Recall Antigen Responses Research Articles

Immunogenicity of PE18, PE31, and PPE26 proteins from Mycobacterium tuberculosis in humans and mice.

The large family of PE and PPE proteins accounts for as much as 10% of the genome of Mycobacterium tuberculosis. In this study, we explored the immunogenicity of three proteins from this family, PE18, PE31, and PPE26, in humans and mice. The investigation involved analyzing the immunoreactivity of the selected proteins using sera from TB patients, IGRA-positive household contacts, and IGRA-negative BCG vaccinated healthy donors from the TB endemic country Mozambique. Antigen-recall responses were examined in PBMC from these groups, including the evaluation of cellular responses in healthy unexposed individuals. Moreover, systemic priming and intranasal boosting with each protein, combined with the Quil-A adjuvant, were conducted in mice. We found that all three proteins are immunoreactive with sera from TB patients, IGRA-positive household contacts, and IGRA-negative BCG vaccinated healthy controls. Likewise, antigen-recall responses were induced in PBMC from all groups, and the proteins stimulated proliferation of peripheral blood mononuclear cells from healthy unexposed individuals. In mice, all three antigens induced IgG antibody responses in sera and predominantly IgG, rather than IgA, responses in bronchoalveolar lavage. Additionally, CD4+ and CD8+ effector memory T cell responses were observed in the spleen, with PE18 demonstrating the ability to induce tissue-resident memory T cells in the lungs. Having demonstrated immunogenicity in both humans and mice, the protective capacity of these antigens was evaluated by challenging immunized mice with low-dose aerosol of Mycobacterium tuberculosis H37Rv. The in vitro Mycobacterial Growth Inhibition Assay (MGIA) and assessment of viable bacteria in the lung did not demonstrate any ability of the vaccination protocol to reduce bacterial growth. We therefore concluded that these three specific PE/PPE proteins, while immunogenic in both humans and mice, were unable to confer protective immunity under these conditions.

Anti CD161 Antibody IMT-009 Is a Novel Immunotherapeutic Agent That Effectively Blocks the Inhibitory CLEC2D/CD161 Axis in CLEC2D+ B Cell Hematological Malignancies Reinvigorating T and NK Cell Function Leading to Anti-Tumor Benefit

Background: The CLEC2D/CD161 axis is a ligand-receptor pathway that has been shown to inhibit both T and NK cell function 1,2. CD161 is a C-type lectin-like receptor, which is expressed on NK cells and subsets of both CD4 + and CD8 + T cells. The tumor infiltrating CD161+ T cells have high cytotoxic potential and display an NK cell-like signature 2. Its ligand CLEC2D is expressed on the surface of both malignant cells and immune cells including germinal center B cells, activated T cells and tumor associated macrophages. Owing to its expression in germinal center B cells, CLEC2D is highly expressed in multiple types of B cell lymphomas including Diffuse Large B Cell Lymphoma (DLBCL), Follicular Lymphoma and Burkitt lymphoma 3. Furthermore, subset of infiltrated T cells in these lymphomas are CD161+ making this axis a promising novel target for immunotherapeutic intervention in hematological malignancies (Fig1). Immunitas has developed IMT-009, a monoclonal, Fc silent, fully human IgG1, high affinity antibody to CD161 that selectively blocks its interaction with CLEC2D. We show that IMT-009 effectively reverses CLEC2D-mediated inhibition and restores NK cell and T cell functions, including antigen specific T cell mediated cytotoxicity to lymphoma cell lines. Furthermore, IMT-009 shows monotherapy benefit in reducing tumor burden in a human B cell lymphoma model engrafted in humanized mice in vivo. IMT-009 is currently being evaluated in a Phase 1/2a clinical trial in both solid tumors and heme malignancies. Results: IMT-009 is a highly selective antibody to CD161 that strongly blocks its interaction with CLEC2D. In presence of CLEC2D-expressing target cells, NK cell degranulation, cytokine production and cellular cytotoxicity towards lymphoma tumor targets is highly suppressed which is overcome by treatment with IMT-009. Similarly, IMT-009 reversed CLEC2D-mediated inhibition and restored T cell activity (TCR signaling, cytokine production) in a Jurkat cell reporter system, and enhanced polyfunctionality of primary antigen-specific human T cells shown by secretion of TNF-α, IL-2, and IFN-γ, and direct T cell mediated cytotoxicity. IMT-009 also released CD161-mediated suppression of antigen recall response by PBMC derived effector memory CD161+ CD4+ T cells, resulting in an increased frequency of CD161+ IFN-γ+ cells and an increase in their proliferation. These data together show that IMT-009 is highly effective in overcoming CLEC2D mediated suppression of both T and NK cell functions. To prioritize indications that will likely benefit from IMT-009, we performed IHC for CLEC2D in a diverse set of tumor indications and found that B cell lymphomas of various subtypes have high CLEC2D protein expression by H score. Also, DLBCL was found to have the second highest median RNA expression of CLEC2D in TCGA. Furthermore, analysis of publicly available single-cell RNA-sequencing (scRNA-seq) data from B cell lymphoma including CNS lymphoma revealed CLEC2D to be highly expressed within the B lineage cells including the abundant malignant cell population (Fig2). KLRB1 (gene encoding for CD161) was also found to be expressed on a subset of tumor infiltrating CD4 and CD8 T cells in these datasets (Fig2). To confirm this, we tested CD161 expression in B cell lymphomas by immunofluorescence. Results showed CD161+ cells infiltrated in Hodgkin Lymphoma, and post-R-CHOP treated DLBCL, highlighting the potential sensitivity of these malignancies to IMT-009 treatment. Together, these results indicate that B cell lymphomas may exhibit an immunosuppressive environment mediated by the CLEC2D/CD161 axis. To test the anti-tumor benefit of IMT-009 in a human B cell lymphoma model, we treated CD34+ humanized mice implanted with Raji, a Burkitt lymphoma cell line with IMT-009. The group of mice treated with IMT-009 had a mean tumor growth inhibition of 35% and the anti-tumor response correlated with increased frequency of CD161+ T and NK cells. We further show that IMT-009 can synergize with Rituximab in NK cell mediated killing of B cell lymphoma cell lines in vitro. Conclusion: B cell malignancies tend to respond poorly to anti-PD-1 therapy 4,5. This lack of efficacy could be driven by a highly immunosuppressive environment mediated by the CLEC2D/CD161 axis. Our results support evaluation of IMT-009 as a novel cancer immunotherapy to disrupt CLEC2D/CD161 signaling, with strong therapeutic potential in lymphomas.

A173 ELUCIDATING THE EFFECTS OF NOD2-MEDIATED SIGNALLING ON INTESTINAL RESIDENT-MEMORY T-CELL FORMATION AND FUNCTION

Abstract Background Aberrant resident memory T-cell (TRM) responses have been associated with increased intestinal inflammation and Crohn’s disease (CD) pathology in humans. Intestinal TRM cells are not only important for maintaining the integrity of the intestinal epithelial barrier, but also for the rapid clearance of pathogens in the intestine during infection. Understanding the signals received by the intestinal immune system to generate TRM responses is paramount to elucidating treatments for CD. Genetic mutations in NOD2 are associated with the highest risk of CD development. As a host intracellular sensor of bacterial peptidoglycan, NOD2 is critical for initiating both innate and adaptive immune responses. Furthermore, work from our lab as well as those of our collaborators suggest that NOD2 deficiency reduces systemic memory B and T-cell responses. However, the role of NOD2 in establishing memory T-cell responses in the intestine remains unclear. This work will therefore establish the role of NOD2 signaling in initiating and maintaining optimal TRM responses to achieve intestinal homeostasis and resilience to intestinal inflammation. Purpose It is the main objective of this project to determine whether NOD2-mediated signalling affects: 1. Antigen-specific T-cell priming in vivo 2. Bona fide intestinal TRM generation 3. Bona fide intestinal TRM function Method To address the effects of NOD2-signalling on intestinal T-cell priming in vivo, wildtype (WT) mice were adoptively transferred 50,000 naïve LCMV-specific (SMARTA) CD4+ T-cells. Mice were subsequently infected with LCMV-Armstrong in the presence or absence of the NOD2 agonist; MDP. 5-days following infection, the numbers and percentage of LCMV-specific T-cells in the mesenteric lymph nodes and spleen were examined. To examine the effects of NOD2 on intestinal TRM generation, littermate WT and NOD2 KO mice were infected with LCMV-Armstrong. Thirty-six-days following infection, the percentage and number of LCMV-specific CD4+ T-cells were profiled in the small and large intestinal lamina-propria by means of gp66-77 class-II MHC-tetramer staining. In another set of experiments, littermate WT and NOD2 KO mice were re-infected with LCMV-C13 30-days following LCMV-Armstrong immunization, and the interferon-response in the small intestine was profiled by quantitative PCR to assess the effect of NOD2-deficiency on antigen recall responses. Result(s) NOD2-stimulation by means of MDP injection increased the percentage and number of adoptively transferred SMARTA CD4+ T-cells in the mesenteric lymph nodes upon LCMV infection. Furthermore, NOD2-deficiency did not alter intestinal LCMV-specific CD4+ TRM seeding in the small and large intestinal lamina propria 36 days after infection. However, in vivo antigen recall experiments showed a decreased intestinal IFN response in NOD2 KO mice. Conclusion(s) Our findings reveal a potential role of NOD2 in the intestinal CD4+ T-cell priming and subsequent Ag-specific memory response. Disclosure of Interest None Declared

Impact of Cryopreservation on Viability, Phenotype, and Functionality of Porcine PBMC.

The use of frozen peripheral blood mononuclear cells (PBMC) is common in immunological studies. The impact of freezing PBMC has been assessed using human and mice cells, but little information is available regarding domestic animals. In the present study, the phenotype and functionality of frozen porcine PBMC were examined. In a preliminary experiment, three freezing media: fetal bovine serum plus 10% dimethyl sulfoxide, PSC cryopreservation kit, and Cryostor CS10, were compared regarding the preservation of cell viability and the response of PBMC to mitogens after thawing. After being stored one month in liquid nitrogen, cell viability was above 89% for all freezing media. The ELISPOT IFN-gamma (IFN-γ) results in response to PHA and of IgG ELISPOT in response to R848+IL-2 were similar to those obtained using fresh PBMC. In the second set of experiments, PBMC were obtained from five pigs vaccinated against Porcine reproductive and respiratory syndrome virus (PRRSV) and then frozen using Cryostor CS10. Recovered cells were phenotyped by flow cytometry using anti-CD3, CD4, CD8, and CD21 antibodies and were used to assess the PRRSV-specific responses in a proliferation experiment, an IFN-γ ELISPOT, and an IgG ELISPOT, and compared to the results obtained with fresh cells. The antigen-specific responses of frozen cells were significantly (p<0.05) impaired in the proliferation assay, particularly for CD4/CD8 double-positive T-cells and for CD21+ cells. Freezing resulted in decreased proliferation when Con A, but not PHA, was used. In ELISPOT, cryopreservation resulted in a decreased frequency of IFN-γ-secreting cells in response to PRRSV (p<0.05) but the response to PHA was not affected. No differences were observed in the IgG ELISPOT after polyclonal activation. Taken together, cryopreservation of porcine PBMC had a significant impact on the magnitude of recall antigen responses and therefore, it may affect the response of effector/memory cells but seems not to have a major impact on naïve T-cells. These results may help to the better use of frozen porcine PBMC, and to the interpretation of the results obtained from them.

IMMU-36. B CELL-VACCINE ELICITS LONG TERM IMMUNITY AGAINST GLIOBLASTOMA VIA ACTIVATION AND DIFFERENTIATION OF TUMOR-SPECIFIC CD8+ MEMORY T CELLS

Abstract While immunotherapy is used clinically to treat many cancers, its translation into brain tumors remains elusive. The importance of B cells in cancer immunity has become increasingly clear, and we previously developed a B cell-based cellular vaccine (BVax) against glioblastoma (GBM) by further activating 4-1BBL+ B cells with CD40 agonism and IFNγ. BVax were characterized as professional antigen-presenting cells (APCs) that promote CD8+ T cell migration and persistence in murine tumor-bearing brains. This study seeks to understand the mechanisms underlying BVax-induced CD8+ T cell fitness in the tumor microenvironment. Initial transcriptomic analysis highlighted that Bvax express high levels of IL15Rα, indicating their potential ability to trans-present IL15. Considering IL15 trans-presentation is fundamental in T-cell memory differentiation, we used BVax to induce T cell activation in the presence of exogenous IL15. BVax were better capable of activating antigen-specific CD8+ T cells and promoting a memory phenotype when compared to other professional APCs such as dendritic cells (DCs). T cell receptor (TCR) CDR3β sequencing showed that BVax expanded a number of TCR clones in-vitro that were found in brains of CT2A tumor-bearing mice in-vivo. These BVax-activated CD8+ T cells displayed a stronger antigen recall response and unique metabolic profile compared to DC-activated CD8+ T cells as shown by metabolomic analysis of tumor-infiltrating CD8+ T cells. When comparing the anti-tumor effects of CD8+ T cells activated by various APCs, BVax with exogenous IL15 promoted CD8+ T cells that displayed the most potent cytotoxicity against GBM cells in-vitro. Collectively, this study suggests that the IL15/IL15Rα axis and interactions with CD8+ T cell are key factors of BVax therapy in promoting a robust survival benefit and long-term immunologic memory against GBM in preclinical models. Additionally, the development of T cell therapies based on B cell licensing can be a promising future approach for glioblastoma therapy.

IMMU-13. CUSTOMIZABLE MULTI-LAMELLAR RNA-NANOPARTICLES FOR PEDIATRIC GLIOMA

BackgroundSince the preponderance of pediatric gliomas are mutationally ‘bland,’ immune checkpoint inhibitors are unlikely to mediate therapeutic benefit. Alternately, immunologic response can be induced de novo against pediatric gliomas with mRNA cancer vaccines. Messenger RNA represents a paradigm shift in vaccinology (i.e. COVID-19) given its flexibility, commercialization, and propensity to confer rapid protection with only a single vaccine. ObjectiveWe sought to develop a new mRNA platform with an optimized backbone for insertion of both personalized and/or “off the shelf’ (i.e. H3K27M) transcripts for rapid induction of anti-tumor activity against pediatric gliomas. ApproachWe synthesized an mRNA backbone with optimized 5’ and 3’ UTRs for delivery of gene transcripts pertinent to pediatric brain tumors using a lipid-nanoparticle (NP) delivery vehicle. This vaccine utilizes a novel engineering design that layers tumor derived mRNA into a lipid-nanoparticle (NP) “onion-like” or multi-lamellar package. ResultsWe demonstrate immunogenicity of RNA-NPs delivering either personalized glioma mRNA or H3K27M mRNA. RNA-NPs localize to myeloid cells in murine KR158b brain tumors and activate dendritic cells that supplant regulatory intratumoral myeloid populations inducing antigen-recall response with long-term survivor benefit. Our optimized mRNA backbone yielded significantly improved anti-tumor efficacy compared with commercial backbones. We have shown this approach can be refined for co-delivery of immunomodulatory RNAs (i.e. GM-CSF) and/or delivery of siRNAs targeting immunoregulatory axes (PD-L1) in murine brain tumors (GL261). We have since established safety of RNA-NPs in acute/chronic murine GLP toxicity studies without cross-reactivity to normal-brain, and launched a large-animal canine brain tumor trial which demonstrated RNA-NPs to be feasible, safe and immunologically active. ConclusionRNA-NPs reprogram the brain tumor microenvironment while inducing a glioma-specific immune response. We have since received FDA-IND approval for first-in-human trials (IND#BB-19304) in pediatric patients with high-grade gliomas (PNOC020 study, NCT04573140).

Full SNO 2021 Abstracts PDF

Background: Since the preponderance of pediatric gliomas are mutationally 'bland,' immune checkpoint inhibitors are unlikely to mediate therapeutic benefit. Alternately, immunologic response can be induced de novo against pediatric gliomas with mRNA cancer vaccines. Messenger RNA represents a paradigm shift in vaccinology (i.e. COVID-19) given its flexibility, commercialization, and propensity to confer rapid protection with only a single vaccine. Objective: We sought to develop a new mRNA platform with an optimized backbone for insertion of both personalized and/or “off the shelf” (i.e. H3K27M) transcripts for rapid induction of anti-tumor activity against pediatric gliomas. Approach: We synthesized an mRNA backbone with optimized 5' and 3' UTRs for delivery of gene transcripts pertinent to pediatric brain tumors using a lipid-nanoparticle (NP) delivery vehicle. This vaccine utilizes a novel engineering design that layers tumor derived mRNA into a lipid-nanoparticle (NP) “onion-like” or multi-lamellar package. Results: We demonstrate immunogenicity of RNA-NPs delivering either personalized glioma mRNA or H3K27M mRNA. RNA-NPs localize to myeloid cells in murine KR158b brain tumors and activate dendritic cells that supplant regulatory intratumoral myeloid populations inducing antigenrecall response with long-term survivor benefit. Our optimized mRNA backbone yielded significantly improved anti-tumor efficacy compared with commercial backbones. We have shown this approach can be refined for co-delivery of immunomodulatory RNAs (i.e. GM-CSF) and/or delivery of siRNAs targeting immunoregulatory axes (PD-L1) in murine brain tumors (GL261). We have since established safety of RNA-NPs in acute/chronic murine GLP toxicity studies without cross-reactivity to normal-brain, and launched a large-animal canine brain tumor trial which demonstrated RNANPs to be feasible, safe and immunologically active. Conclusion: RNA-NPs reprogram the brain tumor microenvironment while inducing a gliomaspecific immune response. We have since received FDA-IND approval for first-in-human trials.

Influenza infection exacerbates disease symptoms in relapsing models of multiple sclerosis

Abstract Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system. Most patients present with a relapsing-remitting form (RRMS), wherein they experience new or exacerbated neurologic symptoms known as relapses followed by periods of partial or complete recovery (remissions). Previous evidence suggest that upper-respiratory infection increases the risk of relapse, but the mechanisms are ill-defined. The aim of this study was to elucidate the effects of upper-respiratory viral infection on disease progression in three models of relapsing experimental autoimmune encephalomyelitis (REAE). Relapsing EAE was induced in SJL/J, C57BL/6J and NOD/SHILtJ mice. At the peak of disease, mice were randomized into treatment groups, then inoculated with saline or mouse-adapted human influenza A virus (strain A/Puerto Rico/8/1934 H1N1; 0.7 HAU) two days later. Weights and scores were recorded daily. At day 42 post induction mice were euthanized and antigen recall responses measured from lymphocytes isolated from the spleen and cervical lymph nodes. Infection exacerbated symptoms of disease in both SJL/J and NOD/SHILtJ and tended to exacerbate disease in C57BL/6 mice. Following antigen stimulation, splenocytes and cervical lymph node lymphocytes from infected mice had increased production of IFNγ compared to cells isolated from saline inoculated controls. There was no effect of infection on IL-17A or GM-CSF production. These data indicate that infection may function to expand myelin-specific Th1 cell clones by an undefined mechanism. Together, these findings indicate influenza infection can exacerbate symptoms of REAE in SJL/J and NOD/SHILtJ mice.

Bovine immune response to leptospira antigen in different novel adjuvants and vaccine delivery platforms

Leptospirosis is a global zoonosis causing significant economic losses for cattle production. Current cattle vaccines against leptospirosis need improvement to provide efficacy against multiple serovars, reduce shedding in urine, and to induce earlier and more robust immune responses. In this study, Leptospira borgpetersenii serovar Hardjo strain 203 antigen was combined with novel adjuvants (a biodegradable polyanhydride compressed rod implant (VPEAR), poly(diaminosulfide) microparticles, a water-oil-water emulsion adjuvant, and aluminum hydroxide) to develop novel vaccines. Cattle were immunized twice, at a 4 week interval, with inoculums containing adjuvants alone or leptospira antigens and immune responses were compared to responses of cattle receiving a commercial monovalent leptospirosis vaccine (Spirovac). All animals were inoculated with a single dose of Spirovac at 20 weeks to assess antigen recall responses. Serum antibody responses were increased (P > 0.05) at 8 and 20 weeks after vaccination in cattle receiving inoculums containing leptospira antigens combined with water-oil-emulsion, poly(diaminosulfide) microparticles (PNSN-MP), or aluminum hydroxide and in cattle vaccinated with Spirovac. Humoral responses were predominantly IgG1 isotypes. Antigen-specific proliferative responses were detected after initial vaccination in cattle vaccinated with Spirovac, PNSN-MP and water-oil-water treatments. Most proliferative responses occurring within CD4+ and gamma delta T cell populations expressing CD45RO and CD25 markers, a response consistent with an effector memory phenotype. Antigen-specific immune responses were not detected in cattle vaccinated with VPEAR after initial inoculation, but were detected in the antigen recall responses. PBMCs from cattle vaccinated with Spirovac, oil-water-oil, or PNSN-MP treatments had increased (P < 0.05) IL-17A release after in vitro stimulation with leptospirosis antigens, whereas all groups produced IFN-γ and IL-17A after in vitro stimulation during the antigen recall response. Our data demonstrates that combining leptospirosis antigens with these adjuvants enhances immunogenicity in cattle.Interpretative Summary: Vaccination of livestock is a key mechanism for minimizing transmission of leptospirosis, a zoonotic disease. Leptospirosis vaccines for cattle need to be improved to provide greater levels of protection from kidney colonization, better immune responses, and protection against multiple serovars. This could be accomplished using new vaccine adjuvants. In this study, several novel adjuvants were evaluated for their ability to induce effective immune responses in cattle to leptospira antigens as compared to currently available vaccines. Data suggested that vaccines containing biodegradable polymer microparticles and oil-emulsion adjuvants induced similar or greater immune responses as compared to a commercial vaccine. Our data suggest these new vaccine formulations warrant further investigation as new vaccine formulations for cattle and other livestock.

The contribution of cyclophilin A to immune-mediated central nervous system inflammation

Cyclophilin A has multiple known functions in inflammation. Intracellular cyclophilin A modulates T helper 2 response (Th2) and extracellular cyclophilin A functions as a leukocyte chemotactic factor. The contribution of cyclophilin A to central nervous system (CNS) inflammation has not been reported. To test the hypothesis that inhibition of cyclophilin A would ameliorate immune-mediated CNS inflammation, we compared the course and neuroimmunology of experimental allergic encephalomyelitis (EAE) in cyclophilin A knockout mice and wild type littermates. There was a trend towards lower incidence of EAE in cyclophilin A knockout mice, but the clinical course of EAE among animals that manifested clinical signs of EAE was similar in cyclophilin A knockout and wild type littermates. Antigen recall response to myelin oligodendrocyte glycoprotein (MOG) peptide showed that interferon-γ release was lower in cyclophilin A knockout mice. Analysis of CNS inflammatory cells showed that CD3+ T cell infiltration into the CNS was lower in cyclophilin A knockout mice. These results showed that the loss of cyclophilin A results in altered peripheral immune activation and CNS leukocyte infiltration, but these changes did not result in a substantial change in the clinical course of EAE.

An immunomodulatory feed additive enhances in vitro viral vaccine recall antigen responses in dairy heifers

Enhancing immunological responses to vaccination is an important goal in many herd health management systems. OmniGen-AF®(OG) is an immunomodulatory feed additive that has been shown to enhance innate immune function in ruminants and its effects on adaptive immunity require additional study. The objective of this study was to evaluate post-vaccine antibody titers and circulating cellular memory development in heifers fed OG and administered a commercially available modified-live bovine respiratory disease (BRD) vaccine. Twenty-four Holstein heifers were assigned to one of two diets for 170 days: Control TMR (CON; n = 11), or TMR plus OG (TRT; 9 g/100 kg BW/day; n = 13). Samples for hematology, serology, and cellular assays were collected on D-110, 0, 21, 42, and 60 of the trial. Heifers were administered two priming doses of a modified-live BRD vaccine, with a third dose given on D0. There were no significant differences in total WBC and absolute number or the percentage of circulating lymphocytes, monocytes, neutrophils, RBC, or platelets on D-110 through D21. On D42 and D60, CON had significantly higher numbers of lymphocytes. On D0, mean serum neutralizing (SN) titer to BHV-1 was significantly higher for CON compared to TRT. SN titers were not significantly different between CON and TRT at any other time point for BHV-1, BVDV type 1, or BVDV type 2. TRT mounted a significantly stronger recall proliferative response to 0.5 multiplicity of infection (MOI) of BHV-1, BVDV type 1 and BVDV type 2 on D42 and D60; 0.25 MOI of BVDV type 1 on D21 and D42; and 0.25 MOI BVDV type 2 on D42 compared to CON. IL-4 production induced by 0.5 and 1.0 MOI BHV-1 (D42 and D60); 0.25 MOI of BVDV type 1 (D21); and 0.25 and 0.5 MOI of BVDV type 2 (D60) were significantly higher for TRT than CON. IL-17 production induced by 0.25 MOI of BVDV type 1 was significantly higher on D60 for TRT compared to CON. IFN-gamma and IL-10 were not significantly different between treatments. These data indicate feeding OG has a beneficial effect on responses to vaccine antigens in Holstein dairy heifers.

Abstract 2696: Genetic and pharmacologic evaluation of the ubiquitin ligase CBL-B as a small-molecule, tumor immunotherapy target

Abstract E3 ubiquitin ligases play critical roles in directing cellular protein fate by controlling the specificity of ubiquitin conjugation to substrate proteins and targeting them for cellular relocalization or degradation by the ubiquitin proteasome system. The E3 ubiquitin ligase CBL-B is expressed in immune cell lineages and negatively regulates activity of the T-cell receptor (TCR) by imposing a requirement for a costimulatory signal to mount a productive immune response upon TCR engagement. Mice deficient in Cbl-b, and more specifically in the RING Zn-finger ligase domain of Cbl-b, demonstrate a tumor rejection phenotype mediated by CD8+ T cells (Paolino et al., JI, 2011). We have reproduced these results and demonstrate that Cbl-b deficient mice show enhanced anti-tumor activity. In addition, we show that CD4+ and CD8+ T cells from mice deficient in Cbl-b have 5 to 10-fold enhanced secretion of IL-2 and IFN γ when stimulated ex vivo. These data provide a genetic rationale for the development of a small molecule inhibitor of CBL-B ligase activity for use in patients with tumor-mediated immune suppression of effector T cells. We have identified a series of small molecule inhibitors of CBL-B activity with biochemical potency at low nanomolar concentrations. CBL-B inhibitors increased cytokine secretion in vitro at low nanomolar concentrations, as measured by IL-2 and IFN γ secretion, in primary human and mouse T cells stimulated with CD3/CD28 or CD3 alone. The compounds also stimulated proliferation and elevated levels of the T cell surface activation markers CD25 and CD69. CBL-B inhibitors enhanced an antigen recall response in human PBMCs ex vivo, as measured by approximately 5-fold higher secretion of GM-CSF, TNF-α and RANTES, and demonstrated effects in an ex vivo model of exhausted T cell function. Oral dosing of an optimized CBL-B inhibitor enhanced anti-CD3 stimulated T cell activation in mouse CD4+ and CD8+ T cells, demonstrating a dose proportional pharmacodynamic effect. Oral administration over 28 days in the syngeneic CT-26 tumor model was well tolerated and resulted in single agent tumor growth inhibition. These data support the continued advancement of small molecule oral CBL-B inhibitors for future development in immuno-oncology. Citation Format: Jennifa Gosling, Ryan Rountree, Asad Taherbhoy, Chenbo Wang, Thomas Cummins, Frederick Cohen, Hiroko Tanaka, Dahlia Weiss, Mario Cardozo, Christopher Karim, May Tan, Joseph Juan, Austin Tenn-McClellan, Szerenke Kiss von Soly, Julie Sheung, Kathleen Boyle, Ketki Dhamnaskar, Katherine Kurylo, Jilliane Bruffey, Jennifer McKinnell, Dane Karr, Andria Christianson, Anne-Renee Van Der Vuurst de Vries, Pallavur Sivakumar, Mark Gallop, Paul A. Barsanti, Anjanabha Saha, Neil F. Bence, Christoph W. Zapf. Genetic and pharmacologic evaluation of the ubiquitin ligase CBL-B as a small-molecule, tumor immunotherapy target [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2696.

Discs large homolog 1 regulates B-cell proliferation and antibody production.

Antibody production results from B-cell activation and proliferation upon antigen binding. Discs large homolog 1 (Dlg1), a scaffold protein from the membrane-associated guanylate kinase family, has been shown to regulate the antigen receptor signaling and cell polarity in lymphocytes; however, the physiological function of Dlg1 in humoral responses is not completely clear. Here, we addressed this question using a conditional knockout (KO) mouse model with Dlg1 deficiency in different B-cell subsets by crossing dlg1fl/fl mice with either mb1cre/+ or aicdacre/+ mice, respectively. In both mouse models, we observed that Dlg1 deficiency in B cells (Dlg1-KO B cells) led to obvious hyper-antibody responses upon immunization, the effect of which was more obvious in antigen-recall responses. Mechanistically, we found that Dlg1-KO B cells exhibited hyper-proliferation compared with wild-type B cells upon antigen stimulation, suggesting that the hyper-antibody responses are likely induced by the hyper-proliferation of Dlg1-KO B cells. Indeed, further studies demonstrated that Dlg1 deficiency in B cells led to the down-regulation of a tumor suppressor, FoxO1. Thus, all these results reveal an unexpected function of Dlg1 in restraining hyper-antibody responses through the inhibition of FoxO1 and thus antigen-binding-induced proliferation in B cells.

Characterization of bovine γδ T cells phenotype following Mycobacterium bovis vaccination or virulent infection

Abstract Bovine tuberculosis caused by Mycobacterium bovis is a significant veterinary health problem. γδ T cells participate in the control of mycobacterial infections. Accumulating data suggest that mycobacterial infection regulates memory/effector phenotype and immune functions of mycobacterium-responsive γδ T cells. To date, the impact of both virulent M. bovis infection and Bacille Calmette-Guerin (BCG) vaccination on bovine γδ T cells memory/effector phenotype remains unknown. In this study, we addressed the functional and phenotypical differences in memory/activation marker, and chemokine receptor expression of M. bovis-specific γδ T cells following BCG vaccination or virulent M. bovis infection. Analysis of γδ T cells in blood, bronchoalveolar lavage fluid, and regional lymph nodes indicated that γδ T cells have phenotypic differences based on the expression of CD27 that suggest distinct functional properties. Here, we show that most peripheral γδ T cells display a CD27+ phenotype. Antigen recall responses showed that expression of CD27 correlates with the expansion of M. bovis-specific γδ T-cells. Thus, the CD27+ subset may comprise the adaptive γδ T cell compartment responding to virulent M. bovis infection and vaccination. Furthermore, we show that γδ T cells from neonatal calves are activated by aerosol BCG vaccination and adopt a lung-homing phenotype that is similar to that observed during virulent M. bovis infection, suggesting an important role for this T cell subset in the response to mucosal vaccination. The ability of γδ T cells to mount a robust local immune response supports the hypothesis that vaccine-elicited γδ T cell immunity might prove beneficial.

Interleukin-7 Unveils Pathogen-Specific T Cells by Enhancing Antigen-Recall Responses.

Interleukin (IL)-7 promotes the generation, expansion, and survival of memory T cells. Previous mouse and human studies showed that IL-7 can support immune cell reconstitution in lymphopenic conditions, expand tumor-reactive T cells for adoptive immunotherapy, and enhance effector cytokine expression by autoreactive T cells. Whether pathogen-reactive T cells also benefit from IL-7 exposure remains unknown. In this study, we investigated this issue in cultures of peripheral blood mononuclear cells (PBMCs) derived from patients infected with various endemic pathogens. After short-term exposure to IL-7, we measured PBMC responses to antigens derived from pathogens, such as Mycobacterium tuberculosis, Candida albicans, and cytomegalovirus, and to the superantigen Staphylococcus aureus enterotoxin B. We found that IL-7 favored the expansion and, in some instances, the uncovering of pathogen-reactive CD4 T cells, by promoting pathogen-specific interferon-γ, IL-2, and tumor necrosis factor recall responses. Our findings indicate that IL-7 unveils and supports reactivation of pathogen-specific T cells with possible diagnostic, prognostic, and therapeutic significance of clinical value, especially in conditions of pathogen persistence and chronic infection.

An Immune-Competent Murine Model to Study Elimination of AAV-Transduced Hepatocytes by Capsid-Specific CD8+ T Cells

Multiple independent adeno-associated virus (AAV) gene therapy clinical trials for hemophilia B, utilizing different AAV serotypes, have reported a vector dose-dependent loss of circulating factor IX (FIX) protein associated with capsid-specific CD8+ T cell (Cap-CD8) elimination of transduced hepatocytes. Hemophilia B patients who develop transient transaminitis and loss of FIX protein may be stabilized with the immune-suppressive (IS) drug prednisolone, but do not all recover lost FIX expression, whereas some patients fail to respond to IS. We developed the first animal model demonstrating Cap-CD8 infiltration and elimination of AAV-transduced hepatocytes of immune-deficient mice. Here, we extend this model to an immune-competent host where Cap-CD8 transfer to AAV2-F9-treated mice significantly reduced circulating and hepatocyte FIX expression. Further, we studied two high-expressing liver tropic AAV2 variants, AAV2-LiA and AAV2-LiC, obtained from a rationally designed capsid library. Unlike AAV2, Cap-CD8 did not initially reduce circulating FIX levels for either variant. However, FIX levels were significantly reduced in AAV2-LiC-F9-treated, but not AAV2-LiA-F9-treated, mice at the study endpoint. Going forward, the immune-competent model may provide an opportunity to induce immunological memory directed against a surrogate AAV capsid antigen and study recall responses following AAV gene transfer.

Reduced immunogenicity of Plasmodium falciparum gamete surface antigen (Pfs48/45) in mice after disruption of disulphide bonds - evaluating effect of interferon-γ-inducible lysosomal thiol reductase.

Sexual stages of Plasmodium are critical for malaria transmission and stage-specific antigens are important targets for development of malaria transmission-blocking vaccines. Plasmodium falciparum gamete surface antigen (Pfs48/45) is important for male gamete fertility and is being pursued as a candidate vaccine antigen. Vaccine-induced transmission-blocking antibodies recognize reduction-sensitive conformational epitopes in Pfs48/45. Processing and presentation of such disulphide-bond-constrained epitopes is critical for eliciting the desired immune responses. Mice lacking interferon-γ-inducible lysosomal thiol reductase (GILT), an enzyme that mediates reduction of S-S bonds during antigen processing, were employed to investigate immunogenicity of Pfs48/45. It has been well established that the ability to reduce S-S bonds in antigens guides effective T-cell immune responses; however, involvement of GILT in the induction of subsequent B-cell responses has not been explored. We hypothesized that the ability to reduce S-S bonds in Pfs48/45 will impact the generation of T-cell epitopes, and so influence helper T-cell responses required for specific B-cell responses. Non-reduced and reduced and alkylated forms of Pfs48/45 were employed to evaluate immune responses in wild-type and GILT knockout mice and studies revealed important differences in several immune response parameters, including differences in putative T-cell epitope recognition, faster kinetics of waning of Pfs48/45-specific IgG1 antibodies in knockout mice, differential patterns of interferon-γ and interleukin-4 secretions by splenocytes, and possible effects of GILT on induction of long-lived plasma cells and memory B cells responsible for antigen-recall responses. These studies emphasize the importance of antigen structural features that significantly influence the development of effective immune responses.

Abstract A51: Intradermal delivery of polymeric nanoparticle based vaccine formulation using a hollow microneedle system

Abstract Introduction: Nanoparticle (NP)-based anticancer vaccine delivery systems provide a number of advantages including spatial repetitive display of the antigen, size dependent lymphatic and intracellular trafficking, and the depot effect of persistence and sustained release of antigens/adjuvants. However, the optimal NP delivery mode and route of administration need additional investigation. To achieve reproducible intradermal PLGA NP vaccine delivery using an in-vivo rat model, we used a hollow microneedle array system (1). The profile of the resulting humoral and cellular immune responses was compared to that resulting from conventional intramuscular (IM) injection of the same formulation. Methods: Immunostimulatory Nanoparticles (ISNPs) loaded with imiquimod and monophosphoryl Lipid A (MPL-A) were created using emulsion- solvent evaporation technique. All vaccine formulations for the in vivo study contained 2 mg of ISNPs as the adjuvant component. Antigen was given either in the form of soluble OVA solution or OVA encapsulated PLGA NPs at equivalent doses of OVA (16 µg). Two doses of vaccine (prime and boost) were administered to Crl:CD(SD) rats three weeks apart via IM or ID delivery. ID delivery was enabled by a plastic, hollow microneedle array with 18 microneedles, each &amp;lt;1mm in length, similar to those referenced previously (1). Blood collection was carried out three weeks after each dose. Serially-diluted serum was incubated with OVA coated plate for IgG1/IgG2a titer test by ELISA. Spleens were harvested at the endpoint of the experiment (three weeks after the booster dose) for splenocyte isolation. The T cell response was determined by pulsing the splenocyte culture with OVA antigen using a Mabtech rat Interferon gamma ELISpot kit. Results and Discussion: ISNPs promoted high antigen-specific antibody response in both IM and ID vaccination groups. Compared to the soluble form, antigen encapsulated in NPs triggered significantly stronger IgG1 antibody response three weeks after priming dose regardless of administration route. Not only did the NP formulation demonstrate faster kinetics in mounting antibody response, but the antibody avidity, determined by Urea ELISA, was also better in the OVA NP formulation when compared to the soluble OVA formulation, indicating quicker progress of antibody affinity maturation. However, three weeks after the boost, IgG1 level of all groups were comparable, reaching a titer plateau. In contrast to the IgG1 response, antigen OVA encapsulated in NPs and delivered intradermally via the microneedles resulted in the highest IgG2a titer among all experimental groups at both time points after both priming and booster doses. IgG2a is widely considered a surrogate biomarker for Th1 response, while IgG1 is a biomarker for Th2 response. The relative strength of Th1/Th2 response could be assessed from IgG1/IgG2a ratio (2). Among the different groups, ID delivery of the OVA NP formulation demonstrated the highest relative strength of Th1 response. This result was further confirmed by the secretion of Th1 cytokine Interferon gamma by splenocytes upon antigen recall response. These data suggest that for a NP vaccine carrier, the skin is a vaccination priming locus that enhances Th1 response, which is considered essential for effective induction of anti-tumor immunity. Conclusion: A polymer-based NP vaccine formulation delivered intradermally via hollow microneedles elicited robust humoral and cellular immunity. When compared to IM injection, the unique combination of the polymeric NP formulation and the ID route of administration led to an optimal Th1 immune response.

PWE-123 Adaptive immune responses in ALF

<h3>Introduction</h3> Acute liver failure (ALF) is characterised by an overwhelming hepatic necrosis, imuneparesis and susceptibility to infections. To date, a number of defects in innate immune responses have been identified but defects in adaptive immune responses remain unexplored. The aims of this study were to evaluate whether impairment in adaptive immune responses could also account for the immuneparesis and infection susceptibility in ALF. <h3>Method</h3> CD4⁺T-cell-subsets in peripheral blood mononuclear cells (PBMCs) from 20 ALF patients and 10 healthy controls (HC) were determined using multicolor flow cytometry. CD45RA/RO were used to differentiate naïve and memory CD4⁺T-cells. Expression or lack of expression of CD45RO/CCR7 was used to determine effector memory (T_EM) subset. Negative regulators of T cell activation (CTLA-4) were analysed. Specific CD4⁺T-cell antigen-recall responses to a pool of HLA class II-restricted T cell epitopes were examined <i>in vitro </i>in the presence or absence of anti-CTLA-4 blocking antibody. CD4⁺T-cell proliferation was assessed by flow cytometry and IL-2 secretion by ELISA. Soluble CTLA-4 (sCTLA-4) in sera samples were measured using ELISA. <h3>Results</h3> Whilst overall percentages of circulating CD4+ T lymphocytes were expanded in ALF (p=0.03), we report a significant reduction in the frequency of memory CD4⁺T-cell population (p=0.01). MELD score correlated negatively with the proportion of CD4⁺T-cells (r=-0.462, p=0.06). Furthermore, T_EMpopulation was significantly reduced in ALF in comparison to HC (p=0.04). This reduction was highly significant in patients with a poor outcome (p=0.002). CTLA-4 expressing CD4⁺T-cells were significantly higher in ALF compared to HC (p=0.0001). Similarly, levels of sCTLA-4 in the sera of ALF were significantly elevated (p= 0.02). ALF (n = 4) showed a defect in proliferative responses to recall antigens compared to HC (n = 3) (p = 0.05). However, blockade of CTLA-4-4 was sufficient to restore antigen-recall responses defined by increased T cell proliferation (p=0.05) and IL-2 secretion (0 vs 20.84 pg/ml). <h3>Conclusion</h3> Our novel data identify defects in function of circulating CD4⁺T-cell subsets in ALF with a significant increase in the CTLA-4 expressing CD4⁺T-cells, a phenotype with an inhibitory functional aspect. Proof-of-principle experiments indicate that blocking CTLA-4, a crucial immune checkpoint, may be a promising immunotherapeutic target to boost adaptive immune responses and reduce susceptibility to infection in ALF patients. <h3>Disclosure of interest</h3> None Declared.

Microchimerism and regulation in living related kidney transplant families.

Long-term harmful effects of immunosuppressive drugs and chronic rejection are a persistent impetus to establish methods to induce immunological tolerance to allografts. PCR-based studies have found evidence that humans and other placental mammals can have prolonged extremely low levels of maternal cells as well as other non-self cells, referred to as microchimerism. The persistence of these cells suggests a mechanism for the maintenance of the regulatory T-cell (Treg) responses frequently detected in offspring to non-inherited maternal antigens. We test the hypothesis that the detection of very low copy levels of insertion/deletion (Indel) alleles consistent with non-inherited maternal genes, will correlate with immune regulation to non-inherited maternal antigens as detected by a trans-vivo Delayed-Type Hypersensitivity (tvDTH) assay in kidney transplant recipients, normal donors and their immediate biological family members. Preliminary data reported here compares qPCR amplification of rare DNA templates in the peripheral blood polymorphonuclear (PMN) fraction of cells, with the results of tvDTH assays for linked suppression of recall antigen responses in the presence of non-inherited maternal antigens [NIMA]. The two assays do not show a definitive correlation.