Transcription Factor BCL6 Research Articles

PF356 CHRONIC LYMPHOCYTIC LEUKEMIA INDUCES SKEWING OF (ANTIGEN‐SPECIFIC) T CELL RESPONSES TOWARDS A DYSFUNCTIONAL SHORT‐LIVED EFFECTOR PHENOTYPE

Background:In recent years, CAR T cells showed to be highly effective in B cell malignancies such as acute lymphocytic leukemia. However, for chronic lymphocytic leukemia (CLL) efficacy of autologous T cell therapies is rather limited. T cells of CLL patients have a dysfunctional, terminally differentiated phenotype, which might be linked with the lack of success of T cell therapies. Recently it was shown that presence of memory T cell subsets in the CAR T cell infusion product associated with complete responses in CLL (Fraietta et al., 2018).Aims:It remains to be elucidated how CLL cells influence T cell skewing and whether this affects T cell responses in vivo. In this context, we functionally investigated acute T cell responses in the TCL1 mouse model, which features similar T cell dysfunction as observed in CLL patients (Gorgun et al., 2009). We applied a set‐up that allows to study the response against acute infection of (1) endogenous CD8+ T cells, which have been exposed to leukemic cells during CLL development, and (2) the antigen‐specific response of naïve, transferred OT‐I T cells.Methods:C57BL/6J (CD45.2+) mice were injected i.p. with 20x106 TCL1 splenocytes or PBS. When CD5+ leukemia reached 70% cells in blood, 50.000 naïve ovalbumin‐specific CD8+ T cells (OT‐I, CD45.1+) were injected i.v. Next, all mice were infected with mCMV‐OVA and sacrificed 7 days later. Splenocytes were analyzed directly or after in vitro ovalbumin stimulation by FACS.Results:(1) Already prior to infection, we found a skewing towards antigen‐experienced cells in the endogenous CD8+ compartment of leukemic mice. After mCMV‐OVA infection, a population of naïve CD8+ T cells persisted in control mice, but in leukemic mice the complete CD8+ compartment differentiated into effector cells. Within the effector compartment, we found a skewing to the short‐lived effector cell (SLEC) phenotype in the leukemic mice, while memory precursor effector cells (MPEC) were reduced. This skewing towards SLEC associated with increased effector‐associated T‐bet expression.(2) Analysis of the OT‐I cells allowed to perform detailed study of the impact of leukemia on an antigen‐specific response. Seven days after infection, spleens of leukemic and control mice contained similar frequencies of differentiated OT‐I cells, that had an effector phenotype. Also here, TCL1‐derived OT‐I cells showed a decreased percentage of MPEC, while SLECs were increased (Figure 1), indicating impaired memory formation. In correlation with skewing towards SLECs in leukemic mice, these OT‐I cells showed reduced expression of memory‐associated transcription factor Bcl6, while T‐bet was also increased. Earlier it was shown that Bcl6 is able to repress glycolysis, essential for T cell effector function (Oestreich et al., 2014). In line with reduced Bcl6 expression, we found increased glucose uptake in TCL1‐derived OT‐I cells. Despite the skewing towards SLEC phenotype, the TCL1‐derived OT‐I cells showed decreased production of IFNγ and TNFα and decreased degranulation (CD107a expression) upon in vitro restimulation.Summary/Conclusion:In this murine model of CLL, both bystander T cells as well as acute antigen‐specific T cell responses are driven towards an SLEC phenotype with signs of functional impairment, and metabolic alterations. This approach may provide crucial insight into the interplay between CLL and T cells, and the underlying failure of cancer immune surveillance.image

Immune Complex (ICs) generate PD1int CD4+ T cells which are Bcl6+IFN-γ+ unlike exhausted PD1high cells

Abstract PD1 is targeted for cancer therapy, however the mechanisms that drive its expression are poorly recognized. Role for Fc-receptors in CD4+ T-cells responses has been ignored in the last decade. We and others have now shown the activation induced expression of CD16a (FcγRIIIa) and CD32 (FcγRIIa) (doi:10389/fimmu.2018.02814). Three groups have now shown that CD32 expression in CD4+ T cells mark HIV-1 provirus enrichment. Thus, we examined whether immune complexes (ICs) by engaging CD16a play a role in PD1 expression and Tfh cell development. Using flow cytometry, biochemical techniques and RNA-seq, we establish a new subset of CD4+ PD1intCD16a+ Tcells. These cells are present in vivo and can be generated in vitro upon IC signaling. These cells express Bcl6+ and produce IFN-γ, IL-17A, and IL-21. A second population of terminally exhausted PD1highCD16a−Bcl6− do not produce cytokines also exist. CD16a ligation by ICs generates CD4+ T effector cells and relocate endosomal NATLRs to the cell surface for joint signaling with FcRs (JI, 198: 4596, 2017). CD16a signaling up-regulate Tfh transcription factors c-Maf, FOXO, Bcl6, also lincRNA and microRNA associated with TFH/T cell differentiation i.e. miR155, 17HG, 22 and HOTIP. Upregulation of IL-27 and ICOS via CD16a could induce c-Maf expression. CD16a signal in CD4+ T cells showed up-regulation of ubiquitination, heat shock proteins, proteasome assembly, GPCR signaling genes and 1139 genes from GO: 007062 category of extracellular exosomes. Our findings establish a new mechanism that drives PD1 expression. Tfh cells are enriched in HIV-1 provirus and expression of FcRs in such cells suggest their role in viral enrichment. Does CD16a+ mark non-exhausted CD4+ Tfh cells remains an open question.

IL-12 signaling drives the differentiation and function of a TH1-derived TFH1-like cell population

Abstract CD4+ T follicular helper (TFH) cells play key roles in the generation of humoral immunity, as they provide T cell help to B cells that ultimately results in the production of pathogen-specific antibodies. A subset of TFH cells that shares features with TH1 cells, referred to as (TH1)-biased TFH (TFH1) cells, has recently been shown to play key roles in responses to chronic infections including HIV and malaria, as well as the onset of some autoimmune diseases. Despite the apparent importance of these cells to comprehensive immune responses and human health, many questions remain regarding their origin, including the molecular mechanisms that regulate their differentiation. Here, we describe a population of TH1-derived TFH1-like cells that produce both the TH1 cytokine interferon-γ and the TFH-associated cytokine interleukin-21 (IL-21). Interestingly, this TFH1-like population exhibits increased B cell helper activity compared to IL-6 derived TFH-like cells. We further demonstrate that IL-12 signaling is required for both the differentiation and functional properties of the TFH1-like cell population. Specifically, IL-12-dependent activation of STAT4, and unexpectedly STAT3, result in both the upregulation of the TFH lineage-defining transcription factor Bcl-6, as well as the increased production of IL-21. Finally, we show that IL-12 signaling is required for TFH1-like cell expression of Icos, a receptor that is critical for the ability of T cells to provide help to B cells. Taken together, our findings provide potential insight into the genesis and regulatory requirements of recently described TFH1 cells with emerging roles in human health and disease.

Enhancing the anti‐tumor efficacy of chimeric antigen receptor‐expressing T cells with naturally occurring plant stilbenes

Despite advances in treatment options, cancer remains the second leading cause of death in the United States. Many therapies use immune cells, specifically T cells, for cancer therapy. To enhance tumor recognition by T cells, chimeric antigen receptors consisting of signaling domains fused to receptors that recognize tumor antigens can be created and expressed in T cells. One receptor that is a prospective target for a chimeric antigen receptor is PD1 because the ligands for the PD1 receptor are expressed on many cancer types. Our lab has genetically engineered a chimeric PD1 (chPD1) receptor and has shown that chPD1‐expressing T cells kill tumor cells, secrete inflammatory cytokines, reduce tumor burden, and increase survival in multiple tumor types. A current focus of cancer research is the development of combination therapies that enhance anti‐tumor efficacy without increasing harmful side effects. Recently, a family of naturally occurring plant compounds called stilbenes have been shown to enhance T cell function. Therefore, we tested if stilbenes enhanced chPD1 T cell function in breast cancer. Addition of resveratrol, a stilbene found in berries, grapes, peanuts, and other plants, decreased proliferation, enhanced tumor cell killing, and increased secretion of proinflammatory cytokines (IFNγ, GM‐CSF, TNFα, IL‐17, IL‐2, and IL‐21) from chPD1 T cells. Presence of resveratrol also increased skewing of T cell differentiation to a central memory phenotype through reduction of AKT and mTORc1 signaling, reduced expression of T‐bet and BLIMP‐1 transcription factors, and increased expression of Bcl‐6 and Eomes transcription factors. Hence, combination of resveratrol and chimeric antigen receptor‐expressing T cells may enhance anti‐tumor immune responses through inhibition of the AKT and mTORc1 pathways and skewing to a long‐lived central memory phenotype.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

MEF2B is a member of the BCL6 gene transcriptional complex and induces its expression in diffuse large B-cell lymphoma of the germinal center B-cell-like type

Myocyte enhancer-binding factor 2B (MEF2B) has been implicated as a transcriptional regulator for BCL6. However, details about the interaction between MEF2B and BCL6 during expression, as well as the relationship of MEF2B to the expression of other germinal center (GC) markers, have not yet been fully explained. Using germinal center B-cell-like diffuse large B-cell lymphoma (GC-DLBCL) and activated B-cell diffuse large B-cell lymphoma (ABC-DLBCL) cell lines, we analyzed the expression of MEF2B and its associations with BCL6, CD10, and ERK. Furthermore, small interfering RNA (siRNA) was used to study the possible effects of MEF2B knockdown on these proteins and cell growth. Analysis of the BCL6 transcriptional complex was performed using electrophoretic mobility shift assay. The correlation between MEF2B expression and the genetic type of DLBCL was assessed using immunohistochemistry on 111 patient samples, and via in silico analysis of publicly available microarray (Gene Expression Omnibus (GEO)) datasets. Our results indicate that the expression of MEF2B protein is important for the growth of GC-DLBCL cells, as evidenced by MEF2B knockdown inhibition of cell growth and the subsequent suppression of BCL6, CD10, and ERK phosphorylation. Analysis of BCL6 transcription factors in nuclear extracts of MEF2-expressing DLBCL cells showed involvement of MEF2B with AP-2α and BCL6 proteins in the formation of the BCL6 gene transcriptional complex. Indeed, differential expression of MEF2B in the GC-DLBCL is statistically significant compared to the ABC-DLBCL in the GEO datasets, as well as in tissue microarray, as indicated via immunohistochemistry (Visco–Young algorithm). Our findings indicate that MEF2B is an essential component of the BCL6 gene transcriptional complex for the regulation of DLBCL growth via the promotion of BCL6 expression. Beyond its regulatory role in DLBCL growth, MEF2B expression correlated positively with BCL6 and CD10 expression, and was preferentially expressed in the GBC-DLBCL group.

Establishment of Systems to Enable Isolation of Porcine Monoclonal Antibodies Broadly Neutralizing the Porcine Reproductive and Respiratory Syndrome Virus.

The rapid evolution of porcine reproductive and respiratory syndrome viruses (PRRSV) poses a major challenge to effective disease control since available vaccines show variable efficacy against divergent strains. Knowledge of the antigenic targets of virus-neutralizing antibodies that confer protection against heterologous PRRSV strains would be a catalyst for the development of next-generation vaccines. Key to discovering these epitopes is the isolation of neutralizing monoclonal antibodies (mAbs) from immune pigs. To address this need, we sought to establish systems to enable the isolation of PRRSV neutralizing porcine mAbs. We experimentally produced a cohort of immune pigs by sequential challenge infection with four heterologous PRRSV strains spanning PRRSV-1 subtypes and PRRSV species. Whilst priming with PRRSV-1 subtype 1 did not confer full protection against a subsequent infection with a PRRSV-1 subtype 3 strain, animals were protected against a subsequent PRRSV-2 infection. The infection protocol resulted in high serum neutralizing antibody titers against PRRSV-1 Olot/91 and significant neutralization of heterologous PRRSV-1/-2 strains. Enriched memory B cells isolated at the termination of the study were genetically programmed by transduction with a retroviral vector expressing the Bcl-6 transcription factor and the anti-apoptotic Bcl-xL protein, a technology we demonstrated efficiently converts porcine memory B cells into proliferating antibody-secreting cells. Pools of transduced memory B cells were cultured and supernatants containing PRRSV-specific antibodies identified by flow cytometric staining of infected MARC-145 cells and in vitro neutralization of PRRSV-1. Collectively, these data suggest that this experimental system may be further exploited to produce a panel of PRRSV-specific mAbs, which will contribute both to our understanding of the antibody response to PRRSV and allow epitopes to be resolved that may ultimately guide the design of immunogens to induce cross-protective immunity.

B cell primary immune responses.

B cell primary immune responses.

Germinal center-derived lymphomas: The darkest side of humoral immunity.

SummaryOne of the unusual features of germinal center (GC) B cells is that they manifest many hallmarks of cancer cells. Accordingly, most B‐cell neoplasms originate from the GC reaction, and characteristically display abundant point mutations, structural genomic lesions, and clonal diversity from the genetic and epigenetic standpoints. The dominant biological theme of GC‐derived lymphomas is mutation of genes involved in epigenetic regulation and immune receptor signaling, which come into play at critical transitional stages of the GC reaction. Hence, mechanistic studies of these mutations reveal fundamental insight into the biology of the normal and malignant GC B cell. The BCL6 transcription factor plays a central role in establishing the GC phenotype in B cells, and most lymphomas are dependent on BCL6 to maintain survival, proliferation, and perhaps immune evasion. Many lymphoma mutations have the commonality of enhancing the oncogenic functions of BCL6, or overcoming some of its tumor suppressive effects. Herein, we discuss how unique features of the GC reaction create vulnerabilities that select for particular lymphoma mutations. We examine the interplay between epigenetic programming, metabolism, signaling, and immune regulatory mechanisms in lymphoma, and discuss how these are leading to novel precision therapy strategies to treat lymphoma patients.

OX40 stimulation and PD-L1 blockade synergistically augment HBV-specific CD4 T cells in patients with HBeAg-negative infection

Current antiviral therapies lack the potential to eliminate persistent hepatitis B virus (HBV) infection. HBV-specific T cells are crucial for HBV control and have recently been shown to be protective in patients following discontinuation of antiviral therapy. Thus, T cell-based approaches may greatly improve the therapeutic landscape of HBV infection. We aimed to augment HBV-specific CD4 T cells from chronically infected patients by targeting different immunological pathways. Expression of various co-stimulatory and inhibitory receptors on HBV- and influenza-specific CD4 T cells was analyzed directly ex vivo by MHC class II-tetramers. Patients infected with HBV genotype D were screened for CD4 T cell responses by IFN-γ ELISpot and intracellular cytokine staining following stimulation with overlapping peptides (OLPs) spanning the HBV-polyprotein. Stimulation with recombinant IL-7, an agonistic OX40-antibody or blockade of PD-L1 was performed in antigen-specific in vitro cultures. Cytokine secretion and expression of transcription factors were analyzed by flow cytometry. Responses targeting influenza, Epstein-Barr virus and tetanus toxoid served as controls. Tetramer-staining revealed that the IL-7 receptor-alpha (CD127), OX40 and PD-1 constitute possible therapeutic targets as they were all strongly expressed on HBV-specific CD4 T cells ex vivo. The HBV-specific CD4 T cell responses identified by OLP screening targeted predominantly the HBV-polymerase and core proteins. Combined OX40 stimulation and PD-L1 blockade significantly augmented IFN-γ and IL-21 producing HBV-specific CD4 T cells in vitro, suggesting active T helper type 1 cell and follicular T helper cell programs. Indeed, transcription factors T-bet and Bcl6 were strongly expressed in cytokine-producing cells. Combined OX40 stimulation and PD-L1 blockade augmented secretion of the helper T cell signature cytokines IFN-γ and IL-21, suggesting that immunotherapeutic approaches can improve HBV-specific CD4 T cell responses. CD4 T cells are important in controlling viral infections but are impaired in the context of chronic hepatitis B virus (HBV) infection. Therapeutic approaches to cure chronic HBV infection are highly likely to require an immune-stimulatory component. This study demonstrates that HBV-specific CD4 T cells can be functionally augmented by combined stimulation of the co-stimulatory molecule OX40 and blockade of the inhibitory PD-1 pathway.

BCL6-Mediated Silencing of PD-1 Ligands in Germinal Center B Cells Maintains Follicular T Cell Population.

The programmed cell death protein 1 (PD-1) ligands PD-L1 and PD-L2 on germinal center (GC) B cells deliver coinhibitory signals to follicular T cells. The PD-L1/L2-PD-1 axis modulates the quality and quantity of follicular T cells and has been shown to influence the GC responses. However, the transcriptional control of PD-1 ligands on GC B cells remains largely unknown. In this study, we report that the transcription factor BCL6 is a key negative regulator of the PD-1 ligands PD-L1 and PD-L2 in GC B cells. Acute deletion of Bcl6 in mature GC B cells resulted in marked upregulation of mRNA and protein abundance of PD-1 ligands. Moreover, the expression levels of BCL6 and PD-1 ligands were inversely correlated during GC B cell development and in human GC-derived lymphoma specimens. Mechanically, BCL6 directly bound to the promoter region of PD-L1 and intron 2 of PD-L2 to suppress their transcription. In addition, BCL6 indirectly inhibited the transcription of PD-1 ligands by repressing the expression of STAT1/STAT3 and IRF1. Moreover, BCL6 exerted these effects via its BTB domain. Finally, PD-1 blockade promoted cell survival to sustain the follicular T cell pool in the presence of Bcl6-deficinet GC B cells. In summary, B cell-specific expression of BCL6 dampens the PD-L1/L2-PD-1 signaling to maintain the size of follicular T cells during GC development.

Di-(2-ethylhexyl)-phthalate interferes with T-follicular helper cell differentiation and cytokine secretion through signaling lymphocytic activation molecule family member-1

Exposure to the widely-used phthalate plasticizer di-(2-ethylhexyl)-phthalate (DEHP) has been shown to be closely related to an increased prevalence of allergic diseases in infants and juveniles. Earlier work in our laboratory found that DEHP-related anaphylactic responses could be ascribed to T-follicular helper (Tfh) cell hyperfunction directly. The Tfh cell, a newly identified CD4+ TH cell subset, until recently has been considered as a key player in humoral immunity. Tfh cells can respond to stimulation through various receptors. Signaling lymphocytic activation molecule family member-1 (SLAMF1, CD150) is a surface co-stimulatory receptor that can bind to an intracytoplasmic adaptor signaling lymphocytic activation molecule-associated protein (SAP) to initiate downstream signaling cascades, regulating some events of immune response. The present study explored the role of SLAMF1 in Tfh cell differentiation and cytokine secretion under the condition of DEHP exposure. Using a weanling mice model of DEHP gavage with ovalbumin (OVA) sensitization, it was found that DEHP acted as an immunoadjuvant to elevate SLAMF1 and SAP expression in host Tfh cells. Ex vivo studies of effects from DEHP exposure on Tfh cells from OVA-sensitized hosts showed that DEHP acted in an adjuvant-like manner to promote the expression of adaptor protein SAP, transcription factors Bcl-6 and c-MAF, and cytokines interleukin (IL)-21 and IL-4 in Tfh cells. Transfection of these Tfh cells with Slamf1 small interfering RNA prior to exposure to the DEHP attenuated the over-expression of these molecules that was caused by the DEHP. In conclusion, this study demonstrated that DEHP, via a SLAMF1-mediated pathway, can impact on Tfh cell differentiation and their ability to form select cytokines.

B cells turn on, tune in with LSD1.

The histone demethylase LSD1 partners with the transcription factor BCL6 to initiate the formation of germinal centers.

First Evidence of Dysfunctional Antigen-Specific T Cell Responses in Experimental CLL As a Model for Studies of Autologous T Cell-Based Therapies

Background - Tumor immunosuppression is a major cause for treatment failure and disease relapse, both in solid tumors and in leukemia. New T cell mediated therapeutic modalities (such as CAR T cells, bispecific antibodies and immune checkpoint blockade) can be highly effective, but depend on autologous T cell functionality. In chronic lymphocytic leukemia (CLL), ex vivo patient studies have shown that T cells are skewed toward a terminally differentiated, dysfunctional phenotype, which may contribute to the disappointing results of T cell therapies in this disease. Recently, it has been shown that for CAR T cell efficacy in CLL, the presence of memory subsets both prior to CAR T cell generation and in the infusion product, is instrumental for CAR T cell persistence and complete responses (Fraietta et al., 2018). However, it is currently unknown how the presence of CLL impacts T cell skewing and whether it affects acute antigen-specific immune responses in vivo. As dynamic immune studies are limited in the human setting, we investigated whether acute antigen-mediated T cell responses are affected by CLL using the TCL1 adoptive transfer mouse model. Earlier T cell studies in this model revealed similar changes as seen in the human disease, both with respect to gene expression profiles as well as phenotypic and functional characteristics.Methods - C57BL/6J (CD45.2+) mice were injected i.p. with 20x106 TCL1 transgenic splenocytes (CD45.2+) or PBS. During development of CLL mice were monitored for T cell subset differentiation. When CLL mice reached over 70% CLL in blood, 50.000 OT-I cells (CD45.1+), which specifically recognize ovalbumin) were adoptively transferred (i.v.) into all mice, directly followed by infection with 100.000 PFU mCMV-OVA. This model allowed us to study the endogenous (CD45.2+) T cell compartment during CLL development, as well as the acute response of naïve, antigen-specific (CD45.1+) T cells that had not been influenced by CLL prior to antigen exposure. Seven days after infection the mice were sacrificed and splenocytes were analyzed by flow cytometry either directly or after re-stimulation with OVA-peptide to assess cytokine production.Results - Using this adoptive transfer model of CLL, we found that CLL mice showed a decrease in naïve CD8 T cells, an increase of antigen-experienced cells, and a reversed CD4/CD8 ratio compared to control mice, as reported earlier (McClanahan et al., 2015). At day 7 after injection of naïve OT-I cells and infection with mCMV-OVA, percentages of OT-I cells were similar in CLL and control mice, and all OT-I cells showed an effector phenotype (CD44+CD62L-). However, in this effector OT-I pool CLL mice showed an increased frequency of KLRG1+CD127- short-lived effector cells (SLEC), while KLRG1-CD127+ memory precursor effector cells (MPEC) were decreased compared to control mice. This was associated with an enhanced expression of the effector-associated transcription factor T-bet, and reduced expression of the memory-associated transcription factor Bcl-6 in total OT-I cells and within the OT-I SLEC and MPEC populations of CLL mice. Since poor responses to CAR T cell therapy are associated with effector phenotypes and higher rates of glycolysis (Fraietta et al., 2018), and Bcl-6 directly represses glycolysis (Oestreich et al., 2014), we analyzed glucose uptake by OT-I cells. In line with reduced expression of Bcl-6, we found increased levels of glucose uptake in CLL derived OT-I cells. Despite the skewing towards a more short-lived effector phenotype of the CLL derived OT-I cells, in vitro re-stimulation with OVA peptide resulted in decreased production of IFN-γ and decreased degranulation as measured by CD107a (LAMP-1) expression compared to control derived OT-I cells.Conclusion - Our findings show that in this mouse model CLL development skews the T cell compartment towards more antigen-experienced cells. In addition to this, our results suggest that there is a CLL-antigen independent effect on acute antigen-specific immune responses. This CLL mediated effect drives T cells towards an effector phenotype, that is functionally impaired. This study provides clues for better understanding T cell responses in CLL, and may lead to new strategies to improve T cell mediated therapies in CLL, which are currently being exploited. DisclosuresEldering:Celgene: Research Funding. Kater:Acerta: Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche/Genentech: Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Research Funding.

Increased Age-Related B-Cells in Patients with Aplastic Anemia

Increased Age-Related B-cells in Patients with Aplastic AnemiaIntroduction:Aplastic anemia is a rare disease characterized by immune dysregulation. T cells in aplastic anemia are characterized by various intrinsic defects leading to increased IFN-g levels and Fas-mediated apoptosis of hematopoietic stem cells. We and others, have previously shown that the transcription factor T-bet is over-expressed in T cells from patients with aplastic anemia. Recently it was shown that a subpopulation of B cells also express T-bet; these cells are characterized as age-related B cells (ABCs) and express high levels of CD11c and CD19, they are CD21 negative and express T-bet. These T-bet+ ABCs are found increased in patients with autoimmune diseases (i.e. systemic lupus erythematosus, rheumatoid arthritis, and multiple sclerosis). Stimulation of B cells with antigens, Toll-like receptors and IFN-g leads to the formation of ABCs, which in turn “talk” to the T cells and stimulate them. Stimulation of T cells leads to IFN-g production, and this IFN-g may lead to further induction of T-bet expressing B cells (ABCs).Specific aim: In this study we wanted to examine the expression of ABCs in patients with aplastic anemia. We isolated peripheral blood mononuclear cells (PBMCs) from patients with aplastic anemia (n=10) and eight healthy, age- matched controls. Written informed consent was obtained from all study subjects. Cells were stained with the surface markers CD11c, CD19 and CD21, and subsequently analyzed using flow cytometry. An anti-Tbet antibody was also used after cell permeabilization. The CD11c-high, CD19 positive, CD21 negative, T-bet positive population represent the ABCs.Results: Patients with aplastic anemia at presentation showed increased numbers of circulating ABCs compared to healthy controls (29,08±1,62% vs 4,06 ±0,4 % respectively, p<0.001).(Fig. 1). Patients who responded to treatment showed comparable levels of ABCs to those of healthy individuals (5,93±0,6% vs 4,06 ±0,4 % respectively). Non-responders had statistically significant increased levels of ABCs compared to healthy controls (p<0,001). Also, non-responders had ABCs levels similar to those of aplastic anemia patients at presentation (29,08±1,62% vs 31,7 ±2,97 % respectively, p=0,44).Conclusion: Although the number of patients analyzed is limited, our preliminary results suggest that aplastic anemia patients show expanded ABCs compared to age-matched control subjects, and these numbers are related to disease status. Further analysis of a larger pool of subjects is underway along with the examination of the specific transcription factor Bcl-6, that is implicated in T-bet expression in ABCs. These results will reveal the role of ABCs in the immune pathogenesis of aplastic anemia. DisclosuresKattamis:CELGENE: Consultancy, Honoraria; Vifor Pharma: Consultancy; Novartis: Consultancy, Honoraria; ApoPharma: Honoraria.

Role of TRAFs in Signaling Pathways Controlling T Follicular Helper Cell Differentiation and T Cell-Dependent Antibody Responses.

Follicular helper T (TFH) cells represent a highly specialized CD4+ T cell subpopulation that supports the generation of germinal centers (GC) and provides B cells with critical signals promoting antibody class switching, generation of high affinity antibodies, and memory formation. TFH cells are characterized by the expression of the chemokine receptor CXCR5, the transcription factor Bcl-6, costimulatory molecules ICOS, and PD-1, and the production of cytokine IL-21. The acquisition of a TFH phenotype is a complex and multistep process that involves signals received through engagement of the TCR along with a multitude of costimulatory molecules and cytokines receptors. Members of the Tumor necrosis factor Receptor Associated Factors (TRAF) represent one of the major classes of signaling mediators involved in the differentiation and functions of TFH cells. TRAF molecules are the canonical adaptor molecules that physically interact with members of the Tumor Necrosis Factor Receptor Superfamily (TNFRSF) and actively modulate their downstream signaling cascades through their adaptor function and/or E3 ubiquitin ligase activity. OX-40, GITR, and 4-1BB are the TRAF-dependent TNFRSF members that have been implicated in the differentiation and functions of TFH cells. On the other hand, emerging data demonstrate that TRAF proteins also participate in signaling from the TCR and CD28, which deliver critical signals leading to the differentiation of TFH cells. More intriguingly, we recently showed that the cytoplasmic tail of ICOS contains a conserved TANK-binding kinase 1 (TBK1)-binding motif that is shared with TBK1-binding TRAF proteins. The presence of this TRAF-mimicking signaling module downstream of ICOS is required to mediate the maturation step during TFH differentiation. In addition, JAK-STAT pathways emanating from IL-2, IL-6, IL-21, and IL-27 cytokine receptors affect TFH development, and crosstalk between TRAF-mediated pathways and the JAK-STAT pathways can contribute to generate integrated signals required to drive and sustain TFH differentiation. In this review, we will introduce the molecular interactions and the major signaling pathways controlling the differentiation of TFH cells. In each case, we will highlight the contributions of TRAF proteins to these signaling pathways. Finally, we will discuss the role of individual TRAF proteins in the regulation of T cell-dependent humoral responses.

A Rare Case of Plasmablastic Lymphoma with Gastric Involvement

Plasmablastic lymphoma (PBL) is a relatively rare, but highly aggressive lymphoma commonly found in the setting of HIV disease.PBL is defined by the World Health Organization as a “diffuse proliferation of large neoplastic cells most of which resemble B-cell immunoblasts, but in which all tumor cells have a plasma cell immunophenotype.”Here we present a unique case of Ann Arbor Stage IV PBL with extranodal gastric involvement diagnosed in an immunocompetent male. An 80-year-old Hispanic male presented to the emergency department with nausea and epigastric pain for four days duration. The patient also endorsed progressive dysphagia to solids and liquids over the past several weeks prior to presentation. Significant findings on physical exam included a pale, well-nourished male with mild epigastric tenderness on light palpation. CT scan revealed diffuse thickening of the gastric wall with a 7.4cm x 6.5cm intraluminal infiltrating mass with luminal narrowing. An EGD was subsequently performed for better visualization and biopsy (Figure 1). Histopathologic studies ultimately identified the mass as malignant plasmablastic lymphoma with aberrant cytokeratin expression. HIV testing was negative but EBV IgG antibody testing was abnormally high. Immunophenotype analysis revealed that CD-138 was strongly positive, CD-20 was negative, CD-45 was weakly positive, and Ki-67 expression was greater than 90% (Figure 2). The patient was started on CHOP chemotherapy regimen.2659_A Figure 1. Large, 5cm, semi-circumferential, easily friable and fungating mass as observed from GE junction during EGD2659_B Figure 2. Histology and Immunophenotype Analysis. (A) High-power magnification showing atypical plasma cells. (B) CD-20 negative. (C) CD-138 positive. (D) Ki-67 Index > 90%.Recent meta-analysis has shown that the median age of diagnosis for HIV-negative PBL is 55 years. Although the pathogenesis of PBL remains incompletely understood, the association with EBV infection and the virus' role in prevention of apoptosis of B cells is well documented.EBV positivity and CD45 expression represent good prognostic indicators for this patient; on the other hand, Ann Arbor Stage IV classification and Ki-67 expression > 80% are typically associated with poor outcome. After the oral mucosa, the GI tract is the most common site of involvement for HIV-negative PBL cases. This case represents a rare phenotypic variant in which the BCL-6 transcription factor is positive, suggesting that perhaps this case is a hybrid of diffuse large B-cell lymphoma (DLBCL) and PBL. Despite the recent advances in the treatment for PBL, prognosis remains poor with the median overall survival of nine months and a 2-year overall survival rate of 10% for HIV-negative PBL.

Transient T-bet expression functionally specifies a distinct T follicular helper subset.

T follicular helper (Tfh) cells express transcription factor BCL-6 and cytokine IL-21. Mature Tfh cells are also capable of producing IFN-γ without expressing the Th1 transcription factor T-bet. Whether this IFN-γ-producing Tfh population represents a unique Tfh subset with a distinct differentiation pathway is poorly understood. By using T-bet fate-mapping mouse strains, we discovered that almost all the IFN-γ-producing Tfh cells have previously expressed T-bet and express high levels of NKG2D. DNase I hypersensitivity analysis indicated that the Ifng gene locus is partially accessible in this "ex-T-bet" population with a history of T-bet expression. Furthermore, multicolor tissue imaging revealed that the ex-T-bet Tfh cells found in germinal centers express IFN-γ in situ. Finally, we found that IFN-γ-expressing Tfh cells are absent in T-bet-deficient mice, but fully present in mice with T-bet deletion at late stages of T cell differentiation. Together, our findings demonstrate that transient expression of T-bet epigenetically imprints the Ifng locus for cytokine production in this Th1-like Tfh cell subset.

Human Extrafollicular CD4+ Th Cells Help Memory B Cells Produce Igs.

Follicular helper T (Tfh) cells are necessary for germinal center B cell maturation during primary immune responses; however, the T cells that promote humoral recall responses via memory B cells are less well defined. In this article, we characterize a human tonsillar CD4+ T cell subset with this function. These cells are similar to Tfh cells in terms of expression of the chemokine receptor CXCR5 and the inhibitory receptor PD-1, IL-21 secretion, and expression of the transcription factor BCL6; however, unlike Tfh cells that are located within the B cell follicle and germinal center, they reside at the border of the T cell zone and the B cell follicle in proximity to memory B cells, a position dictated by their unique chemokine receptor expression. They promote memory B cells to produce Abs via CD40L, IL-10, and IL-21. Our results reveal a unique extrafollicular CD4+ T cell subset in human tonsils, which specialize in promoting T cell-dependent humoral recall responses.

Abstract 3354: Chemically induced degradation of the transcription factor BCL6

Abstract The transcription factor BCL6 is a known driver of oncogenesis in lymphoid malignancies, including diffuse large B-cell lymphoma (DLBCL). Disruption of its interaction with transcriptional repressors interferes with the oncogenic effects of BCL6. We have used structure-based drug design to develop highly potent compounds that block this interaction. A subset of these inhibitors also cause rapid ubiquitylation and degradation of BCL6 in cells. These compounds display significantly stronger induction of expression of BCL6-repressed genes and antiproliferative effects than compounds that merely inhibited co-repressor interaction. The fact that the magnitude of effects elicited by this class of BCL6 degrading compounds greatly exceeds that of our equipotent non-degrading inhibitors offers exciting opportunities for the development of BCL6-based lymphoma therapeutics. To support further research, the most potent BCL6 degrading inhibitor is made freely available to the research community as an in vitro tool compound. Please see http://www.opnMe.com for further infos. Citation Format: Nina Kerres, Steffen Steurer, Stefanie Schlager, Gerd Bader, Maureen Caligiuri, Christian Dank, John R. Engen, Peter Ettmayer, Daniel Gerlach, Thomas Gerstberger, Bingsong Han, Roxana E. Iacob, Dirk Kessler, David R. Lancia, Mayer Moriz, Nikolai Mischerikow, Klaus Rumpel, Renate Schnitzer, Tilman Voss, Xiaozhang Zheng, Andreas Zoephel, Norbert Kraut, Darryl McConnell, Mark Pearson, Manfred Koegl. Chemically induced degradation of the transcription factor BCL6 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3354.

CD8 Follicular T Cells Promote B Cell Antibody Class Switch in Autoimmune Disease.

CD8 T cells can play both a protective and pathogenic role in inflammation and autoimmune development. Recent studies have highlighted the ability of CD8 T cells to function as T follicular helper (Tfh) cells in the germinal center in the context of infection. However, whether this phenomenon occurs in autoimmunity and contributes to autoimmune pathogenesis is largely unexplored. In this study, we show that CD8 T cells acquire a CD4 Tfh profile in the absence of functional regulatory T cells in both the IL-2-deficient and scurfy mouse models. Depletion of CD8 T cells mitigates autoimmune pathogenesis in IL-2-deficient mice. CD8 T cells express the B cell follicle-localizing chemokine receptor CXCR5, a principal Tfh transcription factor Bcl6, and the Tfh effector cytokine IL-21. CD8 T cells localize to the B cell follicle, express B cell costimulatory proteins, and promote B cell differentiation and Ab isotype class switching. These data reveal a novel contribution of autoreactive CD8 T cells to autoimmune disease, in part, through CD4 follicular-like differentiation and functionality.