T-cell Dependent Immune Response Research Articles

The role of ERK2 in dendritic cell function

Abstract Dendritic cells (DCs) are an important link between the innate and adaptive immune systems, specifically by serving as key regulators of T cell-dependent (TD) immune responses. In DCs, several processes critical for initiating TD immune responses, such as migration, provision of costimulatory signals, and antigen uptake and presentation, utilize the mitogen-activated protein kinase (MAPK) signaling pathway. The dysregulation of these processes can result in the aberrant activation of autoreactive CD4+ T cells, resulting in autoimmune disease. Extracellular signal-regulated kinase 1 and 2 (ERK1/2) are key effectors of the MAPK pathway, and while ERK1 has been shown to be a critical regulator of DC migration and TLR-induced IL-10 secretion, the function of ERK2 remains unknown. Preliminary data has shown that Erk2Δ bone marrow-derived DCs (BMDCs), as well as Erk2Δ splenic DCs, exhibit altered expression of surface markers associated with T cell costimulation, antigen presentation, and migration compared with DCs from littermate control. Taken together, this suggests that ERK2 plays a unique role in the regulation of several DC functions, potentially opening a new approach to treating CD4+ T cell-mediated autoimmune diseases. Supported by grant R21AI44731 from the NIH/NIAID (TGF)

Genetic Perturbation of CD70/CD27 Co-Stimulation Promotes the Development of Bcl6-Driven Diffuse Large B-Cell Lymphoma

Multiple co-stimulatory and co-inhibitory pathways modulate T-cell dependent anti-tumor immune responses in lymphoid malignancies. We recently defined the recurrent genetic alterations and associated substructure of diffuse large B-cell lymphoma (DLBCL), including five distinct clusters (1-5), and identified potential genetic bases for immune evasion [Nature Medicine 2018; 24:679-690]. In our series, 26% of tumors had inactivating somatic mutations or copy loss of CD70 and likely disruption of CD70/CD27 co-stimulation.CD70 and CD27 are homotrimer type II and homodimer type I transmembrane proteins and members of the TNF and TNF receptor superfamilies, respectively. CD70 is transiently expressed on certain normal B-cell and dendritic cell populations upon activation and constitutively expressed on multiple B-cell tumors. CD70 activation of CD27 + T cells promotes antigen-dependent T-cell expansion and immune surveillance of normal and malignant B cells. Patients with germline deficiencies of either CD70 or CD27 have an increased incidence of EBV-associated lymphoid malignancies, underscoring the importance of this co-stimulatory pathway.In our series, CD70 alterations were most common in Cluster 1 DLBCLs, which also exhibited recurrent BCL6 chromosomal translocations. The co-occurrence of CD70 and BCL6 genetic alterations was noteworthy because of the established role of CD8 + T-cell dependent immune surveillance in murine models of Bcl6-driven DLBCL [Nature Medicine 2014; 20:283-290]. To assess the consequences of Cd70 deficiency and perturbed CD70/CD27 co-stimulation on Bcl6-driven lymphomagenesis, we crossed the previously described Bcl6tg/+ and Cd70-/- mice to generate Cd70-/-; Bcl6tg/+ animals. In the aging cohorts, Cd70-/-; Bcl6tg/+ mice were more likely than Bcl6tg/+ animals (or the Cd70-/- or wild-type [WT] groups) to be euthanized for symptoms before the study endpoint (18 months [mo]) (5 of 18 Cd70-/-; Bcl6tg/+ euthanized for symptoms prior to the first of 9 Bcl6tg/+). Additionally, significantly greater total numbers and percentages of Cd70-/-; Bcl6tg/+ mice, in comparison to Bcl6tg/+ or Cd70-/- animals, were euthanized for symptoms (64% Cd70-/-; Bcl6tg/+ vs. 29% Bcl6tg/+, p=0.005 and 64% Cd70-/-; Bcl6tg/+ vs. 11% Cd70-/-, p=0.002). Almost all euthanized Cd70-/; Bcl6tg/+ and Bcl6tg/+ micehad massively enlarged spleens infiltrated with histopathologically confirmed DLBCLs characterized by clonal Ig gene rearrangements. Our findings indicate that genetic perturbation of Cd70 accelerates the onset and significantly increases the incidence of Bcl6-driven DLBCL.To characterize potential differences in the anti-tumor immune responses in Cd70-/-; Bcl6tg/+ and Bcl6tg/+ mice (and Cd70-/- and WT controls), we also harvested spleens from 6 animals in each of the aging cohorts at predetermined intervals (2, 6, 14 and 18 mo). We analyzed the composition of splenic-cell suspensions by flow cytometry and evaluated the intact splenic architecture and morphology with expert hematopathologists (VS, KW and SR). At 14 and 18 mo, spleens from WT and Cd70-/- animals were largely normal in appearance and size. In contrast, spleens from 14 and 18 mo Bcl6tg/+ and Cd70-/-; Bcl6tg/+ mice exhibited disordered architecture and abnormal pre-malignant lymphoid proliferation with expanded white pulp including morphologically and immunophenotypically aberrant B cells of small to large size and increased infiltrating T-cells. Additionally, significantly higher fractions of splenic CD8 + T cells from 14 and 18 mo Bcl6tg/+ and Cd70-/-; Bcl6tg/+ animals expressed the CD69 activation marker and exhibited terminal differentiation, consistent with an ongoing anti-tumor immune response. Interestingly, Bcl6tg/+ animals had significantly higher percentages of splenic TCRβ + T cells at the earlier time point (14 mo) with delayed-onset splenomegaly (at 18 mo), which is in marked contrast to the Cd70-/-; Bcl6tg/+ mice that had significantly lower percentages of splenic TCRβ + T cells at the earlier time point (14 mo) and early-onset splenomegaly (14 mo). These findings suggest that the initial T-cell mediated immune response was more effective in Bcl6tg/+ than Cd70-/-; Bcl6tg/+ animals. Taken together, our data indicate that genetic perturbation of CD70/CD27 co-stimulation limits the development of an effective anti-tumor immune response in Bcl6tg/+-driven DLBCL. DisclosuresNeuberg: Madrigal Pharmaceuticals: Other: Stock ownership; Pharmacyclics: Research Funding. Rodig: Affimed: Research Funding; Bristol-Myers-Squibb: Research Funding; Merck: Research Funding; Immunitas: Membership on an entity's Board of Directors or advisory committees; KITE/Gilead: Research Funding. Shipp: Bristol Myers Squibb: Research Funding; Immunitas Therapeutics: Consultancy; AstraZeneca: Consultancy, Research Funding; Merck: Research Funding; Abbvie: Other: Institution: Research Grant/Funding; Bayer: Other: Institution: Research Grant/Funding.

Abstract 527: A new generation of PARP interfering drug candidates for cancer treatment

Abstract Purpose: Therapeutic strategies targeting DNA repair defects have been widely explored, but often restricted to a specific population of patients and tackled with resistance issues. We pioneered a new approach of anti-cancer treatment to tackle emergence of resistance: the decoy agonist mechanism of action. Drugs based on this mechanism hijack and hyperactivate therapeutic targets leading to an impairment of the repair signaling. This breakthrough decoy agonist action has already shown, using our lead compound AsiDNA™, target engagement, excellent safety profile in humans, and importantly lack of acquired resistance. Here, we describe mechanistically the immunomodulatory properties and metabolic effects of a new generation product OX401, generated using the proprietary PlatON™ platform of oligonucleotides designed to trap PARP proteins. Experimental design: OX401-induced PARP activation, NAD+ consumption and cell cytotoxicity were monitored using tumor and non-tumor cells. DNA repair abrogation was monitored by analyzing repair protein recruitment to damage sites. Accumulation of cytoplasmic DNA fragments was monitored after DNA staining and microscopy analysis. OX401 effect on the innate immune response was assessed by following STING pathway activation and T-cell mediated anti-tumor cytotoxicity. OX413, a “super” OX401 was designed to be more stable and less prone to intracellular enzymatic degradation. In a cell-derived xenograft model of mouse breast cancer, OX413-induced PARP activation and percentages of tumor-infiltrating leucocytes (CD45+: CD3+, CD8+, NK, DCs) were analyzed ex-vivo by flow cytometry. Results: Using different tumor models, we showed that OX401 binds and hyper-activates PARP1 with a high affinity and in a dose-dependent manner. As a consequence, OX401 impaired DNA repair due to PARP sequestration leading to an accumulation of cytoplasmic chromatin fragments (CCFs). In line with this, we demonstrated that OX401 displayed cell-autonomous immunomodulatory properties. Mechanistically, the generated CCFs triggered an innate immunity activation through cGAS/STING pathway, downstream CCL5 secretion and potentiation of the anti-tumor T-cell dependent immune response. Through PARP hyper-activation, OX401 also induced a rapid NAD+ consumption (below the viability threshold). This metabolic exhaustion caused selective tumor cell death while sparing healthy cells. These effects were enhanced using the optimized OX413 molecule, and at doses approximately 10 to 100-fold lower compared to OX401. In-vivo, OX413 triggered PARP1 target engagement and STING pathway activation, correlated to an increased innate and adaptive immune cells infiltration. Conclusions: Our results provide a preclinical rationale for using OX413 as an immunomodulatory and “metabolic exhauster” agent, especially in appropriately molecularly selected patients with tumors showing metabolic deficiencies. Citation Format: Wael Jdey, Christelle Zandanel, Véronique Trochon-Joseph, Chloé Doizelet, Vincent Hayes, Marie-Christine Lienafa, Richard Tripelon, Françoise Bono. A new generation of PARP interfering drug candidates for cancer treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 527.

Modelling the interplay between the CD4^{+}/CD8^{+} T-cell ratio and the expression of MHC-I in tumours

Describing the anti-tumour immune response as a series of cellular kinetic reactions from known immunological mechanisms, we create a mathematical model that shows the CD4^{+}/CD8^{+} T-cell ratio, T-cell infiltration and the expression of MHC-I to be interacting factors in tumour elimination. Methods from dynamical systems theory and non-equilibrium statistical mechanics are used to model the T-cell dependent anti-tumour immune response. Our model predicts a critical level of MHC-I expression which determines whether or not the tumour escapes the immune response. This critical level of MHC-I depends on the helper/cytotoxic T-cell ratio. However, our model also suggests that the immune system is robust against small changes in this ratio. We also find that T-cell infiltration and the specificity of the intra-tumour TCR repertoire will affect the critical MHC-I expression. Our work suggests that the functional form of the time evolution of MHC-I expression may explain the qualitative behaviour of tumour growth seen in patients.

CD40 plays roles in T cell-dependent response and B cell activation and differentiation in Nile tilapia (Oreochromis niloticus)

CD40 is a member of the TNF receptor family and plays critical roles in T cell-dependent (TD) immune response in mammals. However, limited information on CD40 functional characterization is known in teleost fish. In this study, Nile tilapia (Oreochromis niloticus) CD40-like (OnCD40) was cloned and characterized with its expression patterns in TD immune response, including the process of B cell activation and differentiation. The OnCD40 has a 948 bp length open reading frame and was expressed wildly in immune tissues, especially in peripheral blood. In addition, the OnCD40 expression level was much higher in IgMhi naïve/activated B cells than that of IgMlo plasma-like B cells. Further, the expression of OnCD40 was significantly up-regulated after in vivo challenge of keyhole limpet hemocyanin (KLH), and the increased level of OnCD40 expression was evidently higher in the second stimulation than the first one. The significant up-regulation of OnCD40 transcript also appeared following KLH immunization in vitro. Moreover, by in vitro stimulation of recombinant Oreochromis niloticus interleukin 6 ((r)OnIL-6), recombinant Oreochromis niloticus interleukin 10 ((r)OnIL-10), or induction of anti-OnIgM monoclonal antibody (mAb), the OnCD40 expression was markedly up-regulated following the B cell activation or differentiation. Taken together, the findings in this study suggest that OnCD40 might be involved in TD humoral immune response and plays roles in B cell activation and differentiation in Nile tilapia.

Suppression of IL-23-mediated psoriasis-like inflammation by regulatory B cells

Psoriasis is an inflammatory cutaneous disease mediated by T-cell dependent immune responses; however, B cells are also considered to play an important role its development. Regulatory B cells (Bregs) regulate immune responses negatively through interleukin-10 (IL-10) production. This study aimed to investigate the role of Bregs in IL-23-mediated psoriasis-like inflammation in mice. Psoriasis-like inflammation was induced in B cell-specific phosphatase and tensin homolog (PTEN)-deficient mice, in which Bregs were significantly expanded, and in their controls, by intradermal injection of 20 μL phosphate-buffered saline (PBS) containing 0.5 μg rmIL-23 into one ear, every other day for 16 days. IL-23-mediated psoriasis-like inflammation was suppressed in B cell-specific PTEN-deficient mice along with decreased ear thickness and epidermal thickness on day 15. Moreover, adoptive transfer of B1 B cells suppressed IL-23-mediated psoriasis-like inflammation. rmIL-23-injected B cell-specific PTEN-deficient mice showed expanded regulatory T cells (Tregs) in the spleen and draining lymph nodes along with increased Bregs. Further, T helper (Th) 17 differentiation in the rmIL-23-injected ear was suppressed in B cell-specific PTEN-deficient mice. Overall, these results indicate that increased Bregs suppress IL-23-mediated psoriasis-like inflammation through Treg expansion and inhibition of Th17 differentiation. Thus, targeting Bregs may be a feasible treatment strategy for psoriasis.

Targeted In Vivo Delivery of NF-κB Decoy Inhibitor Augments Sensitivity of B Cell Lymphoma to Therapy

Despite recent advances, non-Hodgkin's B cell lymphoma patients often relapse or remain refractory to therapy. Therapeutic resistance is often associated with survival signaling via nuclear factor κB (NF-κB) transcription factor, an attractive but undruggable molecular target. In this study, we describe a bipartite inhibitor comprising a NF-κB-specific decoy DNA tethered to a CpG oligodeoxynucleotide (ODN) targeting Toll-like receptor-9-expressing B cell lymphoma cells. The Bc-NFκBdODN showed efficient uptake by human diffuse large B cell (U2932, OCI-Ly3), Burkitt (RaJi), and mantle cell (Jeko1) lymphomas, respectively. We confirmed that Bc-NFκBdODN inhibited NF-κB nuclear translocation and DNA binding, resulting in CCND2 and MYC downregulation. Bc-NFκBdODN enhanced radiosensitivity of lymphoma cells invitro. In xenotransplanted human lymphoma, local injections of Bc-NFκBdODN reduced NF-κB activity in whole tumors. When combined with a local 3-Gy dose of radiation, Bc-NFκBdODN effectively arrested OCI-Ly3 lymphoma progression. In immunocompetent mice, intratumoral injections of Bc-NFκBdODN suppressed growth of directly treated and distant A20 lymphomas, as a result of systemic CD8 Tcell-dependent immune responses. Finally, systemic administration of Bc-NFκBdODN to mice bearing disseminated A20 lymphoma induced complete regression and extended survival of most of the treated mice. Our results underscore clinical relevance of this strategy as monotherapy and in support of radiation therapy to benefit patients with resistant or relapsed B cell lymphoma.

Exploration of Recombinant Fusion Proteins YAPO and YAPL as Carrier Proteins for Glycoconjugate Vaccine Design against Streptococcus pneumoniae Infection.

Pneumolysin (Ply), pneumococcal surface protein A (PspA), and pneumococcal surface adhesin A (PsaA) are promising cell surface protein antigen targets for Streptococcus pneumoniae (Spn) vaccine development. Herein, we designed and recombined two fusion proteins, named YAPO and YAPL, which contained the main antigenic epitopes of Ply, PspA, and PsaA. In-depth immunological evaluations revealed that YAPO and YAPL had strong immunocompetence to be well-qualified potential carrier proteins. To verify this possibility, a serotype 3 Spn (ST3) CPS pentasaccharide was conjugated to each fusion protein to generate the resultant glycoconjugates. Immunological studies in mice revealed that, as compared with TT conjugate, YAPO and YAPL conjugates provoked robust T-cell dependent immune responses that could provide better recognition, in vitro efficient opsonophagocytosis, and in vivo effective protection against various serotypes of Spn. Collectively, YAPO and YAPL were identified as immunopotentiating carriers that could help convert immunologically inactive ST3 pentasaccharide into a T cell-dependent antigen and provide efficient and broad spectrum of immunoprotection coverage so as to formulate functional glycoconjugate vaccines against Spn infections.

Hemophilia A Inhibitor Subjects Show Unique PBMC Gene Expression Profiles That Include Up-Regulated Innate Immune Modulators

Formation of pathological anti-FVIII antibodies, or “inhibitors,” is the most serious complication of therapeutic FVIII infusions, affecting up to 1/3 of severe Hemophilia A (HA) patients. Inhibitor formation is a classical T-cell dependent adaptive immune response. As such, it requires help from the innate immune system. However, the roles of innate immune cells and mechanisms of inhibitor development vs. immune tolerance, achieved with or without Immune Tolerance Induction (ITI) therapy, are not well-understood. To address these questions, temporal transcriptomics profiling of FVIII-stimulated peripheral blood mononuclear cells (PBMCs) was carried out for HA subjects with and without a current or historic inhibitor using RNA-Seq. PBMCs were isolated from 40 subjects in the following groups: HA with an inhibitor that resolved either following ITI or spontaneously; HA with a current inhibitor; HA with no inhibitor history and non-HA controls. PBMCs were stimulated with 5 nM FVIII and RNA was isolated 4, 16, 24, and 48 h following stimulation. Time-series differential expression analysis was performed and distinct transcriptional signatures were identified for each group, providing clues as to cellular mechanisms leading to or accompanying their disparate anti-FVIII antibody responses. Subjects with a current inhibitor showed differential expression of 56 genes and a clustering analysis identified three major temporal profiles. Interestingly, gene ontology enrichments featured innate immune modulators, including NLRP3, TLR8, IL32, CLEC10A, and COLEC12. NLRP3 and TLR8 are associated with enhanced secretion of the pro-inflammatory cytokines IL-1β and TNFα, while IL32, which has several isoforms, has been associated with both inflammatory and regulatory immune processes. RNA-Seq results were validated by RT-qPCR, ELISAs, multiplex cytokine analysis, and flow cytometry. The inflammatory status of HA patients suffering from an ongoing inhibitor includes up-regulated innate immune modulators, which may act as ongoing danger signals that influence the responses to, and eventual outcomes of, ITI therapy.

Immunogenicity of Bioproducts: Cellular Models to Evaluate the Impact of Therapeutic Antibody Aggregates.

Patients treated with bioproducts (BPs) frequently develop anti-drug antibodies (ADAs) with potential neutralizing capacities leading to loss of clinical response or potential hypersensitivity reactions. Many factors can influence BP immunogenicity and could be related to the patient, the treatment, as well as to the product itself. Among these latter factors, it is now well accepted that BP aggregation is associated with an increased potential for immunogenicity, as aggregates seem to be correlated with ADA development. Moreover, the presence of high-affinity ADAs suggests a CD4 T-cell dependent adaptive immune response and therefore a pivotal role for antigen-presenting cells (APCs), such as dendritic cells (DCs). In this review, we address the in vitro methods developed to evaluate how monoclonal antibodies could trigger the immunization process by focusing on the role of aggregated antibodies in the establishment of this response. In particular, we will present the different cell-based assays that have been used to assess the potential of antibodies and their aggregates to modulate cellular mechanisms leading to activation and the biological parameters (cellular activation markers, proliferation and secreted molecules) that can be measured to evaluate the different cell activation stages and their consequences in the propagation of the immune response. Indeed, the use of such strategies could help evaluate the risk of BP immunogenicity and their role in mitigating this risk.

The role of ERK2 in dendritic cell function

Abstract Dendritic cells (DCs) are an important link between the innate and adaptive immune systems, specifically by serving as key regulators of T cell-dependent (TD) immune responses. In DCs, several processes critical for initiating TD immune responses, such as migration, provision of costimulatory signals, and antigen uptake and presentation, utilize the mitogen-activated protein kinase (MAPK) signaling pathway. The dysregulation of these processes can result in the aberrant activation of autoreactive CD4+ T cells, resulting in autoimmune disease. Extracellular signal-regulated kinase 1 and 2 (ERK1/2) are key effectors of the MAPK pathway, and while ERK1 has been shown to be a critical regulator of DC migration and TLR-induced IL-10 secretion, the function of ERK2 remains unknown. Our lab has developed a novel mouse model in which the inducible Cre-mediated deletion of Erk2 is accompanied by the expression of an eYFP reporter protein. This provides us the unique ability to circumvent the embryonic lethality of deleting Erk2 in the germline while also allowing the detection of viable Erk2-deficient cells via flow cytometry. Preliminary data using this model has shown that Erk2Δ bone marrow-derived DCs (BMDCs), as well as Erk2Δ splenic DCs, exhibit altered expression of surface markers associated with T cell costimulation, antigen presentation, and migration compared with DCs from littermate controls. Taken together, this suggests that ERK2 plays a unique role in the regulation of several DC functions, potentially opening a new approach to treating CD4+ T cell-mediated autoimmune diseases.

Hemophilia A Inhibitor Subjects Show Unique PBMC Gene Expression Profiles That Include up-Regulated Innate Immune Modulators

The formation of pathological anti-FVIII antibodies, referred to as "inhibitors", is the most serious complication of therapeutic FVIII infusions, affecting up to one third of severe Hemophilia A (HA) patients. Intensive FVIII therapy, i.e. "Immune Tolerance Induction" (ITI), enables ~2/3 of treated patients to achieve peripheral tolerance to FVIII. FVIII inhibitor formation is a classical T-cell dependent adaptive immune response. As such, it requires help from the innate immune system. However, the roles of innate immune cells and mechanisms of inhibitor development versus immune tolerance, achieved with or without ITI therapy, are not well understood. To address these questions, we carried out temporal transcriptomics profiling of FVIII-stimulated peripheral blood mononuclear cells (PBMCs) from HA subjects with and without a current or historic inhibitor using RNA-seq. PBMCs were isolated from 40 subjects in the following groups: (A) HA with an inhibitor that resolved either following ITI or spontaneously; (B) HA with a current inhibitor; (C) HA with no inhibitor history and (D) non-HA healthy controls. PBMCs were rested overnight and then stimulated with 5 nM FVIII, and total RNA was isolated 4, 16, 24 and 48 hours following stimulation. RNA from unstimulated cells at t = 4 hrs served as a negative control. Time-series differential expression analysis was performed with DESeq2 and genes with a log likelihood ratio test FDR <0.05 and an absolute fold change >1.25 at at least one stimulation time point compared to control were deemed significant. Subjects with a resolved past inhibitor (Group A) showed differential expression of only 15 genes. In contrast, subjects with a current inhibitor (Group B) showed differential expression of 56 genes. A clustering analysis divided the temporal trajectories of Group B genes into 3 distinct clusters. Twenty-three genes were up-regulated at 16 hr and 21 genes at 48 hr post-stimulation, respectively. Interestingly, gene ontology (GO) enrichment analysis of these genes revealed enrichments for innate immune modulators, including NLRP3, TLR8, IL32, CLEC10A and COLEC12.NLRP3 and TLR8 are associated with enhanced secretion of the pro-inflammatory cytokines IL-1beta and TNF-alpha, while IL32, which has several isoforms, has been associated with both inflammatory and regulatory immune processes. Expression levels of NLRP3, TLR8, CLEC10A and IL32 transcripts were validated by real time PCR, and changes in RNA transcript abundances correlated well with the RNA-seq results. IL-32 results were validated by both RT-qPCR on an aliquot of the original RNA sample and ELISA to measure the cytokine in supernatants at t=48 hrs. HA subjects with no inhibitor history (Group C) had 195 differentially expressed genes whose temporal profiles fell into 4 distinct clusters. GO enrichment analysis revealed biological processes related to epithelial cell proliferation, responses to toxic substances, and positive/negative regulation of cytokine secretion (TNF, NQO1, PMEPA1). The non-HA healthy control subjects (Group D) also showed cellular responses to ex vivo FVIII stimulation. A total of 63 differentially regulated genes fell into 4 distinct clusters. GO analysis identified expression patterns associated with leukocyte-mediated immunity, T-cell activation, and a hypoxia response. Overall, distinct transcriptional signatures were identified for each of the four groups, providing clues as to cellular mechanisms leading to or accompanying their disparate anti-FVIII antibody responses. We are currently characterizing PBMC immune cell subsets, e.g. macrophages and CD4+ T cells, to identify specific cell types responsible for the differentially regulated genes. Cellular responses of tolerized HA subjects and healthy non-HA controls were consistent with the known immunogenicity of FVIII, including persistence of FVIII-specific CD4+ T cells even in individuals with no measurable FVIII inhibitor. The inflammatory status of HA patients suffering from an ongoing inhibitor clearly includes up-regulation of innate immune modulators, some of which may act as ongoing danger signals that influence the responses to, and eventual outcome of, ITI therapy. Disclosures Pratt: Grifols, Inc: Research Funding; Bloodworks NW: Patents & Royalties: inventor on patents related to FVIII immunogenicity.

EZH2 Gain-of-Function Mutations Generate a Lymphoma-Permissive Immune Niche

The polycomb histone methyltransferase protein EZH2 is highly expressed and mutated in approximately 25% of the two most common subtypes of non-Hodgkin's lymphoma: follicular lymphoma (FL) and diffuse large B cell lymphoma (DLBCL). Both of these tumor types originate from germinal center (GC) B-cells, which are highly proliferative cells that form transiently during the T-cell dependent humoral immune response. Wild type EZH2 plays a critical role in the formation of GC B-cells and mutant EZH2 drives malignant transformation of these cells in vivo. These lymphoma-associated mutations of EZH2 are gain-of-function and enhance EZH2 methyltransferase activity, which cause abnormally efficient H3K27me3, enhancing repression of transcription. Of note, EZH2 mutant lymphomas manifest aberrant suppression of antigen presentation pathways, such as loss or reduced expression of MHC-I and MHC-II, and reduced T-cell infiltrates. We show that mice engineered to conditionally express mutant Ezh2Y641F specifically in GC B-cells exhibited massive GC hyperplasia with expansion of the "light zone" -the GC niche where B-cells are selected based on the affinity of their antibodies- and extended follicular dendritic cells network. With chimeric bone marrow transplantation experiments we found that Ezh2Y641F provides a competitive advantage to activated B-cells in seeding and expanding the T-cell dependent GC reaction. Liquid chromatography separation and mass spectrometry of histone tryptic peptides revealed a massive increase in H3K27me3 in Ezh2Y641F GC B-cells, at the expense of a reduction in H3K27me2. Remarkably, we observed increased unmodified and decreased H3K27me1, indicating a loss-of-function mono-methylase activity of Ezh2Y641F. We confirmed an extensive global gain of H3K27me3 by ChIP-seq, with massive spreading of the mark both upstream and downstream TSS. RNA-seq analysis showed differential expression of genes involved in antigen processing and presentation and immune response, suggesting that B-cell selection process is altered in Ezh2Y641F GC light zone. Therefore, we hypothesized that, through an epigenetic mechanism, Ezh2 mutation makes B-cells less dependent of T-cells by aberrantly suppressing genes involved in the immune synapse. Indeed, inhibition of the interaction between TFH and GC B-cell in vivo using anti CD40L antibody, abrogated GC reaction in WT but not in Ezh2Y641F mice. Of note, EZH2 mutant GCs manifest a massive expansion of the follicular dendritic cell network in spite of lack of T-cell dependence, a finding that reflects low grade FL histology. Hence, EZH2 mutant lymphomas might progress due to acquired immune escape, which is a critical consideration when designing precision, tailored immune-therapy regimens. Disclosures Melnick: Constellation: Consultancy; Janssen: Research Funding; Epizyme: Consultancy.

Growth Hormone Aggregates Activation of Human Dendritic Cells Is Controlled by Rac1 and PI3 Kinase Signaling Pathways.

The presence of protein aggregates in biological products is suggested to promote immunogenicity, leading to the production of anti-drug antibodies with neutralizing capacities. This suggests a CD4+ T-cell dependent adaptive immune response, thus a pivotal role for antigen-presenting cells, such as dendritic cells (DCs). We previously showed that human growth hormone aggregates induced DC maturation, with notably an increase in CXCL10 production. DC phenotypic modifications were sufficient to promote allogeneic CD4+ T-cell proliferation with Th1 polarization. In this work, we identified the main intracellular signaling pathways involved in DC activation by human growth hormone aggregates, showing that aggregates induced p38 mitogen-activated protein kinase, extracellular signal-regulated kinase, and c-Jun N-terminal kinase phosphorylation, as well as nuclear factor κB subunit p65 nuclear translocation. Next, investigating the implication of Rho GTPases and phosphoinositide 3-kinase (PI3K) in activated DC showed that Rac1 and Cdc42 regulated the phosphorylation of MAP kinases, whereas PI3K was only implicated in c-Jun N-terminal kinase phosphorylation. Furthermore, we showed that Rac1 and PI3K pathways, but not Cdc42, regulated the production of CXCL10 via the MAP kinases and nuclear factor κB. Taken together, our results bring new insight on how protein aggregates could induce DC activation, leading to a better understanding of aggregates role in therapeutic proteins immunogenicity.

BOB.1 controls memory B-cell fate in the germinal center reaction

During T cell-dependent (TD) germinal center (GC) responses, naïve B cells are instructed to differentiate towards GC B cells (GCBC), high-affinity long-lived plasma cells (LLPC) or memory B cells (Bmem). Alterations in the B cell-fate choice could contribute to immune dysregulation leading to the loss of self-tolerance and the initiation of autoimmune disease. Here we show that mRNA levels of the transcription regulator BOB.1 are increased in the lymph node compartment of patients with rheumatoid arthritis (RA), a prototypical autoimmune disease caused by the loss of immunological tolerance. Investigating to what extent levels of BOB.1 impact B cells during TD immune responses we found that BOB.1 has a crucial role in determining the B cell-fate decision. High BOB.1 levels promote the generation of cells with phenotypic and functional characteristics of Bmem. Mechanistically, overexpression of BOB.1 drives ABF1 and suppresses BCL6, favouring Bmem over LLPC or recycling GCBC. Low levels of BOB.1 are sufficient for LLPC but not for Bmem differentiation. Our findings demonstrate a novel role for BOB.1 in B cells during TD GC responses and suggest that its dysregulation may contribute to the pathogenesis of RA by disturbing the B cell-fate determination.

Abstract B087: MHC class II on cancer cells—Role in response to BCG therapy of bladder cancer

Abstract BCG, a live mycobacterium also used worldwide as a vaccine against tuberculosis, is one of the first successful immunotherapies of cancer. Despite being in clinical use for the treatment of non-muscle-invasive bladder cancer (NMIBC) for 4 decades, the mechanism by which BCG activates the immune system to destroy cancer cells is poorly understood and there are no reliable methods to predict a patient’s response to BCG treatment. Current data suggest that the efficacy of BCG depends on initial attachment of BCG to bladder cancer cells leading to activation of an immune response that is T-cell dependent. It is not known whether bladder cancer cells have a direct role in activating T-cells. Using an orthotopic mouse model of BCG treatment of bladder cancer we show that: 1) BCG induces tumor-specific immunity; 2) CD4+ T-cells are the main requirement for BCG efficacy and for the tumor-specific immunity engendered by BCG therapy;3) bladder cancer cells can directly activate CD4+ T-cells; 4) the efficacy of BCG depends on MHC class II expression on the surface of bladder cancer cells. These data suggest that bladder cancer cells directly activate and drive a CD4+ T-cell dependent immune response that is required for the efficacy of BCG. Current work in the lab is focused on characterizing MHC class II expression on human NMIBC specimens and determining whether expression of MHC class II on bladder cancer cells predicts clinical outcomes after BCG therapy. Citation Format: Gil Redelman-Sidi, Michael Glickman, Anna Binyamin, Anthony Antonelli. MHC class II on cancer cells—Role in response to BCG therapy of bladder cancer [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B087.

The Role of Interleukin-10 in Mediating the Effect of Immune Challenge on Mouse Gonadotropin-Releasing Hormone Neurons In Vivo.

Immune challenge alters neural functioning via cytokine production. Inflammation has profound impact on the central regulation of fertility, but the mechanisms involved are not clearly defined. The anti-inflammatory cytokine interleukin (IL)-10 is responsible for balancing the immune response in the brain. To examine whether IL-10 has an effect on the function of the gonadotropin-releasing hormone (GnRH) neurons, we first examined the effect of immune responses with distinct cytokine profiles, such as the T cell-dependent (TD) and T cell-independent (TI) B-cell response. We investigated the effect of the TD and TI immune responses on ERK1/2 phosphorylation in GnRH neurons by administering fluorescein isothiocyanate/keyhole limpet hemocyanin (KLH-FITC) or dextran-FITC to female mice. Although dextran-FITC had no effect, KLH-FITC induced ERK1/2 phosphorylation in GnRH neurons after 6 d. KLH-FITC treatment increased the levels of IL-10 in the hypothalamus (HYP), but this treatment did not cause lymphocyte infiltration or an increase in the levels of proinflammatory cytokines. In IL-10 knock-out (KO) mice, KLH-FITC-induced ERK1/2 phosphorylation in the GnRH neurons was absent. We also showed that in IL-10 KO mice, the estrous cycle was disrupted. Perforated patch-clamp recordings from GnRH-GFP neurons, IL-10 immunohistochemistry, and in vitro experiments on acute brain slices revealed that IL-10 can directly alter GnRH neuron firing and induce ERK1/2 phosphorylation. These observations demonstrate that IL-10 plays a role in influencing signaling of GnRH neurons in the TD immune response. These results also provide the first evidence that IL-10 can directly alter the function of GnRH neurons and may help the maintenance of the integrity of the estrous cycle.

Impact of Chronic Viral Infection on T-Cell Dependent Humoral Immune Response.

During the last decades, considerable efforts have been done to decipher mechanisms supported by microorganisms or viruses involved in the development, differentiation, and function of immune cells. Pathogens and their associated secretome as well as the continuous inflammation observed in chronic infection are shaping both innate and adaptive immunity. Secondary lymphoid organs are functional structures ensuring the mounting of adaptive immune response against microorganisms and viruses. Inside these organs, germinal centers (GCs) are the specialized sites where mature B-cell differentiation occurs leading to the release of high-affinity immunoglobulin (Ig)-secreting cells. Different steps are critical to complete B-cell differentiation process, including proliferation, somatic hypermutations in Ig variable genes, affinity-based selection, and class switch recombination. All these steps require intense interactions with cognate CD4+ helper T cells belonging to follicular helper lineage. Interestingly, pathogens can disturb this subtle machinery affecting the classical adaptive immune response. In this review, we describe how viruses could act directly on GC B cells, either through B-cell infection or by their contribution to B-cell cancer development and maintenance. In addition, we depict the indirect impact of viruses on B-cell response through infection of GC T cells and stromal cells, leading to immune response modulation.

Novel Insights Into the Immunoregulatory Function and Localization of Dendritic Cells.

Dendritic cells (DCs) are antigen-presenting cells that normally play a critical role in stimulating T-cell-dependent immune responses. However, tolerogenic DCs (CD11cMHC-IICD80CD86) induce immune tolerance by stimulating regulatory T cells (Tregs: CD4CD25Foxp3). Although tolerogenic DCs are used to treat autoimmune diseases and to prevent transplantation rejection, the mechanisms by which they regulate alloimmunity are poorly understood. Here, we review our previous studies aiming to elucidate the mechanisms involved in immune rejection of corneal allografts using a corneal transplant model. We found that donor-derived tolerogenic DCs significantly prolonged corneal allograft survival by suppressing indirect allosensitization. We also reported the precise distribution of intraepithelial corneal DCs, termed Langerhans cells (LCs: CD11cLangerinMHC-II) in the cornea, which we maintain play a critical role in regulating corneal immunity. By confocal microscopy, we constructed 3-dimensional images of corneal LCs, which demonstrated that their cell bodies are present in the basal cell layer of the corneal epithelium. Furthermore, LC dendrites extend toward the ocular surface, but do not connect to epithelial tight junctions, indicating that they cannot directly interact with ocular surface antigens. We confirm the potential of DC therapy for corneal graft rejection and report the function of intraepithelial DCs (LCs) in the normal cornea.

Genetic diversity of the Pneumococcal CbpA: Implications for next-generation vaccine development

Pneumococci are capable of vaccine escape by genetic recombination at the targeted capsular locus, significantly reducing long-term vaccine effectiveness. Recently, efforts have been redirected to understanding pneumococcal biology related to potential next-generation vaccine candidates. A variety of serotype-independent protein antigens capable of inducing protective immune responses in tissue culture and animal models of infection have been identified. However, ideal vaccine candidates that are conserved across all genotypes, provide broad population coverage, and induce T-cell dependent immune responses are still under investigation. We examined whether immune responses due to the highly polymorphic CbpA antigen are due to a conserved domain capable of evoking specific immune “memory” across all genotypes of pneumococci. We defined the genotypes in a global dataset of 213 pneumococcal isolates. This isolate collection was genotypically diverse and ideal for establishing the presence of conserved CbpA epitopes as potential protein vaccine candidates. Examination of the CbpA locus sequence was highly polymorphic at both the nucleic acid and amino acid level. Despite this high polymorphism some domains are broadly conserved and consist of different amino acid residues with the same physicochemical properties, and therefore have similar tertiary structures. The two most common domains identified in the CbpA gene are modular teichoic acid phosphorylcholine esterase Pce (2bib:A), and R2 domain (1w9r:A). These conserved domains are immunogenic, therefore capable of inducing long-term host immune responses; moreover they are extracellularly located and thus accessible. We proposed their evaluation as suitable next-generation CbpA-fusion protein vaccine candidates.