Germinal Center Phenotype Research Articles

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.

Burkitt-like lymphoma with 11q aberration: a germinal center-derived lymphoma genetically unrelated to Burkitt lymphoma.

Burkitt-like lymphoma with 11q aberration is characterized by pathological features and gene expression profile resembling those of Burkitt lymphoma but lacks the MYC rearrangement and carries an 11q-arm aberration with proximal gains and telomeric losses. Whether this lymphoma is a distinct category or a particular variant of other recognized entities is controversial. To improve the understanding of Burkitt-like lymphoma with 11q aberration we performed an analysis of copy number alterations and targeted sequencing of a large panel of B-cell lymphoma-related genes in 11 cases. Most patients had localized nodal disease and a favorable outcome after therapy. Histologically, they were high grade B-cell lymphoma, not otherwise specified (8 cases), diffuse large B-cell lymphoma (2 cases) and only one was considered as atypical Burkitt lymphoma. All cases had a germinal center B-cell signature and phenotype with frequent LMO2 expression. The patients with Burkitt-like lymphoma with 11q aberration had frequent gains of 12q12-q21.1 and losses of 6q12.1-q21, and lacked common Burkitt lymphoma or diffuse large B-cell lymphoma alterations. Potential driver mutations were found in 27 genes, particularly involving BTG2, DDX3X, ETS1, EP300, and GNA13. However, ID3, TCF3, or CCND3 mutations were absent in all cases. These results suggest that Burkitt-like lymphoma with 11q aberration is a germinal center-derived lymphoma closer to high-grade B-cell lymphoma or diffuse large B-cell lymphoma than to Burkitt lymphoma.

Low Relapse Rate after 2 Years in Patients with Diffuse Large B-Cell Lymphoma (DLBCL) Treated with R-CHOP and Achieved Complete Response

Introduction: Patients with DLBCL usually relapse early after the first treatment but some relapse after 2 years (defined as late relapses). In a previous study, patients with DLBCL who achieved event free survival at 24 months had a subsequent overall survival equivalent to the general population. We analyzed the time of relapse and clinical characteristics of late relapse in patients with DLBCL who achieved complete response (CR) after frontline R-CHOP.Methods: We retrospectively reviewed prospectively collected lymphoma registry of the Asan Medical Center in Seoul, Korea, between 2002 and 2015. We found 1,047 DLBCL patients who have been treated with R-CHOP as the first line modality. Among 1,047 patients, 859 (82.0%) patients achieved CR. Chi-square or Fisher's exact test was used for comparison of categorical variables and Student's t-test or Mann-Whitney U-test for continuous variables, as appropriate. Two-sided p-value of <0.05 was considered significant.Results: Among 859 patients, 182 (21.2%) patients relapsed and 51 (5.9%) patients had late relapse after 2 years from the end of treatment evaluation that confirmed CR. For patients who had a late relapse, median time from diagnosis to relapse was 4.0 years (range, 2.0 to 10.0 years), median age was 61 years old (range, 39-78), and 27 (52.9%) patients were male. Twenty-six (51%) patients had stage I-II, and 36 (70.6%) patients had low/low-intermediate International Prognostic Index (IPI) score at diagnosis. Compared with early relapses (n=131, 12.5%), late relapses were associated with low IPI score (median, 2 vs. 3; p = 0.001), low LDH (median, 244 vs. 337 IU/L; p < 0.001), low beta-2-microglobulin (median, 2.4 vs. 2.6 mg/L p = 0.002), higher germinal center phenotype rate (30.6% vs. 15%; p=0.05). Median overall survival from the end of treatment evaluation that confirmed CR was significantly higher in the late relapse group compared with early relapse group [112.6 months (95% CI 73.1-152.0) vs. 19.9 months (95% CI 15.3-24.4); p<0.001 by log rank].Conclusion: Our study demonstrated that relapse rate after 2 years was low (5.9%) in patients with DLBCL who achieved CR after R-CHOP. Patient with late relapse showed favorable clinical characteristics and survival outcomes compared with patients with early relapse. DisclosuresNo relevant conflicts of interest to declare.

A Rare and Aggressive Lymphoma; Burkitt Lymphoma in the Terminal Ileum

Burkitt Lymphoma (BL) is an aggressive form of high-grade B-cell neoplasm that is rarely identified in the adult population. The World Health Organization (WHO) describes three clinical variants; endemic, sporadic, and immunodeficiency associated.The following case is a rare presentation of Primary small intestine Burkitt lymphoma in an adult male presenting with fatigue, dyspnea on exertion, and unexplained weight loss. We present a case of an 81-year-old male with a past medical history of stage I Hodgkin's lymphoma treated with chemotherapy(may be state chemo regime) approximately 8 years ago, non-insulin-dependent diabetes, hypertension, hyperlipidemia, chronic kidney disease who presented to our a tertiary center with the complaints of increasing weakness, fatigue, and unexplained weight loss. In the emergency department the patient was found to have acute kidney injury and hypercalcemia 14.1mg/dL (Ca 8.4 - 10.3 mg/dL). Due to concern for malignancy with hypercalcemia and the patient's history of Hodgkin's lymphoma a computed tomography (CT) chest, abdomen and pelvis was obtained. Imaging revealed concerning findings of peritoneal carcinomatosis with gastrointestinal malignancy as possible source. Gastroenterology was consulted and an EGD as well as colonoscopy were performed.Colonoscopy revealed diverticulosis in the sigmoid colon and in the descending colon, a malignant-appearing tumor in the distal ileum that was biopsied. Pathology revealed an aggressive B-cell lymphoma with Burkitt like morphology and a germinal center phenotype. The infiltrate was positive for CD 20, CD 10, BCL 2, BCL 6, and MUM1. The Ki 67 proliferation rate was greater than 90%. Overall, the morphology and high proliferation rate were consistent with Burkitt lymphoma. The patient chose to follow up at an alternative facility for his ongoing oncology care. Burkitt lymphoma is a very aggressive form of B-cell lymphoma the sporadic form occurring worldwide with a predilection for children. Clinical symptoms depend on the site of involvement. There is frequently extranodal involvement commonly occurring in the gastrointestinal tract. CT findings can include peritoneal thickening, as well as ascites, and omental involvement. Often times when there is diffuse peritoneal disease, it is usually associated with a focal gastrointestinal mass. Although rare, Burkitt lymphoma should be kept on the differential when investigating CT findings of what appears to be peritoneal carcinomatosis.2530_A Figure 1. peritoneal carcinomatosis2530_B Figure 2. Malignant appearing tumor in the distal ileum

Lymphomas with pseudo–double-hit BCL6-MYC translocations due to t(3;8)(q27;q24) are associated with a germinal center immunophenotype, extranodal involvement, and frequent BCL2 translocations

High-grade B-cell lymphomas with MYC, BCL2, and/or BCL6 rearrangements, "double-hit" or "triple-hit" lymphomas (DTHL), are aggressive neoplasms associated with a poor prognosis. A t(3;8)(q27;q24) rarely occurs in B-cell lymphomas that results in a unique "pseudo-double-hit" BCL6-MYC fusion, indistinguishable by interphase fluorescence in situ hybridization (FISH) from more conventional DTHL with independent MYC and BCL6 translocations. Reports of t(3;8)(q27;q24) lymphomas are sparse, and to better characterize their pathologic, cytogenetic, and clinical features, 6 new cases from 2 institutions and 19 previously published cases were reviewed. All new cases displayed aggressive morphologic features, and most previously published cases were classified as aggressive lymphomas. Collectively, all t(3;8)(q27;q24) cases had a germinal center (GC) phenotype, and most had complex karyotypes (22/24, 92%), including frequent concomitant BCL2 rearrangements (17/24, 71%). When compared to two large published DTHL cohorts, t(3;8)(q27;q24) lymphomas less often expressed BCL2 (P < .01), had a greater likelihood of extranodal involvement (P < .01), and more frequently appeared triple-hit by FISH analysis (P < .01). Despite presenting with aggressive clinicopathologic features, 100% (6/6) of t(3;8;)(q27;q24) patients achieved complete remission after intensive induction regimens, and 2- and 3-year overall survival rates were 63% (10/16) and 57% (8/14), respectively. These findings suggest that lymphomas with t(3;8)(q27;q24) may represent a subset of GC B-cell lymphomas distinct from conventional DTHL. Our results further highlight the value of routine karyotype assessment in aggressive B-cell lymphomas, and the importance of recognizing the t(3;8)(q27;q24) so that its clinical significance can be more fully explored.

Abstract 30: SOX9 enhancer regulator may play an oncogenic role in B-cell lymphomas

Abstract A majority of B-cell lymphomas, including diffuse large B-cell lymphomas (DLBCLs) and follicular lymphomas (FLs), arise from germinal center (GC) B cells. GC B cells are highly proliferative and manifest genomic instability as a byproduct of immunoglobulin affinity maturation. These characteristics make GC B cells particularly prone to malignant transformation such that DLBCLs and FLs inherit and are dependent on mutations in proteins that are also required for normal GC B cells. For example, the BCL6 transcriptional repressor is required to establish the GC phenotype in both normal B-cell development and GC B-cell malignancy through its effect on silencing expression of plasma cell differentiation and checkpoint genes. GC B cells feature a unique transcriptional program featuring upregulation of genes involved in cell proliferation and other growth pathways. Cell context-specific gene expression is largely mediated through gene enhancers. Hence we hypothesized that transcription factors that activate GC specific gene enhancers would play a critical role in formation of these cells, and would likely be required in turn to maintain the survival of GC-derived lymphoma cells. To address this question we first mapped gene enhancers in primary human resting B cells and purified GC B cells by performing ChIP-seq to map the distribution of H3K27Acetyl, H3K4me1, and H3K4me3. This procedure allowed us to identify several thousand GC-specific enhancers. We next examined these enhancers using bioinformatic approaches to identify transcription factor binding sites and identified a list of fifteen putative GC enhancer regulators. Of these potential GC enhancer regulators, only SOX9 was highly induced in GC B cells as compared to resting B cells, pointing to SOX9 as a candidate master enhancer regulator of the GC transcriptome with potential relevance to lymphomas. Indeed, in addition to the well-known role of SOX9 as a stem cell self-renewal and cell-differentiation regulator during development, SOX9 has also been described as playing a role in colon, breast, and prostate carcinogenesis through inhibition of apoptosis, and promoting proliferation, invasion, and metastasis. We confirmed that SOX9 is highly induced in GC cells using QPCR and Western blots. To determine whether SOX9 would indeed bind to these enhancer sites, we next performed SOX9 ChIP-seq in GC B-cell derived lymphoma cell lines. These experiments validated and showed that SOX9 binds to 1,668 upstream distal enhancer regions (-5 to -100 kb upstream from TSS) associated with 963 genes. These target genes were significantly enriched in cell cycle regulation (CCND2, CDC25B, CDK1), transcription regulation (BCOR, NCOR2), epigenetic regulation (BMI1, DNMT3A, MLL2, SUZ12, TET3), and MAPK signaling (MAP2K3, MAP3K7) and also for B-cell activation and BCL6 repressed pathways (p&amp;lt;0.001). We next wished to determine whether SOX9 was also expressed in GC-derived lymphoma clinical samples. Therefore we next examined RNA-seq gene expression profiles derived from cohorts of FL and DLBCL patients. We found that SOX9 is expressed at higher levels in FL patient samples, but lower levels in most DLBCLs compared to naïve B cells. These data raise the possibility that SOX9 might be differentially relevant to FL more than DLBCLs, perhaps contributing to the biologic distinction between these two GC-derived lymphomas. To determine if SOX9 protein was also expressed in these tumors, we examined protein expression by immunohistochemistry using tissue microarrays (TMAs) containing both FLs and DLBCLs. We found that 88 of the 242 FL TMA samples (36.4%) were positive for SOX9 staining. Eleven of the 114 DLBCL TMA samples (10%) were positive for SOX9 staining, among which 80% (9/11) were GC-type DLBCL and 45% (5/11) had a prior FL history. To determine if any of the currently available GC derived lymphoma cell lines manifest high SOX9 levels, we performed a series of Western blot experiments. These studies show high levels of SOX9 protein in the GC-type DLBCL cell lines HT, Karpas422, DB, and Farage, but not in any of the eight ABC-DLBCLs subtypes. Ongoing studies are assessing the biologic relevance of SOX9 for the GC reaction and possible contribution to lymphomagenesis using gain- and loss-of-function studies in human cells and mouse models. These data will determine whether this apparently key enhancer regulatory protein is a novel lineage factor for follicular lymphoma and potentially suitable as a therapeutic target. Citation Format: Angela A. Fachel, Yanwen Jiang, Wayne Tam, Ashlesha S. Muley, Cem Meydan, Xabier Agirre, Ari M. Melnick, Kristy L. Richards. SOX9 enhancer regulator may play an oncogenic role in B-cell lymphomas [abstract]. In: Proceedings of the Second AACR Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; May 6-9, 2017; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(24_Suppl):Abstract nr 30.

Mutational profile of primary breast diffuse large B-cell lymphoma.

Primary breast lymphoma is a rare form of extra-nodal lymphoid neoplasm. The most common histological type is the diffuse large B-cell lymphoma, which represents 60–80% of all the cases. Our study analyzes the mutational profile of the primary lymphoma of the breast through targeted massive sequencing with a panel of 38 genes in a group of 17 patients with primary breast diffuse large B-cell lymphoma. Seventy-point-five percent of the patients presented with stage IE and 29.5% with stage IIE. 44% of the cases correspond to lymphomas with germinal center phenotype and 33.3% to activated B-cell. The genes with a higher mutational frequency include PIM1 (in 50% of the analyzed samples), MYD88 (39%), CD79B, PRDM1 and CARD11 (17%), KMT2D, TNFIAP3 and CREBBP (11%). The profile of mutant genes involves mostly the NFκB signaling pathway. The high frequency of mutations in PIM1 compared with other lymphomas may have implications in the clinical presentation and evolution of this type of lymphoma.

Phenotyping of autoreactive B cells with labeled nucleosomes in 56R transgenic mice

The phenotypic characterization of self-reactive B cells producing autoantibodies is one of the challenges to get further insight in the physiopathology of autoimmune diseases. We took advantage of our previously developed flow cytometry method, using labeled nucleosomes, prominent autoantigens in systemic lupus erythematosus, to analyze the phenotype of self-reactive B cells in the anti-DNA B6.56R mouse model. We showed that splenic anti-nucleosome B cells express mostly kappa light chains and harbor a marginal zone phenotype. Moreover, these autoreactive B cells fail to acquire a germinal center phenotype and are less abundant in the transitional T3 compartment. In conclusion, the direct detection of autoreactive B cells helped determine their phenotypic characteristics and provided a more direct insight into the B cell tolerance process in B6.56R mice. This method constitutes an interesting new tool to study the mechanisms of B cell tolerance breakdown in B6.56R mice crossed with autoimmune prone models.

Toll-like receptor 9 suppresses lupus disease in Fas-sufficient MRL Mice.

Genetic deficiency in TLR9 accelerates pathogenesis in the spontaneous polygenic MRL.Faslpr murine model of systemic lupus erythematosus, despite the absence of anti-nucleosome autoantibodies. However, it could be argued that this result was dependent on Fas-deficiency rather than lupus-promoting genes in the MRL genetic background. Here we report the effects of TLR9 deficiency on autoimmune disease independent of the lpr mutation in Fas by characterizing Tlr9-/- and Tlr9+/+ mice on the Fas-intact MRL/+ genetic background. By 30 weeks of age, Tlr9-deficient MRL/+ had more severe renal disease, increased T cell activation, and higher titers of anti-Sm and anti-RNA autoantibodies than Tlr9-intact animals, as had been the case in the MRL.Faslpr model. In addition, Tlr9-deficient MRL/+ mice had increased numbers of germinal center phenotype B cells and an increase in splenic neutrophils and conventional dendritic cell populations. Thus, the disease accelerating effects of Tlr9 deficiency are separable from those mediated by the Fas mutation in the lupus-prone MRL genetic background. Nonetheless, disease acceleration in Tlr9-deficient MRL/+ mice was phenotypically distinct from that in Fas-deficient counterparts, which has important implications.

Differing Requirements for MALT1 Function in Peripheral B Cell Survival and Differentiation.

During a T cell-dependent immune response, formation of the germinal center (GC) is essential for the generation of high-affinity plasma cells and memory B cells. The canonical NF-κB pathway has been implicated in the initiation of GC reaction, and defects in this pathway have been linked to immune deficiencies. The paracaspase MALT1 plays an important role in regulating NF-κB activation upon triggering of Ag receptors. Although previous studies have reported that MALT1 deficiency abrogates the GC response, the relative contribution of B cells and T cells to the defective phenotype remains unclear. We used chimeric mouse models to demonstrate that MALT1 function is required in B cells for GC formation. This role is restricted to BCR signaling where MALT1 is critical for B cell proliferation and survival. Moreover, the proapoptotic signal transmitted in the absence of MALT1 is dominant to the prosurvival effects of T cell-derived stimuli. In addition to GC B cell differentiation, MALT1 is required for plasma cell differentiation, but not mitogenic responses. Lastly, we show that ectopic expression of Bcl-2 can partially rescue the GC phenotype in MALT1-deficient animals by prolonging the lifespan of BCR-activated B cells, but plasma cell differentiation and Ab production remain defective. Thus, our data uncover previously unappreciated aspects of MALT1 function in B cells and highlight its importance in humoral immunity.

Modular Immune Organoids with Integrin Ligand Specificity Differentially Regulate Ex Vivo B Cell Activation.

Germinal centers are dynamic structures within lymphoid tissues, which develop once B cells receive activating signals from surrounding immune cells. Germinal center B cells are small in number, heterogeneous, and prone to rapid apoptosis unless selected by the body to form memory B cells. Despite extensive research in the B cell differentiation process, the role of the lymphoid niche, in particular integrin ligands, in the development of early germinal center-like phenotype remains unclear. Here, we report a biomaterials-based modular immune organoid that enables development of early germinal-center phenotype in an integrin ligand-specific manner. We demonstrate the differential role of integrin α4β1- and αvβ3-binding ligands in the induction of GL7+ (GC-like) and GL7- (non-GC-like) phenotype in differentiating B cells while in the presence of CD40 ligand and interleukin-4. We further demonstrate the role of integrin ligand specificities in clustering of β3 integrin and B cell receptor on the surface of differentiated B cells in 3D organoids as compared to the classic 2D cocultures. The study demonstrates that biomaterials-based immune organoids represent an ex vivo platform technology, which recapitulates certain aspects of GC biology to understand the process of B cell differentiation and induction of immunological responses. This platform is particularly useful in understanding the role of selective biomolecular signals and the temporal dependency of immune responses to these signals.

Novel Near-Infrared Heptamethine Carbocyanine Fluorescent Dye-Cisplatin Conjugate Demonstrates Significant Antitumor Activity and Overcomes Cisplatin Resistance in MYC-Driven TP53 Mutated Aggressive B-Cell Burkitt's Lymphoma

Introduction: Burkitt’s lymphoma (BL) is is composed of highly proliferating mature B cells expressing a germinal center phenotype (Cai, Oncotarget 2015). It is one of the fastest growing human tumors and presents in extranodal as well as nodal sites. The genetic hallmark of BL is the MYC translocation with MYC and TP53 mutations in ∼60% and 40% of the cases, respectively (Ott, Blood 2014). While prompt administration of multiagent immunochemotherapy regimens is associated with favorable outcomes for the majority of patients, prognosis of refractory or relapsed disease is poor (Jacobson, Blood 2014). We previously identified a novel near-infrared heptamethine carbocyanine fluorescent dye (NIR-dye) that has tumor homing property through cell surface transporters, organic anion transporting peptides (OATPs), and shows preferential uptake in tumor but not normal cells as demonstrated in cell culture, mouse and dog tumor models, patient-derived xenografts (PDXs), and perfused kidney cancers in patients (Wu, Biomaterials 2014). Here, we describe the effects of a newly synthesized NIR-dye-cisplatin conjugate (NIR-dye-Cis) on MYC-driven TP53 mutated aggressive B-cell BL cell lines in culture and tumors in mice.Methods:The growth and viability of immortalized B-cell lymphoma cell lines, Namalwa, Raji, CA46, Ramos, Daudi, were evaluated by MTT upon 72 h exposure to conventional cisplatin (Cis) and NIR-dye-Cis. Induction of apoptosis was assesed using a caspase 3/7 luminescent assay. Cleavage of apoptosis substrates and inhibition of c-myc was determined by western blotting, with images quantified by ImageJ after normalization with GAPDH. NIR-dye-Cis uptake in cells and tumors were imaged by NIRF microscopy and IVIS Lumina XR Imaging System, respectively. Male NCr nude and NOD/SCID mice bearing s.c. Namalwa tumors were injected i.p. with equimolar doses of NIR-dye-Cis and conventional Cis 3 times a week for 4 weeks. Formalin-fixed tumor and kidney sections were stained with hematoxylin and esosin and kidney sections scored using a semiquantitative scale designed to assess AKI-associated tubular injury (tubular epithelial cell loss, necrosis, tubular epithelial simplification, intratubular debris, and casts).Results: 1) All BL cell lines tested are sensitive to growth inhibition by NIR-dye-Cis but not Cis with IC50 ranging between 720 nM - 2.53 µM and >64 µM (p<0.0001), respectively. 2) NIR-dye-Cis induces apoptosis after 24 hours of drug exposure as assessed by luminescent assay of caspase 3/7 and western blot analyses of apoptosis related markers (caspase 9, caspase 3 and PARP) in extracts of Namalwa and Raji cell lines treated with 8, 16, 32 µM of concentrations of NIR-dye-Cis with the levels of significance at p=0.0001 to p=0.01; no sign of increased apoptotic activity was observed when these cells were exposed to the conventional Cis. 3) NIR-dye-Cis, but not Cis, downregulates the anti-apoptotic protein c-myc, which is highly expressed in BL cell lines; no significant changes of minimally expressed bcl-2, an antiapoptotic protein, was noted in NIR-dye-Cis and Cis treated BL cell lines. 4) NIR-dye-Cis was found to accumulate 7 to 15 times more in cell line-derived xenografts than mouse kidneys (p<0.0001) and lungs (p<0.0001), and these results were confirmed by NIRF microscopy and IVIS Lumina XR Imaging. 5) NIR-dye-Cis when injected in mice bearing Namalwa cell line-derived xenografts, shows tumor growth inhibiton and apoptotic nuclear lobulation/fragmentation when compared to vehicle- or Cis-treated group (p<0.05). 6) Whereas NIR-dye-Cis preserved the integrity and normal morphology of mouse kidneys, by contrast, Cis was observed to induce marked kidney damage as assessed by the histopathology of Cis-treated mice.Conclusion: Aggressive B-cell BL can be treated safely by NIR-dye-Cis. This agent was found to accumulate in BL cells and tumor xenografts and in contrast to Cis, NIR-dye-Cis induced apoptosis and shrinked BL tumor xenografts in mice. NIR-dye-Cis induced apoptosis, downregulated c-myc and overcame Cis-resistance in MYC-driven TP53 mutated aggressive B-cell BL. This agent could have potential clinical benefit in relapsed/refractory heavily pretreated patients for improved efficacy and reduced kidney toxicity. We proposed NIR-dye-Cis could be employed as the secondary agent for the treatment of relapsed/refractory agressive B-cell non Hodgkin lymphomas. DisclosuresNo relevant conflicts of interest to declare.

Loss of TOSO Promotes Richter's Transformation of TCL1A Driven CLL

Background: The immunoglobulin-like protein TOSO, which has been found to serve as Fc receptor for IgM (FcµR), was shown by us and others to be overexpressed on CLL cells and only weakly expressed on more aggressive B-NHL. However the functional role of TOSO on lymphomagenesis has not been explored so far.Methods: To determine the role of TOSO on lymphoma development, we took advantage of the Eµ-TCL1 transgenic mice, which usually end up with an aggressive (IgVH unmutated) CLL-like phenotype. We generated a novel B cell-specific conditional knockout (KO) mouse model in which EµTCL1 mice (TC or control in the following) were crossbred with TOSO-floxed mice, expressing Cre recombinase under the control of the CD19 promoter (EµTCL1;Tosofl/fl;Cd19cre/wt or TCT in the following). TCT mice were further compared with p53 conditional knockout (EµTCL1;Tp53fl/fl;Cd19cre/wt or TCP).Results: In this study, we compared kinetics, overall survival and phenotype of lymphoma/CLL in TC, TCT and TCP mice. Interestingly, TCTmice developed a very aggressive phenotype and resulted in significantly shorter overall survival compared to TC mice (TCT 274 days vs. TC 346 days; p<0.0001). As expected, mice lacking p53 (TCP) died even more rapidly than TCT mice (median survival: TCP 233 days). Initially, all three genotypes (TC, TCT, TCP) developed a CLL phenotype, exhibiting a CD19 and CD5 positive malignant clone. In the TCT mice, shorter overall survival is accompanied by a stronger increase of blood leukocytes. Flow cytometry analysis confirmed a strong increase of leukemic CD19/CD5-positive B cells in the blood of TCT mice. With only 20 weeks of age, leukemic cells already made up 37.5 % (SD ± 15.47; n=14) of lymphocytes (TC: 14.3 % SD ± 9.81; n=31). At the age of 36 weeks, TCT mice showed even a 3.6-fold elevated malignant cell count compared to control mice (n=35 TC, n=14 TCT; p=0.006). All TCT mice developed a splenomegaly, with spleen weight (p=0.01) and size (p=0.018) significantly increased in 36 week old TCT mice (n=7) compared to TC mice (n=7) and comparable to those from TCP mice. Interestingly, between week 28 and 36, we could observe that most of the TCT mice start losing CD19+ cells in the blood in contrast to TC and TCP mice. Immunohistochemistry revealed the expansion of malignant cells with pleomorphic nuclei and abundant cytoplasm in the spleen and bone marrow, as we know it from Richter‘s transformation. To understand the rapid development of leukemia in TCT mice, we first determined the role of the BCR in this model. Interestingly, flow cytometry revealed a higher surface IgM expression (MFI: TCT 9,27; TC 2,05). In addition, in vitro assays revealed a significantly higher resistance of TCT cells towards PI3K inhibition (Idelalisib and Duvelisib) compared to TC cells.To further rule out the role of TOSO under "germinal-center conditions", we stimulated primary human CLL cells with CD40L expressing feeder cells and IL-4. Interestingly, both stimuli (either alone or in combination) resulted in almost complete loss of TOSO on CLL cells. Moreover, we uncovered, that the TOSO promoter is counteractively regulated by NF-κB and BCL6. Furthermore, our data illustrate that DNA hypomethylation of the TOSO promoter is a discriminating characteristic in CLL patients compared to healthy donors, thus explaining the significantly enhanced expression levels. Thus, both, epigenetic regulation and altered NF-κB/ BCL6 expression are critical pathogenetic steps in the development of CLL and aggressive B-NHL by regulating TOSO expression.Conclusion: The transformation of CLL into more aggressive malignancies is still not fully understood. Our data reveal that the loss of TOSO might play a major role in Richter's transformation by upregulation of the BCR and by mimicking the germinal-center phenotype. DisclosuresFingerle-Rowson:Roche: Employment. Wendtner:Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Mundipharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Hoffmann-La Roche: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Servier: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Morphosys: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Hallek:GSK: Research Funding; Mundipharma: Research Funding; Janssen: Research Funding; Celgene: Research Funding; Gilead: Research Funding; AbbVie: Consultancy, Honoraria, Research Funding; Roche: Consultancy, Honoraria, Research Funding; Genentech: Consultancy, Honoraria.

Abstract LB-020: IP3 3-kinase B suppresses B-cell lymphoma by antagonizing PI3K/mTOR in B cells

Abstract Accounting for ∼4% of all cancers in the US, Non-Hodgkin Lymphoma (NHL) is the most prevalent blood cancer. Diffuse large B-cell Lymphoma (DLBCL) is the most common and aggressive type of NHL. Here, we identify inositol-trisphosphate (IP3) 3-kinase B (Itpkb) as a novel tumor suppressor whose deficiency triggers DLBCL in mice. We found that aging Itpkb-/- mice die prematurely with anemia and splenomegaly. 15% of these mice showed multiorgan-infiltration with neoplastic germinal-center phenotype B cells reminiscent of DLBCL. Itpkb-/- B cell lymphomas are mono-or oligoclonal, transplantable, and constitutively hyperactivate the oncogenic phosphoinositide 3-kinase (PI3K) signaling pathway. Growth of Itpkb-/- B cell lymphoma xenografts in immunodeficient mice is sensitive to treatment with PI3K or mTOR inhibitors. In humans, subsets of DLBCL patients carry Itpkb missense mutations, deletions or copy number reductions. Additionally, Itpkb expression is altered in several other hematopoietic and non-hematopoietic human cancers. We propose that Itpkb suppresses tumors by producing inositol-tetrakisphosphate (IP4), a soluble analog of the PI3K product phosphatidylinositol-trisphosphate (PIP3). We and others have previously shown that IP4 antagonism with PIP3 for Akt effector kinase binding prevents excessive Akt/mTORC1 signaling in hematopoietic cells. Our new results suggest that this mechanism prevents oncogenic PI3K signaling in the B cell lineage and possibly in other cell types. Thus, Itpkb might be a novel biomarker for tumor aggressiveness or prognosis, and Itpkb activating drugs or IP4 might have therapeutic potential. This work was supported by NIH grants AI070845 and GM100785 to KS, The Leukemia &amp; Lymphoma Society Scholar Award 1440-11 to KS, the AAI Careers in Immunology Fellowship Program to SR, DFG Fellowship SI 1547/1-1 to SS, and an ARC fellowship to CC. Citation Format: Karsten Sauer, Claire Conche, Hyun-Yong Jin, Kelly Bethel, Stephanie Rigaud, Luise Westernberg, Sabine Siegemund, Changchun Xiao. IP3 3-kinase B suppresses B-cell lymphoma by antagonizing PI3K/mTOR in B cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-020.

IP3 3-Kinase B Suppresses B Cell Lymphoma by Antagonizing PI3K/mTOR in B cells

Abstract Accounting for ~4% of all cancers in the US, Non-Hodgkin Lymphoma (NHL) is the most prevalent blood cancer. Diffuse large B-cell Lymphoma (DLBCL) is the most common and aggressive type of NHL. Here, we identify inositol-trisphosphate (IP3) 3-kinase B (Itpkb) as a novel tumor suppressor whose deficiency triggers DLBCL in mice. We found that aging Itpkb−/− mice die prematurely with anemia and splenomegaly. 15% of these mice showed multiorgan-infiltration with neoplastic germinal-center phenotype B cells reminiscent of DLBCL. Itpkb−/−B cell lymphomas are mono-or oligoclonal, transplantable, and constitutively hyperactivate the oncogenic phosphoinositide 3-kinase (PI3K) signaling pathway. Growth of Itpkb−/−B cell lymphoma xenografts in immunodeficient mice is sensitive to treatment with PI3K or mTOR inhibitors. In humans, subsets of DLBCL patients carry Itpkb missense mutations, deletions or copy number reductions. Additionally, Itpkb expression is altered in several other hematopoietic and non-hematopoietic human cancers. We propose that Itpkb suppresses tumors by producing inositol-tetrakisphosphate (IP4), a soluble analog of the PI3K product phosphatidylinositol-trisphosphate (PIP3). We and others have previously shown that IP4 antagonism with PIP3 for Akt effector kinase binding prevents excessive Akt/mTORC1 signaling in hematopoietic cells. Our new results suggest that this mechanism prevents oncogenic PI3K signaling in the B cell lineage and possibly in other cell types. Thus, Itpkb might be a novel biomarker for tumor aggressiveness or prognosis, and Itpkb activating drugs or IP4 might have therapeutic potential.

Local T/B cooperation in inflamed tissues is supported by T follicular helper-like cells.

Autoimmune diseases and other inflammatory conditions are characterized by large lymphocytic tissue infiltrates in which T and B cells can be found in close contact. Here, using a murine airway inflammation model, we compare antigen-specific T and B cells in lung tissue versus lung-draining lymph node. In the lung we identify a B-cell population exhibiting a classical germinal centre phenotype without being organized into ectopic lymphoid tissue. By contrast, classical CXCR5+ Bcl-6+ T follicular helper cells are not present. Nevertheless, lung-infiltrating T cells exhibit follicular helper-like properties including the potential to provide help to naive B cells. The lung tissue is also a survival niche for memory T and B cells remaining in residual peribronchial infiltrates after resolution of inflammation. Collectively, this study shows the importance of T/B cooperation not only in lymph nodes but also in inflamed peripheral tissues for local antibody responses to infection and autoimmunity.

Gene Rearrangements and Other Molecular Features in Aggressive B-Cell Lymphomas of Patients with and without HIV-Infection

Aggressive B-cell Lymphomas are the second most frequent AIDS-defining cancers. Few studies have compared the molecular characteristics of aggressive B-cell lymphomas in patients with and without HIV-infection; and to our knowledge, there are no reports comparing the incidence of gene rearrangements between the two groups and their impact on outcome in series treated with RCHOP.We retrospectively studied two series of patients with (N=32) and without HIV-infection (N=43) with diffuse large B-cell lymphomas (DLBCL) NOS (75% and 70%, respectively), T-rich DLCBL (13% and 5%), transformed DLBCL (3% and 14%) and double-hit (DH) DLCBL (9% and 11%) [defined by translocations affecting MYC (TMYC) concomitantly with translocation affecting BCL2 (TBLC2) and/or BCL6 (TBCL6)]. Tissue microarrays were constructed and translocations were studied by fluorescent in situ hybridization. The germinal center (GC) phenotype was defined according to the Hans' algorithm based on the expression of CD10, MUM1 and BCL6. Clinical data was retrieved from records. Continuous and categorical variables are presented using descriptive statistics and compared using Fisher's exact test, χ 2-test, and Mann-Whitney U-test. Survival analyses were performed using the Kaplan-Meier method. P-valuesof less than 0.05 were considered statistically significant.The median follow-up of HIV-infected patients was 6.9 years and of HIV-uninfected was 5 years. HIV-uninfected patients were older than HIV-infected patients, median age (range) 59 years (25-80) and 45 years (37-68) respectively; (P=0.002). There were differences between HIV-infected and HIV-uninfected patients regarding; male gender (81% vs. 54%, P=0.015), ECOG performance status higher than 2 (56% vs. 26%; P=0.018) and elevated ß-2 microglobulin (82% vs. 47%, P=0.005). On the other hand, the percentage of patients with III and IV Ann Arbor stages, two or more extranodal involvement, elevated LDH, 3 to 5 International Prognostic Index (IPI) scores, B-symptoms and bulky disease were similar in both series.The study of molecular features showed that more HIV-infected cases (57%) tended to have a GC phenotype than HIV-uninfected (35%); P=0.093. Regarding gene rearrangements, there was a trend for more HIV-infected patients (30%) to present TBCL2 than HIV-uninfected patients (11%); P=0.056. Of note, only 2 patients of the HIV-infected series were transformed from follicular lymphoma. The frequency of TMYC, TBCL6 and DH was similar in HIV-infected (24%, 28% and 9%, respectively) and HIV-uninfected (15%, 28% and 11%, respectively).HIV-infected patients were treated with RCHOP (N=27), intensive immunochemotherapy for Burkitt Lymphoma (IICT-BL) (N=4), CHOP (N=1) and HIV-uninfected patients with RCHOP (N=39), IICT-BL (N=2), RCOP (N=1), RESHAP (N=1). The complete response rate was not statistically different in HIV-infected and HIV-uninfected patients neither the relapse rate when considered all treatments given.Only patients treated with RCHOP were considered in survival analyses. The overall survival (OS) of HIV-uninfected patients with high IPI scores (3 to 5) (N=15) tended to be shorter than in patients with low IPI scores (0 to 2) (N=24), (5-y OS 60% [35%-85%] vs. 81% [64%-98%], P=0.089) and the progression free survival (PFS) was clearly inferior (5-y PFS 33% [9%-57%] vs. 73% [54%-92%], P=0.004). HIV-infected patients with high IPI scores presented similar OS and PFS than patients with low IPI scores. Contrary to other reports, TMYC had no statistically significant adverse impact on OS and PFS of HIV-uninfected patients, most probably because of small size of our series. The same reasoning could be applied for HIV-infected patients since both OS and PFS of patients with TMYC (N=5) was shorter than in those without TMYC (N=20) (5-year OS 40% [0%-83%] vs. 65% [44%-86%], P=0.365 and 5-year PFS 40% [0%-83%] vs. 60% [38%-82%], P=0.546). TBCL2, TBCL6 and GC phenotype had no impact on OS and on PFS of both HIV-infected and HIV-uninfected patients. Survival analyses of DH were not performed due to the small number of events.In summary, the frequency of TMYC and TBCL6 in aggressive B-cell lymphomas was similar in HIV-infected and HIV-uninfected patients but TBCL2 was less frequent in HIV-infected patients.Supported in part by grants EC11-041 from ISCIII, Spain. DisclosuresNo relevant conflicts of interest to declare.

MYC/BCL2 Double Hit Lymphoma: What Really Matters

Introduction: MYC/BCL2 double hit lymphoma (DHL), defined as a large B-cell lymphoma with concurrent MYC and BCL2 translocations, is the most common type of DHL. Although multiple studies focused on DHL have been published, several issues regarding impact on prognosis remain controversial including: 1) history of low grade B cell lymphoma; 2) morphology (diffuse large B-cell lymphoma [DLBCL] versus B cell lymphoma unclassifiable with features intermediate between DLBCL and Burkitt lymphoma [BCLU)]; 3) Absence or low expression of MYC or BCL2; 4) MYC translocation partner gene; and especially 5) most effective therapy. The aim of this study was to attempt clarify the prognostic importance of these factors in DHL.Methods: 157 patientsdiagnosed with MYC/BCL2 DHL between 2003 and April 2015 at two institutions were included in this study. MYC and BCL2 gene rearrangement were confirmed by FISH using a MYC breakapart probe and BCL2 and IGH dual color dual fusion probes. BCL6/3q27gene status was tested either by FISH using breakapart probe or by karyotype. MYC partner gene was identified by karyotype. MYC/BCL2 DHL cases were identified if they had rearrangements of MYC and BCL2 but not BCL6. Positive for MYC or BCL2 by immunohistochemistry was defined by >40% and >50% of lymphoma cells showed positive expression, respectively. Patient survival was analyzed using the Kaplan-Meier method and compared using the log-rank test. Fisher's exact test was used to compare the clinicopathologic features. Statistical analysis was performed using SPSS 23 software.Results: There were 103 men and 54 women with a median age of 61 years (range, 18-87). 110 patients had de novo disease and 47 patients had a history of low-grade B-cell NHL, mostly follicular lymphoma. The clinicopathologic features were similar (P>0.05) between patients with a history of low-grade B-cell NHL and patients with de novo NHL, and therefore analysis was performed on all 157 DHL cases. Using the 2008 WHO classification, there were 91 DLBCL, 61 BCLU, and 5 composite lymphoma (4 DLBCL + follicular lymphoma and 1 DLBCL + B-lymphoblastic lymphoma). 99% of cases had a germinal center B-cell immunophenotype by the Hans algorithm. MYC expression was observed in 39/47 (83%) and BCL2 in 129/141(91%) of cases. MYC and BCL2 dual expression was present in 34/46(74%) cases. Of the 39 cases assessed, the MYC translocation partner was IGH in 13, IG light chain in 19, and a non-IG gene in 7 cases. 144 patients had complete treatment information: 61 received the R-CHOP regimen initially, 31 R-EPOCH, 29 R-HCVAD, and 23 various other chemotherapy regimens. 39 patients also received stem cell transplant (SCT) including 31 autologous and 8 allogeneic. 62 patients reached complete remission (CR) after initial chemotherapy. The median overall survival was 19 months. In a univariate analysis that evaluated 17 clinicopathologic parameters including those mentioned in introduction, extranodal sites of disease, bone marrow involvement, CNS involvement, advanced stage, and high/high-intermediate International Prognostic Index score were associated with a worse overall survival (OS, P<0.05), whereas CR and SCT were associated with a better OS (P<0.05). By multivariate analysis, stage, IPI, SCT, and CR predicted OS (Table 1 and Figure 1).Conclusion: MYC/BCL2 DHL are clinically aggressive B cell lymphomas with a germinal center phenotype and patients have a poor prognosis. In this study, a history of low grade B cell lymphoma, morphology (DLBCL vs BCLU), MYC expression, BCL2 expression, MYC/BCL2 dual expression (double expresser lymphoma), MYC translocation partner gene, and initial chemotherapy regimens were not associated with overall survival. The independent prognostic factors shown by multivariate analysis were IPI score, stage, achievement of CR, and a therapeutic regimen including SCT.Table 1Multivariate analysisFeaturesHR95% CIPBM involvement0.920.48 - 1.800.817Extranodal sites ≥ 21.120.48 - 2.650.793Stage III /IV vs I/II34.454.92 - 241.31<0.001IPI (H/H-I vs L/L-I)3.001.14 - 7.770.026Initial ChemotherapyR-EPOCH vs R-CHOP1.170.49 - 2.840.723R-HCVAD vs R-CHOP0.950.47 - 1.930.894Stem cell transplant0.220.10 - 0.46<0.001CR after initial chemotherapy0.140.07 - 0.30<0.001 [Display omitted] DisclosuresReddy:ImmunoGen: Consultancy; PCYC: Consultancy; Gilead: Other: Speaker; Seattle Genetics: Consultancy; Celgene: Consultancy, Research Funding.

A Chromatin Reader That Acts As a Key to Lock in and Coordinate Recruitment of Transcription Factors and a Novel Polycomb Complex to Bivalent Chromatin Thus Driving Formation of Germinal Centers and B-Cell Lymphomas

The EZH2 Polycomb H3K27 histone methyltransferase and the BCL6 transcriptional repressor are highly expressed in germinal center (GC) B cells and are each targeted by activating somatic mutations in DLBCL. In previous work we demonstrated that i) EZH2 is required for GC formation, ii) EZH2 Y641 somatic mutation drives GC hyperplasia and lymphomagenesis and iii) that WT and mutant EZH2 function by creating bivalent chromatin at promoters of genes that regulate cell cycle and terminal differentiation. In other work we showed that BCL6 represses gene promoters specifically by recruiting BCOR complex (a non-canonical form of PRC1 complex); and that BCL6 and EZH2 bind to many of the same promoters, although they do not physically interact. Finally we reported that the presence of both EZH2 and BCL6 was required for BCOR complex to stably associate with chromatin. This led us to hypothesize that BCL6 and EZH2 may be mutually dependent in normal and malignant B-cells.We found that conditional deletion of EZH2 from already established GC B-cells abrogates the ability of constitutive IµBCL6 expression to induce GC hyperplasia. A similar effect was observed by administering the specific EZH2 inhibitor GSK503. For the reciprocal experiments we developed two new mouse models: a conditional BCL6 knockout and a conditional EZH2Y641F knockin in the EZH2 endogenous locus. We showed that EZH2Y641F knockin mice developed GC hyperplasia, but this effect is abrogated by conditional deletion of BCL6 from established GC B cells. Given cell line data suggesting that BCOR might be important for the effect of EZH2 and BCL6 we next generated GC BCOR conditional knockout mice and observed that loss of BCOR not only disrupts GC formation but also abrogates the GC hyperplasia when crossed into the conditional EZH2Y641F knockin mice. Reversal of the EZH2Y641F knockin GC hyperplasia was also achieved by treating these mice with a novel small molecule called 1085 that potently disrupts BCL6 binding to BCOR. Notably, 1085 synergized with EZH2 inhibitors to kill DLBCL cells in vitro and eradicate DLBCL xenografts in vivo. Collectively these data show that EZH2, BCL6 and BCOR are mutually dependent both in the context of the normal GC reaction as well as in the setting of lymphoma precursor lesions driven by constitutive BCL6 expression or mutant EZH2.We next wished to determine the basic mechanism that explains the functional connection between EZH2 and BCL6-BCOR. Using ChIP-seq in primary human GC B cells we showed that there is significant overlap between BCL6-BCOR complex binding with EZH2 and bivalent chromatin. Using ChIP-re-ChIP we found that EZH2, BCOR and BCL6 are all localized to the same chromatin at bivalent gene promoters. Previously we showed that EZH2 mediated H3K27me3 is required for BCOR recruitment. However it was not clear why or how H3K27me3 is important for this effect. We postulated that a histone reader protein might bind to BCOR and recruit it to H3K27me3 marked chromatin. We therefore performed tandem affinity purification of BCOR followed by mass spectrometry to identify putative partner proteins and identified the chromodomain containing protein CBX8 as a novel BCOR partner. Notably CBX8 is the only H3K27 chromodomain protein that is upregulated in GC B cells. We confirmed endogenous BCOR-CBX8 interaction in human DLBCL cells. We showed that CBX8 is recruited to bivalent chromatin in an H3K27me3 dependent manner, and CBX8 depletion results in loss of BCOR complex assemble. Most strikingly, CBX8 knockdown was a phenocopy of EZH2 shRNA or EZH2 inhibitor. CBX8 loss resulted in derepression of EZH2 targets, and phenocopied the effect of EZH2 shRNA or inhibitors (proliferation arrest followed by plasma cell differentiation and then apoptosis). Therefore, CBX8 is shown for the first time to recruit non-canonical PRC1 and to explain how EZH2 cooperates with BCL6 to repress transcription and mediate the GC and DLBCL phenotype. PRC2 functions in a mutually interdependent manner with BCL6 to jointly enable stable association of non-canonical PRC1 complex with bivalent chromatin.In summary, our data establish a paradigm whereby EZH2 functions in a coordinate manner with transcription factors to transiently suppress genes in a cell context-specific manner relevant to its function in normal immunity as well as DLBCL. Our results also provide a rational basis for clinical translation of combined EZH2 and BCL6 inhibitors. DisclosuresLevine:Foundation Medicine: Consultancy; CTI BioPharma: Membership on an entity's Board of Directors or advisory committees; Loxo Oncology: Membership on an entity's Board of Directors or advisory committees.

Chronic bacterial infection activates autoreactive B cells and induces isotype switching and autoantigen-driven mutations.

The links between infections and the development of B-cell-mediated autoimmune diseases are still unclear. In particular, it has been suggested that infection-induced stimulation of innate immune sensors can engage low affinity autoreactive B lymphocytes to mature and produce mutated IgG pathogenic autoantibodies. To test this hypothesis, we established a new knock-in mouse model in which autoreactive B cells could be committed to an affinity maturation process. We show that a chronic bacterial infection allows the activation of such B cells and the production of nonmutated IgM autoantibodies. Moreover, in the constitutive presence of their soluble antigen, some autoreactive clones are able to acquire a germinal center phenotype, to induce Aicda gene expression and to introduce somatic mutations in the IgG heavy chain variable region on amino acids forming direct contacts with the autoantigen. Paradoxically, only lower affinity variants are detected, which strongly suggests that higher affinity autoantibodies secreting B cells are counterselected. For the first time, we demonstrate in vivo that a noncross-reactive infectious agent can activate and induce autoreactive B cells to isotype switching and autoantigen-driven mutations, but on a nonautoimmune background, tolerance mechanisms prevent the formation of consequently dangerous autoimmunity.