomas. We observed that a 7-day course of orally-administered DAS (0.1 mg/day) combined with VAC against the OVA (257-264) peptide epitope resulted in extended overall survival versus either single modality. Immunotherapeutic benefits associated with the DAS + VAC protocol included reduced levels of suppressor cell (MDSC and Treg) populations in the TME, enhanced production of CXCR3 ligand chemokines in the tumor stroma, recruitment of Type-1, vaccine-induced CXCR3+CD8+ tumor-infiltrating lymphocytes (TIL) and IL12p70+CD11c+ dendritic cells (DC) into the TME, and profound “spreading” in the repertoire of tumor-associated antigens recognized by CD8+ TIL. These results have fueled the development of UPCI 12-048 (NCT01876212), a randomized phase II pilot clinical trial of type I-polarized autologous DC vaccines incorporating tumor blood vessel antigen-derived peptides in combination with DAS that is currently enrolling HLA-A2+ patients with metastatic melanoma.
Engineering AFP and GPC3 to create highly immunogenic gene vaccines to prevent carcinogen-induced murine autochthonous hepatocellular carcinoma (VAC12P.1015)
Authors: He Yukai , Hong Yuan , Peng Yibing , Butterfield Lisa , Guo Sheng , Bartlett David , Munn David , Guevara-Patino Jose , Pang Junfeng , Mivechi Nahid
Published: January 2023

In this study, we investigated whether alpha-fetoprotein (AFP) and glypican 3 (GPC3) could be engineered to create effective vaccines to potently activate CD8 T cells to prevent clinically relevant carcinogen-induced autochthonous hepatocellular carcinoma (HCC) in mice. We found that the approach of epitope-optimization created a highly immunogenic AFP and GPC3 and that immunization with lentivector expressing the epitope-optimized AFP and GPC3, but not wild-type AFP and GPC3, potently activated CD8 T cells. Critically, the activated CD8 T cells not only cross-recognized native AFP peptides, but also recognized and killed tumor cells expressing native AFP and GPC3 protein. Immunization with lentivector expressing optimized AFP, but not native AFP, completely protected mice from tumor challenge and reduced the incidence of carcinogen-induced autochthonous HCC. In addition, prime-boost immunization with the optimized AFP significantly increased the frequency of AFP-specific memory CD8 T cells in the liver that were highly effective against emerging HCC tumor cells, further enhancing the tumor prevention of carcinogen-induced autochthonous HCC. Our data demonstrate that epitope-optimization is required to potently activate AFP- and GPC3- specific CD8 T cells, generating effective antitumor effect to prevent carcinogen-induced HCC in mice. Our study provides a roadmap to develop effective human HCC vaccines that may result in an improved antitumor effect.
Titration of T cell help into epitope-based self-assembling vaccines (VAC12P.1017)
Authors: Pompano Rebecca , Chen Jianjun , Verbus Emily , Han Huifang , Collier Joel , Chong Anita
Published: January 2023

Distinct ratios of T effector cells are necessary for effective protective immunity. T cell epitope content plays a critical role in determining T effector cell outcome, but its effects are nonlinear and difficult to predict when designing vaccines. Here, we developed a precise experimental approach to generate vaccines with optimized antibody and CD4+ T cell responses, using non-covalent self-assembly of peptides to titrate a high-affinity universal T cell epitope (PADRE) into a nanoparticulate vaccine. Co-assembly of a B cell epitope (B peptide) from methicillin-resistant Staphylococcus aureus with PADRE produced a modular vaccine that resembled a traditional protein conjugate, requiring cognate T-B interactions to raise an antibody response. Varying the PADRE concentration over nearly three orders of magnitude, from 5 μM to 1 mM, elicited bell-shaped dose-response curves for the anti-B peptide titers and PADRE-specific CD4+ cells, but these peaked at 10-fold different concentrations (0.05 and 0.005 mM, respectively). The antibody titer correlated with a peak in the T follicular helper response, while the total PADRE-specific T cell count reflected a peak in the Th1 and Th2 cells. Peak titers and affinities were similar to those obtained from an alum-adjuvanted B peptide-conjugate vaccine, indicating that titrating the T cell epitope provides a means to optimize the T effectors cells and generate strong responses in chemically precise vaccines.
HPV based photodynamic therapy: a new approach for anti-cancer therapy (VAC12P.1019)
Authors: Kines Rhonda , Çuburu Nicolas , Kobayashi Hisataka , MacDougall John , de los Pinos Elisabet , Schiller John
Published: January 2023

A new method of targeted photodynamic therapy (PDT) is proposed for potentiating anti-tumor immunity. Human papillomavirus (HPV) virus-like particles (VLPs) bind a broad range of tumor types both, in vitro and in vivo, but do not bind healthy, intact tissues. We have linked a photosensitizer dye, IR700, to the HPV VLPs and applied the dye-coupled particles (IR700-VLP) to tumor cells. When exposed to 690nm light, the bound conjugates induced immediate necrotic-like cell death in ovarian, lung, melanoma and cervical cancer cell lines in vitro, and in melanoma and lung tumors in vivo. We are currently expanding these studies to examine long-term tumor growth/survival and the generation of anti-tumor immunity in C57Bl/6 mice using the TC-1 tumor model, which expresses the HPV oncogenes, E6 and E7. This model allows us to track the T-cell responses generated against the HPV oncogenes, in both primary and metastatic tumor settings. Treatment with IR700-VLP and light exposure induced an influx of CD8+ and CD4+ T-cells into the treated tumors. Ex vivo cultures of whole tumor cell suspensions showed IFN-γ production in PMA/ionomycin-stimulated CD8+ and CD4+ T cells and to a lower extent without stimulation, suggesting that these T-cells may have been primed against the tumor antigens. Our data suggest that local anti-tumor immunity can be induced through the rapid tumor cell death caused by IR700, in conjunction with the ability of HPV VLP to induce an innate immune response.
Absence of organized lymphoid tissues in the intestine of the freshwater fish, C. commersonii (VET2P.1042)
Authors: Taylor Rebekah , Kleponis Jennifer , Tatum Russia , Terrero Fernando
Published: January 2023

Microscopic organized lymphoid tissues in the small intestine, such as cryptopatches (CP) and isolated lymphoid follicles (ILF), have been well characterized in the laboratory mouse. Descriptions of these structures have also been reported in rat and human, but the presence or absence of CP and ILF in other animals has not been explored. Given the evolutionary development of the adaptive immune system beginning in jawed fish, we hypothesized that CP and ILF would be present in the intestines of fish. We examined the intestines of 10 wild-caught Catostomus commersonii, a bottom-feeding freshwater fish found in local streams in Western Maryland and throughout the United States. The intestines of these fish were opened longitudinally and arranged in flattened layers for horizontal sectioning. Samples of mouse small intestine were prepared simultaneously as a positive control. After quick-freezing and sectioning, the tissue was stained with hematoxylin and eosin. Interestingly, a complete absence of organized lymphoid tissue was noted, suggesting that CP and ILF, in addition to the well-known Peyer’s patches, are limited to higher-order animals and potentially only mammals. Plans to study other fish species, especially carnivorous varieties (since the composition of the intestinal immune system can change in response to diet) are currently underway to further support this finding.
Effects of regulatory T cells on peripheral blood mononuclear cell responses to live Mycobacterium avium subspecies paratuberculosis in cows with Johne’s disease. (VET2P.1035)
Authors: Roussey Jonathan , Coussens Paul
Published: January 2023

Johne’s disease (paratuberculosis) is a chronic wasting disease of wild and domestic ruminants caused by infection with Mycobacterium avium subspecies paratuberculosis (MAP). Previously, we proposed that the chronic nature of Johne’s disease may result in development of a regulatory T cell (Treg) population in the host. These cells function to shift the immune balance away from a pro-inflammatory Th1 response to an unproductive Th2 response. Due to the inherently low abundance of Tregs in the periphery and the relatively low proportion of Tregs reactive toward any one particular infectious agent, we have developed an in vitro method to stimulate expansion of MAP-reactive Treg populations from CD4+CD25+ peripheral blood mononuclear cells (PBMCs). Enriched Tregs may subsequently be used in Treg-mediated suppression assays. A simplified version of this procedure is also used to determine differences in naïve T cell responses to MAP antigen stimulation, including induction of FoxP3 expression. Following PBMC stimulation with live MAP bacteria and with MAP-reactive Tregs, real-time PCR is used to analyze expression of Th1, Th2, and Treg transcription factors as well as pro- and anti-inflammatory cytokines including. Results are compared between cattle that are test-negative and test-positive for Johne’s disease, with results from test-positive cattle further subdivided between animals exhibiting symptoms of clinical vs subclinical disease.
B cell subpopulations alteration in pathogenic and nonpathogenic SIV infections (VIR7P.1061)
Authors: Brocca Cofano Egidio , Siewe Basile , Kurt Dave , Xu Cui Ling , Kristoff Jan , Ma Dongzhu , Haret-Richter George Sebastian , Montefiore David , Landay Alan , Pandrea Ivona , Apetrei Cristian
Published: January 2023

SIV/HIV infection is associated with B cell dysfunction, the significance of which is not known. We compared the B cell dynamics in blood, lymph nodes (LNs) and gut during SIVsab pathogenic infection of pigtailed macaques (PTMs) and nonprogressive SIVsab infection of African green monkeys (AGMs) by monitoring the B cell subsets, B regulatory cells (Bregs), CD4+ T follicular helper (Tfh) cells, B cell immune activation, apoptosis, exhaustion and homing to the intestine. Binding anti-gp41 and neutralizing antibodies (Nabs) were also measured. Acute SIV infection induced a transient but significant loss of total circulating B cells in PTMs only. In AGMs, total B cell from LNs increased during chronic infection. B cells transiently increased in the gut and naïve subset decreased in all three compartments in both species. Memory B cell subsets showed a different dynamic for both species. Bregs increased in PTM, correlated with IL-10 expression. B cell proliferation occurred in both species. PTMs expressed high baseline levels of α4β7, high degree of B cell activation and apoptosis and high frequency of Tfh. The dynamics of Ab response was similar in both species, but rapid progressor PTMs lacked the ability to mount anti-gp41. Our data show that B cell dysfunction only occurs in pathogenic SIV infection. While B cell changes were not correlated with production of Nabs, rapid disease progression associated a severe incapacity to mount an anti-SIV Ab response.
Identification of PRRSV cross-neutralizing memory B cells (VIR7P.1062)
Authors: Murtaugh Michael , Rahe Michael , Robinson Sally , Taylor Justin , Jenkins Marc
Published: January 2023

Porcine reproductive and respiratory syndrome virus (PRRSV) is widely considered to be the most important pathogen of swine. Yet, there are no vaccines that produce effective broadly neutralizing antibodies to genetically diverse viral genotypes. We recently showed that adult sows develop cross-neutralizing antibodies following infection outbreaks. However, the relevant B-cells producing the cross-neutralizing antibodies have not been identified. Thus, we have characterized surface markers that differentiate porcine B-cell subpopulations and determined their antibody secretion characteristics. Culture of CD21+ B-cells sorted by surface Ig expression showed that surface IgM- and IgG-positive cells proliferated in response to mitogenic stimulation. Sequencing of light and heavy chain variable region mRNA revealed more extensive somatic mutation in the order of plasma cell < IgM-positive < IgG- positive complexity. The CD21+, IgG+ B-cells are putative class-switched peripheral memory B-cells. The isolation of class-switched memory B cells that display cross-neutralizing activity to PRRSV and the subsequent characterization of class-switched memory B cell genetic sequences will allow us to ascertain the necessary somatic mutations that result in the production of PRRSV cross-neutralizing antibodies.
Vaccine induced immunopathology mediated by memory CD4 T cells following chronic viral infection (VIR7P.1054)
Authors: Penaloza-MacMaster Pablo , Barber Daniel , Wherry John , Provine Nicholas , Teigler Jeffrey , Blackmore Steven , Borducchi Erica , Bronson Roderick , Barouch Dan
Published: January 2023

CD4 T cells are important for promoting functional adaptive immune responses following viral infections, but the specific contribution of vaccine-elicited CD4 T cell responses to immune protection remains unclear. We interrogated whether selective induction of virus-specific CD4 T cell responses by vaccination, in the absence of other adaptive immune responses, would improve immune protection against a chronic lymphocytic choriomeningitis virus (LCMV) challenge in mice. Surprisingly, we observed that immunization of mice with a vaccine that induced predominantly CD4 T cell responses resulted in severe immunopathology and mortality following chronic LCMV clone 13 challenge. Vaccinated mice exhibited impaired adaptive responses, severe inflammation, accelerated destruction of lymphoid tissues, and fulminant mortality as compared with naïve mice following challenge. This immunopathology was caused by uncontrolled antigen-driven stimulation of memory CD4 T cells that resulted in cytokine storm and widespread inflammation. The adverse effects of vaccination were abrogated by co-immunization with vaccines that generated CD8 T cell responses, adoptive transfer of virus-specific CD8 T cells, or passive immunization with virus-specific immunoglobulins. Altogether, these data raise important concerns for vaccines that predominantly elicit CD4 T cell responses.
Reversible nerve damage regulates pathology in murine herpes simplex stromal keratitis. (VIR7P.1056)
Authors: Yun Hongmin , Rowe Alexander , Lathrop Kira , Harvey Stephen , Hendricks Robert
Published: January 2023

Herpes simplex type 1 (HSV-1) keratitis (HSK) is characterized by recurrent bouts of immunopathology consisting of corneal opacity, vascularization, and progressive scarring that is triggered by viral reactivation from a latent state and release from sensory nerves in the cornea. Nerve damage and loss of corneal blink reflex (BR) accompanies HSK recurrences, but testing BR is usually not part of the clinical HSK workup in humans or mouse models. Although loss of BR can lead to desiccation of the corneal surface, especially in mice that lack consensual blink reflex, a possible contribution of nerve damage to HSK immunopathology has not been explored. Here we show that chronic HSK in mice is associated with loss of corneal BR, and that preventing corneal desiccation through tarsorrhaphy (eyelid stitching) dramatically reduces HSK severity and resolves existing severe HSK. Tarsorrhaphy dramatically reduced the neutrophilic infiltrate and expression of the neutrophil chemotactic factor CXCL1 in infected corneas. Mice that were depleted of CD4+ T cells exhibited transient severe HSK, with concurrent resolution of HSK, recovery of corneal BR, and regeneration of corneal nerves. Thus, CD4-mediated chronic, but reversible nerve damage and the accompanying corneal desiccation accounts for much of the inflammation associated with HSK in mice.
Hydrophobicity is a hallmark of immunogenic MHC class I T cell epitopes (APP2P.111)
Authors: Chowell Diego , Krishna Sri , Blattman Joseph , Anderson Karen
Published: January 2023

The molecular mechanisms underlying MHC class I-peptide immunogenicity are not well defined. Here, we investigated the biochemical properties that are associated with immunogenicity from immunogenic (n=5035) and non-immunogenic (n=4853) MHC-I peptides curated from the Immune Epitope Database (IEDB). Using a bioinformatic comparative analysis, we evaluated three amino acid properties (hydrophobicity, side chain mass, and polarity) for an association with immunogenicity within the pMHC dataset. We found a strong bias towards hydrophobic amino acid usage within immunogenic peptides (Spearman ρ = 0.71, P = 4.24 x 10^-4); this difference in hydrophobicity is due to preferential usage of non-polar hydrophobic amino acids in TCR-contact residues for peptides restricted by HLA-A2, H-2Db, H-2Kb (P-values < 0.01). We used amino acid hydrophobicity to design an artificial neural network (ANN) model of immunogenicity for MHC-I peptides. We trained this model to predict mouse H2Db-restricted immunogenic epitopes for 4 different antigens from LCMV, Adenovirus, and Influenza A virus. Our findings suggest that hydrophobicity of TCR contact residues may be a fundamental biochemical hallmark that favors immunogenicity of MHC/peptide complexes. Incorporating hydrophobicity into epitope prediction algorithms may improve prediction of immunogenic CTL epitopes.
Contribution of the lymph antigens to the MHC II self-peptidome (APP2P.102)
Authors: Santambrogio Laura , Stern Lawrence
Published: January 2023

Thymic MHC class II peptide presentation shapes the CD4+ T cell repertoire. Conventional migratory dendritic cells (cDC) participate in this process by presenting peripheral self antigens to the developing thymocytes after migration to the thymus. To evaluate the contribution of cDC in central tolerance we analyzed their HLA-DR1 eluted peptidome. Over 3000 peptides with a prediction score above identity, were sequenced. Peptides derived from both intracellular and extracellular sources displayed a snap shot of the HLA-DR1-restricted self peptidome presented under physiological conditions. Processing enzymes responsible for generating these peptides were identified and included cathepsins, metalloproteases, calpains and caspases among others. Comparative analysis between the HLA-DR1 eluted peptidome and naturally processed peptides found in the human lymph identified peptides that shared common sequences, common N or C terminal cleavage site, and few peptides that completely overlapped, indicating that peptides found in biological fluids can contribute to the overall MHC-II peptidome presented by cDC. HLA-DR1 binding affinity and HLA-DM sensitivity was evaluated for several of the mapped peptides. Peripheral injection of the eluted peptides in HLA-DR1 mice identified their role in CD4+ thymocytes selection. Altogether our data indicate that peptides found in biological fluids can shape the thymic T cell repertoire and contribute to the generation of central tolerance.
Effect of pH on cooperativity in peptide binding to MHC class II (APP2P.105)
Authors: Crane Kyle , Castellini J. Margaret , Hoffman Megan , Gotti Marie , Ferrante Andrea
Published: January 2023

Initiation of an adaptive immune response occurs when peptides bound to class II MHC (MHCII) molecules expressed on the surface of Antigen Presenting Cells are recognized by CD4+ T Cells. Formation of peptide-MHCII complexes (pMHCII) occurs within acidic endosomal compartments. The effect of acid pH on peptide binding is well known, and is usually associated with an enhancement of binding and acceleration in kinetics relative to pH 7.4. We have shown that peptide binding to and release from MHCII at extracellular pH are function of structural permissiveness and affected by cooperative effects. To resolve whether cooperativity, thus system flexibility, is a function of pH, we measured the affinity of peptides derived from influenza H3 HA306-319 via cycle-mutation for the human MHCII allele HLA-DR1 (DR1) at pH 5.4, 6.4, and 7.4. In keeping with prior results, cooperativity affects peptide affinity in an exponential fashion, and increasing values of pH are associated to increasing values of cooperativity. This pH-dependence of cooperativity can be observed for destabilizing combinations of mutations, as well as for multiple substituted peptides whose binding is improved as compared to the single substituted sequences. These results indicate that the endosomal pH enhances system permissiveness with respect to extracellular conditions, increasing the probability of peptides with suboptimal chemistry to complex with MHCII.
Identifying the upstream events of innate autoimmunity in intestinal reperfusion injury (BA3P.204)
Authors: Zhang Ming , Lee Haekyung
Published: January 2023

Acute intestinal ischemia is a medical emergency with a high mortality rate, attesting to the need to understand its pathogenesis and develop effective therapies. Recent studies show that innate autoimmune response initiated by natural IgM and complement plays an important role in the reperfusion injury after intestinal ischemia. The goal of this study was to identify the upstream events of innate autoimmunity in intestinal reperfusion injury. To study this, we used a murine model of natural IgM-mediated intestinal I/R. Our results showed that mice overexpressing anti-oxidant enzyme SOD1 had significantly reduced intestinal tissue damage and blocked IgM-mediated complement activation compared with WT controls. To determine if cell membrane instability is an intermediate event between ROS formation and natural IgM-mediated innate autoimmune response, a cell membrane stabilizer was administered to WT mice prior to the induction of intestinal ischemia. Treatment with cell membrane stabilizer significantly protected animals from I/R injury along with inhibition of IgM-mediated complement activation. Stabilizing the cell membrane did not affect ROS formation in the intestinal model. Thus, these data indicate that intracellular ROS formation is an upstream event of cell membrane instability which leads to subsequent innate autoimmune response by natural IgM and complement.
Anti-TRIM21 autoantibody and inflammasome interaction in pathogenesis of Sjogren's syndrome (BA3P.200)
Authors: Deshmukh Umesh , Szczerba Barbara , Kaplonek Paulina , Rybakowska Paulina , Bagavant Harini
Published: January 2023

Sjögren’s syndrome is a chronic autoimmune disorder mainly affecting the exocrine glands and is commonly associated with autoantibodies to TRIM21. This study was undertaken to determine the role of TRIM21-reactive antibodies in pathogenesis of Sjögren’s syndrome. NZM mouse strains (2758, 2328, 2410) were immunized with recombinant TRIM21 protein adsorbed on to alum. Mice were monitored for autoantibodies, serum cytokines and pilocarpine induced salivation. Although all NZM mouse strains generated a robust anti-TRIM21 antibody response, salivary gland dysfunction only occurred in the NZM2758 mice. Moreover, alum immunized NZM2758 mice also displayed some loss of glandular function. Serum levels of IL-1α and CXCL1 were upregulated in alum and alum-TRIM21 treated groups of NZM2758 mice. In contrast, levels of IL-2, IL-5, IL-6 and IL-17 were upregulated, only in the alum-TRIM21 treated mice. Antibodies from TRIM21 immunized mice penetrated live cells in vitro, and showed cytoplasmic staining. Passive transfer of sera from TRIM21 immunized mice induced salivary gland dysfunction in recipient mice, only if the recipients were pre primed with alum. Our study shows that in a genetically susceptible mouse strain, anti-TRIM21 antibodies directly affected salivary gland function and this phenomenon was dependent on the activation of inflammasome pathway. Thus, interaction between innate and adaptive immunity is critical for pathogenesis of Sjögren’s syndrome.
Altered regulation of T-cell signal regulating (TSR) molecules in systemic autoimmunity (BA2P.129)
Authors: Pinkhasov Julia , Acuna Dora , Singh Ram
Published: January 2023

Recent evidence suggests that besides the T-cell receptor (TCR), there are other signal regulating molecules that play a pivotal role in establishing signal threshold during thymocyte selection through a process known as TCR tuning. We examined the expression level of a number of potential T-cell signal regulating (TSR) molecules in the MRL mouse model of multi-system autoimmune disease. We found the expression levels of negative regulators (CD5, CD6, CD2, GITR) that attenuate signal to be reduced, while positive regulators (CD26) that augment signal were increased on thymocytes in MRL mice compared to the C3H control mice. Thymocytes from MRL mice showed increased activation and phosphokinase signal upon ex vivo stimulation. Moreover, TSRs were further reduced on activated peripheral T cells after disease onset in the MRL mice. The reduced levels of these TSR molecules correlated with increased responsiveness to TCR-mediated signal upon ex vivo stimulation and phosphokinase detection. The reduced TSR expression levels were due to increased levels of ubiquitination, which led to targeted protein degradation. Importantly, co-stimulation with agonistic antibody against the TSR molecule CD5 reversed the hyper-responsiveness to TCR stimulation. The ability of T cells to alter their internal signal threshold by altering TSR expression may be a novel mechanism for T cells to escape peripheral tolerance and exacerbate autoimmune disease progression.
Differential contributions for Bim and Nur77 in negative selection against ubiquitous and tissue-restricted self-antigens (BA2P.122)
Authors: Hu Qian , Baldwin Troy
Published: January 2023

The thymic cortex and medulla are both sites of negative selection, where high affinity encounter with ubiquitous (UbA) and tissue-restricted (TRA) self-antigens, respectively, is thought to result in clonal deletion. Our group has shown that Bim is not required for UbA-mediated deletion but is required for TRA-mediated deletion. Nur77 is another protein implicated in thymocyte apoptosis, though its mechanism of action and regulation remain unclear. Using a physiological TCR transgenic model, HYcd4, we found that Nur77 was also dispensable for clonal deletion against UbA. Though Nur77 and its family member Nor-1 were induced during negative selection in HYcd4 Bim-/- mice, caspase-3 activation remained abrogated. Concurrent expression of a TCR transgene with a Nur77 transgene significantly inhibited Nur77-mediated thymocyte apoptosis, suggesting that TCR signalling can impair the pro-apoptotic function of Nur77. In contrast to a recent study using the OT-II Rip-mOva system, we found that Nur77 deficiency only modestly inhibited TRA-mediated clonal deletion in OT-I Rip-mOva chimeras. Furthermore, in polyclonal Bim-/- or Nur77-/- mice, we observed an increase in anergic phenotype CD4+ T cells, which may indicate an increase in self-specificities due to a block in clonal deletion. Collectively, these studies highlight potential differences in the molecular mechanisms of negative selection against UbA versus TRA, as well as of MHC class I versus MHC class II restricted thymocytes.
T cell motility within lymph nodes fits a Levy signature (CAM1P.225)
Authors: Cannon Judy , Asperti-Boursin Francois , Fricke Matthew , Letendre Kenneth , Moses Melanie
Published: January 2023

Naïve T cell motility within lymph nodes (LNs) is crucial for the initiation of the T cell response. Multiphoton imaging has allowed direct visualization of T cell movement in LNs, demonstrating T cell search as a random walk. Different types of random walks exist in biological systems, including Brownian and Levy walks. Levy walks differ from Brownian walks by the presence of large steps, and naïve T cell motility in LNs had been assumed to be a Brownian walk. Recent evidence suggests that the motility of T cells in the brain is characterized by a Levy signature, not a Brownian walk. However, detailed analysis naïve T cells search within LNs has not been done. Here we present evidence that naïve T cells use a Levy signature to search in LNs. Using multiphoton microscopy, we determined displacement and step size taken by naive T cells in LNs. We find that naïve T cell motility show a heavy-tailed distribution characteristic of a Levy signature. However, we do not observe a classical Levy walk as defined by a power-law fit. Instead, the best fit for T cell motility is a log-normal distribution. We then used computer modeling to assess the efficiency of T cell search parameters. We find that heavy-tailed distributions are as efficient as the classical Levy walk at target search. These findings shed new light on the fundamental mechanisms driving T cell search for antigen within lymph nodes.
InsP4 negatively regulates dendritic cell cytokine secretion and migration to peripheral draining lymph nodes. (CAM1P.234)
Authors: Wang Xinxin , Wang Yaming , Xu Xiaolu , Swiecki Melissa , Colonna Marco , Huang Yina
Published: January 2023

Inositol 1,3,4,5-tetrakisphosphate (InsP4) is a novel second messenger that is critical for T and B cell development and activation. However, whether InsP4 has a similar regulatory role in dendritic cell (DC) function is not yet known. We show here that InsP4 differentially regulates ERK and Akt signaling in response to LPS activation of bone marrow derived DCs (BMDCs). InsP4 supports ERK activation but negatively regulates Akt and its down-stream signals. Interestingly, endogenous migratory DCs from InsP4 deficient mice and BMDCs migrated more efficiently to peripheral lymph nodes in vivo. InsP4 deficient BMDCs also have enhanced secretion of pro-inflammatory cytokines/chemokines and mediate stronger antigen presentation to antigen-specific T cells. Our data support an important role for InsP4 in DC migration and cytokine secretion, identifying InsP4 as a dual regulator of Akt and ERK signaling pathways that controls DC function.
Formyl peptide receptor 2 plays a role on the oxidized low-density lipoprotein-induced pathological events of atherosclerosis (CAM1P.238)
Authors: Oh Eunseo , Lee Ha Young , Kim Sang Doo , Seo Jeong Kon , Bae Yoe-Sik
Published: January 2023

During the pathological progress of atherosclerosis, the level of low-density lipoprotein (LDL) and its modification into oxidized LDL (oxLDL) are reported to be increased. Eventhough some scavenger receptors such as CD36 and RAGE have been considered as target receptors for oxLDL, involvement of other receptors should be investigated for oxLDL-induced pathological responses of atherosclerosis. Here, we observed that oxLDL-induced foam cell formation was inhibited by formyl peptide receptor 2 (FPR2) antagonist WRW4. oxLDL stimulated calcium increase and chemotactic migration in FPR2-expressing RBL-2H3 cells but not in vector-expressing RBL-2H3 cells. Moreover, oxLDL stimulated TNF-α production, which was also almost completely inhibited by FPR2 antagonist. Taken together our findings, we suggest that oxLDL stimulates macrophages, resulting in chemotactic migration, TNF-α production, and foam cell formation via FPR2 signaling, contributing to the pathological progress of atherogenesis. We also suggest that FPR2 can be regarded as a therapeutic target for the development of drugs to treat atherosclerosis.
Cancer cell technology as a tool for teaching basic principles of immunology (EDU1P.248)
Authors: Aguilar Robert , Tuohy Vincent
Published: January 2023

Testicular cancer mainly affects men between the ages of 20 and 39, but it is the most common neoplasm in men between the ages of 15 and 34. The National Cancer Institute states that localized testicular cancer has a recurrence rate of 15-20%, and those tumors, including Sertoli or Leydig cell derived, do not respond well to chemotherapy or radiation treatment. My summer research was centered on the effort to better characterize the immune response to a testicular tumor. This was accomplished through the use of different immunology techniques such as proliferation assays, immunohistochemistry, ELISA, and ELISPOT. My lab experience inspired me to design a unit curriculum which will help elucidate the various physiognomies of tumor growth. Students first learn and apply the essential techniques of cancer cell culture. The testicular cancer cell line I-10 (CCL-83, ATCC) is used to familiarize students with routine feeding and maintenance, subculturing, counting, and cryopreservation. I-10 cell RNA is converted to cDNA and qualitatively analyzed for inhibin-alpha gene expression, which serves as a tumor marker, using RT-PCR. Then, they analyze previously formalin-fixed, paraffin embedded murine testes (normal and cancerous) using H&E staining and immunohistochemical staining for CD3, inhibin-alpha, and IgG. Upon completion of this unit, students demonstrate these learned skills in a combined written and practical examination.
Unique progenitors in adult mouse bone marrow expressing CD127 (IL-7Rα) and high level of cKit efficiently develop into all lymphocytes including T cells and innate lymphoid cells (HEM2P.266)
Authors: Ghaedi Maryam , Lichy Nesim , Takei Fumio
Published: January 2023

All lymphocytes are thought to develop from common lymphoid progenitors (CLPs) that are found in the Lin−Sca-1locKitloCD127+Flt3hi bone marrow (BM) cell population. However, only small numbers of T cells and innate lymphoid cells (ILCs) are generated from CLPs transplanted into irradiated mice, suggesting that they might mainly develop from alternative progenitors such as lymphoid-primed multipotent progenitors (LMPPs). We now have identified novel progenitors in adult mouse BM that efficiently develop into all lymphocytes including T cells and ILC2s. The novel progenitors are similar to LMPPs and found in the cKithiSca-1+Flt3+ BM cell subset, but unlike LMPPs they express the lymphoid lineage marker CD127, thus resembling early thymic progenitors (ETPs). Upon transplantation into irradiated mice, CD127+ LMPP-like cells robustly engraft and develop into T, B, NK cells and ILC2s. They are more efficient than LMPPs and CLPs in generating T cells and ILC2s. Similar to LMPPs, they also retain myeloid potentials. Flt3L-deficient mice have greatly reduced LMPPs and CLPs but have almost normal numbers of CD127+ LMPP-like progenitors, pre-ILC2 in the BM and mature ILC2 in the lung. While the exact relationship between CD127+ LMPP-like progenitors, CD127- LMPPs and CLPs are yet to be clarified, these results suggest that CD127+ LMPP-like BM cells are important progenitors for T cells and ILC2s independent of CLPs.
Plasma cell gene expression depends on ELL2 (HEM2P.271)
Authors: Bayles Ian , Milcarek Christine , Park Kyung Soo
Published: January 2023

ELL2, the eleven-nineteen lysine rich leukemia gene, a transcription elongation factor, has been found to play vital roles in Igh mRNA production, myeloma cell survival, and in HIV tar:tat mediated viral RNA transcription. We previously showed that ELL2 is responsible for Igh mRNA being both alternatively processed to the secretory-specific form and increased in its abundance, leading to high levels of secreted Ig protein and a robust immune response. ELL2 associates with pTEFb in a super elongation complex. We made conditional knockouts of ELL2 in mouse B cells using loxp/ CD19 cre. The splenic B cell populations of T3 and mature plasma cells in the whole mouse are reduced in the knockouts. The ELL2 cKO results in decreased serum Ig levels and severe reduction in antigen specific responses against NP-ficoll and NP-KLH. Recall responses are also impaired. LPS stimulation of splenic B cells shows decreased maturation to B220loCD138+ plasma cells with the expression of IRE1, XBP-1 spliced form, and cyclin B2 significantly lower in the cKO than LPS stimulated WT mice. The processing of the Igh mRNA to the secretory-specific form is suppressed in the conditional ELL2 knockouts with a compensatory increase in Igh membrane mRNA. Production of the unfolded protein response is thus impaired. Analysis of the ELL2 knockouts may lend insight into the relationships between Ig secretion, cell growth and the unfolded protein response in plasmablasts and long term plasma cells.
Generation and resolution of bivalent chromatin during hematopoiesis (HEM2P.259)
Authors: Ghosh Amalendu , De Supriyo , Bell Jeremiah , Weber Brittany , Wood William , Becker Kevin , Sen Jyoti , Bhandoola Avinash , Sen Ranjan
Published: January 2023

Bivalent promoters, that are simultaneously marked by activation- and repression-associated histone modifications, have been proposed to maintain ES cell genes in a poised configuration. During differentiation such promoters resolve to fully active or inactive states, with associated changes in gene expression. Here we followed the fate of bivalent promoters during hematopoietic stem cell (HSC) differentiation to T lymphoid lineage committed cells. We show that HSC bivalents interconverted between monovalent and bivalent chromatin states at sequential developmental stages, with only a minority resolving to either monovalent state for the long-term. Conversely, bivalent promoters that were generated de novo interconverted between precursor states and bivalency. Neither generation nor resolution of bivalency was accompanied by changes in gene expression. Moreover, singly marked genes did not transition to the opposing monovalent state via a bivalent intermediate. Our observations reveal unprecedented dynamics of bivalent chromatin during hematopoiesis that suggests a developmental role beyond poising genes for future resolution.
Inhibition of B lymphopoiesis by myeloid-derived suppressor cells (HEM2P.262)
Authors: Kennedy Domenick , Knight Katherine
Published: January 2023

B lymphopoiesis declines with age in humans, mice, and rabbits. In rabbits, loss of B lymphopoiesis occurs as early as two months of age and correlates with an increase in adipose tissue in the bone marrow (BM). Increased adipose tissue has also been observed in the BM of aged humans and mice. We showed previously that adipocyte-derived factors inhibit B lymphopoiesis in vitro; however, the mechanism by which this occurs is unknown. Through co-cultures of mouse BM cells with OP9 stromal cells, we found that adipocyte conditioned medium (ACM) induces the generation or expansion of CD11b+ Gr1+ myeloid cells. Further, we found that these cells inhibit B cell development in vitro. The ACM-induced CD11b+ Gr1+ cells express arginase (Arg1) and iNos (Nos2), and functionally suppress CD4+ T cell proliferation, suggesting that these cells are classical myeloid-derived suppressor cells (MDSCs). Transwell and conditioned medium experiments show that inhibition of B lymphopoiesis by ACM-induced MDSCs is mediated through soluble factors. This study uncovers a previously unrecognized capacity of MDSCs to inhibit B lymphopoiesis and provides a potential target for therapeutics aimed at restoring B lymphopoiesis in aged individuals.
Development of a novel method for isolation of four developmentally distinct myeloid derived dendritic cells pre-cursors: phenotypic and functional analyses (HEM3P.283)
Authors: Rogers Peter , Hiltbold Elizabeth
Published: January 2023

Myeloid cell progenitors have the potential to give rise to dendritic cells which have a high capacity to stimulate adaptive immune responses as well as to cells with potent immunosuppressive activity. However, the stage in their development in which these functions are gained is poorly defined. Here, we developed a method for isolating progenitor populations at specific stages of development. Bone marrow cells were lineage depleted and cultured in GM-CSF to drive their differentiation toward a myeloid lineage for three days. Cells were then sorted based on Ly6C and CSF-1R (CD115) expression, two markers whose expression is known to change throughout myeloid ontogeny. We then monitored the developmental progression of each population based on the expression of these markers as well as other hallmarks of dendritic cell or myeloid suppressor cell phenotypes. We also measured the function of each population relative to antigen uptake and degradation as well as their response to stimulation by TLR agonists. Collectively, our results indicate that myeloid progenitors progress along this pathway: Ly6C-CD115- -> Ly6C+CD115- -> Ly6C+CD115+ -> Ly6C-CD115+ -> Ly6C-CD115+. Phenotypically, these populations represent CMP, MDSC, monocytes, immature DC, and more mature DC, respectively. These findings not only enhance our understanding of myeloid cell development and differentiation driven by GM-CSF, but also inform the design of GM-CSF-derived DC based vaccine strategies.