Germinal center dysregulation by histone methyltransferase EZH2 promotes lymphomagenesis

Protection against deadly pathogens requires the production of high-affinity antibodies by B cells, which are generated in germinal centers (GCs). Alteration of the GC developmental program is common in many B cell malignancies. Identification of regulators of the GC response is crucial to develop targeted therapies for GC B cell dysfunctions, including lymphomas. The histone H3 lysine 27 methyltransferase enhancer of zeste homolog 2 (EZH2) is highly expressed in GC B cells and is often constitutively activated in GC-derived non-Hodgkin lymphomas (NHLs). The function of EZH2 in GC B cells remains largely unknown. Herein, we show that Ezh2 inactivation in mouse GC B cells caused profound impairment of GC responses, memory B cell formation, and humoral immunity. EZH2 protected GC B cells against activation-induced cytidine deaminase (AID) mutagenesis, facilitated cell cycle progression, and silenced plasma cell determinant and tumor suppressor B-lymphocyte–induced maturation protein 1 (BLIMP1). EZH2 inhibition in NHL cells induced BLIMP1, which impaired tumor growth. In conclusion, EZH2 sustains AID function and prevents terminal differentiation of GC B cells, which allows antibody diversification and affinity maturation. Dysregulation of the GC reaction by constitutively active EZH2 facilitates lymphomagenesis and identifies EZH2 as a possible therapeutic target in NHL and other GC-derived B cell diseases.     

The G protein–coupled receptor P2RY8 and follicular dendritic cells promote germinal center confinement of B cells,whereas S1PR3 can contribute to their dissemination

The orphan Gα13-coupled receptor P2RY8 is mutated in human germinal center (GC)–derived lymphomas and was recently found to promote B cell association with GCs in a mouse model. Here we establish that P2RY8 promotes clustering of activated B cells within follicles in a follicular dendritic cell (FDC)–dependent manner. Although mice lack a P2RY8 orthologue, we show that mouse GC B cell clustering is also dependent on FDCs acting to support the function of a Gα13-coupled receptor. Mutations in GNA13 and its downstream effector ARHGEF1 are associated with the development of disseminated GC-derived lymphomas. We find that egress of Gna13 mutant GC B cells from lymph nodes in the mouse depends on sphingosine-1-phosphate receptor-3. These findings provide evidence that FDCs promote GC confinement of both human and mouse GC B cells via Gα13-dependent pathways, and they show that dissemination of Gα13-deficient GC B cells additionally requires an egress-promoting receptor.Germinal centers (GCs) are organized as discrete, tightly confined clusters of GC B cells and T follicular helper cells (Tfh cells) in the center of reactive (secondary) follicles. Confinement of GC B cells to the GC is important for fostering interactions with antigen-loaded follicular DCs (FDCs) and Tfh cells during the selection events necessary for antibody affinity maturation (Victora and Nussenzweig, 2012). The sphingosine-1-phosphate receptor S1pr2 is a G protein–coupled receptor (GPCR) that is highly up-regulated on GC B cells and signals via the G protein Gα13 and the RhoGEF Arhgef1 (also known as p115 RhoGEF or Lsc) to inhibit cell migration in response to chemoattractants (Green et al., 2011; Muppidi et al., 2014). S1P is abundant in lymph and blood, rapidly degraded in interstitial fluids by the action of membrane phosphatases and an intracellular lyase, and thought to exist in a decaying gradient from the outer to the center follicle (Green et al., 2011; Cyster and Schwab, 2012). S1pr2 promotes GC B cell and Tfh cell confinement to the follicle center, most likely by inhibiting migration into regions of high S1P (Green et al., 2011; Moriyama et al., 2014; Muppidi et al., 2014). Maintenance of the follicular S1P gradient appears to occur in the absence of FDCs (Wang et al., 2011). However, S1pr2 deficiency does not lead to a complete dispersal of GC B cells or to their appearance in circulation, suggesting that additional factors act to promote GC B cell confinement.Deficiency in Gα13 and Arhgef1 in the mouse is also associated with loss of local GC B cell confinement, and these deficiencies lead to appearance of GC B cells in lymph and blood (Muppidi et al., 2014). The more penetrant effect of Gα13 deficiency than S1pr2 deficiency led us to identify a second human receptor, P2RY8, that can act via Gα13 to promote B cell association with GCs (Muppidi et al., 2014). Although human P2RY8 is active when expressed in the mouse, the P2RY8 gene is located on a portion of the pseudoautosomal region of the X chromosome that is lost in rodents, and no orthologues have been identified in the mouse. Because Gα13 signaling in B cells inhibits migration, the GC clustering activity of P2RY8 suggests that the P2RY8 ligand might, like S1P, be more abundant in the outer follicle than in the follicle center. Although the identity of the P2RY8 ligand is unknown, in this study, we take advantage of the ability of P2RY8 overexpression to direct cell movements in the mouse to dissect cellular requirements for controlling ligand distribution.Loss of function mutations of S1PR2, P2RY8, GNA13 (encoding Gα13), and ARHGEF1 are frequently found in human lymphomas derived from GC B cells (GC B cell–like diffuse large B cell lymphoma [GCB-DLBCL] and Burkitt lymphoma [BL]; Morin et al., 2011; Lohr et al., 2012; Schmitz et al., 2012; Muppidi et al., 2014). These malignancies are considered systemic diseases in humans, and spread of the malignant GC B cells to distant sites such as BM is associated with poor prognosis (Sehn et al., 2011). It is currently unclear whether Gα13 deficiency itself is sufficient to promote GC B cell egress or whether loss of Gα13 signaling allows other promigratory signals to dominate and promote egress.In this study, we sought to further define how GC B cells are normally confined to the GC niche and to understand how GC B cells carrying mutations typical of GC lymphomas undergo systemic spread. We demonstrate that FDCs are required for the function of human P2RY8 in promoting cell clustering at the center of mouse lymphoid follicles. Although P2RY8 is not present in the mouse, we find that FDCs act via a Gα13-dependent mechanism to promote clustering of mouse GC B cells and prevent their egress into lymph. Finally, we show that egress of Gα13-deficient mouse GC B cells is promoted by the promigratory S1P receptor S1pr3.     

Germinal center reentries of BCL2-overexpressing B cells drive follicular lymphoma progression

It has recently been demonstrated that memory B cells can reenter and reengage germinal center (GC) reactions, opening the possibility that multi-hit lymphomagenesis gradually occurs throughout life during successive immunological challenges. Here, we investigated this scenario in follicular lymphoma (FL), an indolent GC-derived malignancy. We developed a mouse model that recapitulates the FL hallmark t(14;18) translocation, which results in constitutive activation of antiapoptotic protein B cell lymphoma 2 (BCL2) in a subset of B cells, and applied a combination of molecular and immunofluorescence approaches to track normal and t(14;18)⁺ memory B cells in human and BCL2-overexpressing B cells in murine lymphoid tissues. BCL2-overexpressing B cells required multiple GC transits before acquiring FL-associated developmental arrest and presenting as GC B cells with constitutive activation–induced cytidine deaminase (AID) mutator activity. Moreover, multiple reentries into the GC were necessary for the progression to advanced precursor stages of FL. Together, our results demonstrate that protracted subversion of immune dynamics contributes to early dissemination and progression of t(14;18)⁺ precursors and shapes the systemic presentation of FL patients.     

Cyclin D3 drives inertial cell cycling in dark zone germinal center B cells

During affinity maturation, germinal center (GC) B cells alternate between proliferation and somatic hypermutation in the dark zone (DZ) and affinity-dependent selection in the light zone (LZ). This anatomical segregation imposes that the vigorous proliferation that allows clonal expansion of positively selected GC B cells takes place ostensibly in the absence of the signals that triggered selection in the LZ, as if by “inertia.” We find that such inertial cycles specifically require the cell cycle regulator cyclin D3. Cyclin D3 dose-dependently controls the extent to which B cells proliferate in the DZ and is essential for effective clonal expansion of GC B cells in response to strong T follicular helper (Tfh) cell help. Introduction into the Ccnd3 gene of a Burkitt lymphoma–associated gain-of-function mutation (T283A) leads to larger GCs with increased DZ proliferation and, in older mice, clonal B cell lymphoproliferation, suggesting that the DZ inertial cell cycle program can be coopted by B cells undergoing malignant transformation.     

Tetraspanin CD37 protects against the development of B cell lymphoma

Worldwide, B cell non-Hodgkin lymphoma is the most common hematological malignancy and represents a substantial clinical problem. The molecular events that lead to B cell lymphoma are only partially defined. Here, we have provided evidence that deficiency of tetraspanin superfamily member CD37, which is important for B cell function, induces the development of B cell lymphoma. Mice lacking CD37 developed germinal center–derived B cell lymphoma in lymph nodes and spleens with a higher incidence than Bcl2 transgenic mice. We discovered that CD37 interacts with suppressor of cytokine signaling 3 (SOCS3); therefore, absence of CD37 drives tumor development through constitutive activation of the IL-6 signaling pathway. Moreover, animals deficient for both Cd37 and Il6 were fully protected against lymphoma development, confirming the involvement of the IL-6 pathway in driving tumorigenesis. Loss of CD37 on neoplastic cells in patients with diffuse large B cell lymphoma (DLBCL) directly correlated with activation of the IL-6 signaling pathway and with worse progression-free and overall survival. Together, this study identifies CD37 as a tumor suppressor that directly protects against B cell lymphomagenesis and provides a strong rationale for blocking the IL-6 pathway in patients with CD37^– B cell malignancies as a possible therapeutic intervention.     

TGF-β prevents T follicular helper cell accumulation and B cell autoreactivity

T follicular helper (Tfh) cells contribute to the establishment of humoral immunity by controlling the delivery of helper signals to activated B cells; however, Tfh development must be restrained, as aberrant accumulation of these cells is associated with positive selection of self-reactive germinal center B cells and autoimmunity in both humans and mice. Here, we show that TGF-β signaling in T cells prevented Tfh cell accumulation, self-reactive B cell activation, and autoantibody production. Using mice with either T cell–specific loss or constitutive activation of TGF-β signaling, we demonstrated that TGF-β signaling is required for the thymic maturation of CD44⁺CD122⁺Ly49⁺CD8⁺ regulatory T cells (Tregs), which induce Tfh apoptosis and thus regulate this cell population. Moreover, peripheral Tfh cells escaping TGF-β control were resistant to apoptosis, exhibited high levels of the antiapoptotic protein BCL2, and remained refractory to regulation by CD8⁺ Tregs. The unrestrained accumulation of Tfh cells in the absence of TGF-β was dependent on T cell receptor engagement and required B cells. Together, these data indicate that TGF-β signaling restrains Tfh cell accumulation and B cell–associated autoimmunity and thereby controls self-tolerance.     

DUSP4 deficiency caused by promoter hypermethylation drives JNK signaling and tumor cell survival in diffuse large B cell lymphoma

The epigenetic dysregulation of tumor suppressor genes is an important driver of human carcinogenesis. We have combined genome-wide DNA methylation analyses and gene expression profiling after pharmacological DNA demethylation with functional screening to identify novel tumor suppressors in diffuse large B cell lymphoma (DLBCL). We find that a CpG island in the promoter of the dual-specificity phosphatase DUSP4 is aberrantly methylated in nodal and extranodal DLBCL, irrespective of ABC or GCB subtype, resulting in loss of DUSP4 expression in 75% of >200 examined cases. The DUSP4 genomic locus is further deleted in up to 13% of aggressive B cell lymphomas, and the lack of DUSP4 is a negative prognostic factor in three independent cohorts of DLBCL patients. Ectopic expression of wild-type DUSP4, but not of a phosphatase-deficient mutant, dephosphorylates c-JUN N-terminal kinase (JNK) and induces apoptosis in DLBCL cells. Pharmacological or dominant-negative JNK inhibition restricts DLBCL survival in vitro and in vivo and synergizes strongly with the Bruton’s tyrosine kinase inhibitor ibrutinib. Our results indicate that DLBCL cells depend on JNK signaling for survival. This finding provides a mechanistic basis for the clinical development of JNK inhibitors in DLBCL, ideally in synthetic lethal combinations with inhibitors of chronic active B cell receptor signaling.Diffuse large B cell lymphoma (DLBCL) is the most commonly diagnosed lymphoma in adults. It may either arise de novo at nodal or extranodal sites or as a consequence of malignant transformation of indolent lymphomas or leukemias such as follicular lymphoma (FL), chronic lymphocytic leukemia (CLL), and marginal zone lymphoma (MZL; Schneider et al., 2011; Shaffer et al., 2012; Pasqualucci and Dalla-Favera, 2014). DLBCL represents a heterogeneous disease, with molecular subtypes being characterized by distinct gene expression profiles, specific sets of somatic mutations, and differentially active intracellular signaling pathways (Roschewski et al., 2014). Three subtypes of DLBCL can be distinguished based on the presumed normal B cell counterpart, with activated B cell–like DLBCL (ABC-DLBCL) resembling the post–germinal center (GC) plasmablast, GC B cell–like DLBCL (GCB-DLBCL) deriving from GC B cells, and primary mediastinal B cell lymphoma (PMBL) arising in the thymus from a rare subset of thymic B cells (Alizadeh et al., 2000; Rosenwald et al., 2003). The three subtypes of DLBCL differ not only in their pathogenesis, but also in their cure and survival rates (Cultrera and Dalia, 2012). The rational development of more targeted therapies is complicated by the heterogeneity of DLBCL as well as the coexistence of genetic lesions affecting multiple redundant survival pathways. Genetic aberrations in DLBCL either exclusively affect GCB-DLBCL (deregulated c-Myc or Bcl-2 expression, gain of function of the H3K27 methyltransferase EZH2) or ABC-DLBCL (A20 loss, gain of function of MYD88, CD79A/B, or CARD11, all of which promote the constitutive activation of the NF-κB pathway) or are found in both major subtypes (inactivating mutations and deletions in the histone acetyltransferases CBP and p300 as well as the histone methyl transferase MLL2; Schneider et al., 2011; Shaffer et al., 2012; Pasqualucci and Dalla-Favera, 2014).Aberrant changes of the DNA methylation landscape are a hallmark of cancer cells and have been linked to clinical aggressiveness and chemoresistance of DLBCL (Shaknovich et al., 2010; Clozel et al., 2013; De et al., 2013; Chambwe et al., 2014). Examples of tumor suppressor genes known to be silenced by promoter hypermethylation in DLBCL include SMAD1, MGMT, CDKN2A, and the lamin A/C gene (Martinez-Delgado et al., 1997; Esteller et al., 2002; Agrelo et al., 2005; Clozel et al., 2013). We have shown in earlier studies that the epigenetic silencing of the tumor suppressor microRNAs miR-203 and miR-34a contribute to the transformation of gastric MZL to DLBCL and to the deregulated expression of the hematopoietic oncoprotein FoxP1 (Craig et al., 2011a,b). Here, we have conducted a genome-wide analysis of the DNA methylome of gastric DLBCL and MZL and of nodal DLBCL samples and cell lines. The hypermethylated gene loci were further examined by RNA sequencing with respect to their reactivation upon experimental DNA demethylation. Aberrantly silenced genes were ectopically expressed in DLBCL cell lines and assessed for possible effects on cell survival. This unbiased approach uncovered a new tumor suppressor in DLBCL, the dual-specificity phosphatase DUSP4, and introduces the constitutively active JNK signaling pathway as a promising new target in DLBCL treatment.     

B cell–intrinsic TBK1 is essential for germinal center formation during infection and vaccination in mice

The germinal center (GC) is a site where somatic hypermutation and clonal selection are coupled for antibody affinity maturation against infections. However, how GCs are formed and regulated is incompletely understood. Here, we identified an unexpected role of Tank-binding kinase-1 (TBK1) as a crucial B cell–intrinsic factor for GC formation. Using immunization and malaria infection models, we show that TBK1-deficient B cells failed to form GC despite normal Tfh cell differentiation, although some malaria-infected B cell–specific TBK1-deficient mice could survive by GC-independent mechanisms. Mechanistically, TBK1 phosphorylation elevates in B cells during GC differentiation and regulates the balance of IRF4/BCL6 expression by limiting CD40 and BCR activation through noncanonical NF-κB and AKT^T308 signaling. In the absence of TBK1, CD40 and BCR signaling synergistically enhanced IRF4 expression in Pre-GC, leading to BCL6 suppression, and therefore failed to form GCs. As a result, memory B cells generated from TBK1-deficient B cells fail to confer sterile immunity upon reinfection, suggesting that TBK1 determines B cell fate to promote long-lasting humoral immunity.     

Coronavirus-specific antibody production in middle-aged mice requires phospholipase A2G2D

Worse outcomes occur in aged compared with young populations after infections with respiratory viruses, including pathogenic coronaviruses (SARS-CoV, MERS-CoV, and SARS-CoV-2), and are associated with a suboptimal lung milieu (“inflammaging”). We previously showed that a single inducible phospholipase, PLA₂G2D, is associated with a proresolving/antiinflammatory response in the lungs, and increases with age. Survival was increased in naive Pla2g2d^–/– mice infected with SARS-CoV resulting from augmented respiratory dendritic cell (rDC) activation and enhanced priming of virus-specific T cells. Here, in contrast, we show that intranasal immunization provided no additional protection in middle-aged Pla2g2d^–/– mice infected with any of the 3 pathogenic human coronaviruses because virtually no virus-specific antibodies or follicular helper CD4⁺ T (Tfh) cells were produced. Using MERS-CoV–infected mice, we found that these effects did not result from T or B cell intrinsic factors. Rather, they resulted from enhanced, and ultimately, pathogenic rDC activation, as manifested most prominently by enhanced IL-1β expression. Wild-type rDC transfer to Pla2g2d^–/– mice in conjunction with partial IL-1β blockade reversed this defect and resulted in increased virus-specific antibody and Tfh responses. Together, these results indicate that PLA₂G2D has an unexpected role in the lungs, serving as an important modulator of rDC activation, with protective and pathogenic effects in respiratory coronavirus infections and immunization, respectively.     

IL-4–producing CD4+ T cells in reactive lymph nodes during helminth infection are T follicular helper cells

Interleukin (IL)-4 is the quintessential T helper type 2 (Th2) cytokine produced by CD4⁺ T cells in response to helminth infection. IL-4 not only promotes the differentiation of Th2 cells but is also critical for immunoglobulin (Ig) G1 and IgE isotype-switched antibody responses. Despite the IL-4–mediated link between Th2 cells and B lymphocytes, the location of IL-4–producing T cells in the lymph nodes is currently unclear. Using IL-4 dual reporter mice, we examined the Th2 response and IL-4 production in the draining mesenteric lymph nodes during infection with the enteric nematode Heligmosomoides polygyrus. We show that although IL-4–competent Th2 cells are found throughout the B and T cell areas, IL-4–producing Th2 cells are restricted to the B cell follicles and associate with germinal centers. Consistent with their localization, IL-4 producers express high levels of CXCR5, ICOS, PD-1, IL-21, and BCL-6, a phenotype characteristic of T follicular helper (Tfh) cells. Although IL-4 was dispensable for the generation of Th2 and Tfh cells, its deletion resulted in defective B cell expansion and maturation. Our report reveals the compartmentalization of Th2 priming and IL-4 production in the lymph nodes during infection, and identifies Tfh cells as the dominant source of IL-4 in vivo.IL-4 is widely recognized as the canonical marker for Th2-polarized CD4⁺ T cells (1). IL-4 was originally identified by its function as a B cell–stimulating factor (2, 3), and numerous in vivo models of infection concur that IL-4 is critical for the isotype switch of B cells to IgE and IgG1 (4–10). IL-4 also promotes the Th2 polarization of naive CD4⁺ T cells in vitro (11, 12). However, in vivo studies have found that Th2 cells can develop in mice deficient for IL-4, the IL-4Rα chain, or the IL-4R–associated Stat 6 (4–8). Collectively, these observations suggest that IL-4 production in sentinel lymph nodes draining a site of infection may be more important to support type 2 B cell responses rather than to establish the underlying Th2 response. Therefore, it stands to reason that the production of IL-4 by Th2 cells should preferentially occur in B cell follicles to optimize B cell help. However, the localization and characterization of IL-4–producing T cells in the lymph nodes has not been assessed.T follicular helper (Tfh) cells are a subset of CD4⁺ T cells that migrate to B cell follicles after activation and promote germinal center formation and B cell Ig isotype switching in mice and humans (13–16). Phenotypically, Tfh cells are characterized by expression of CXCR5 (an obligatory receptor for the follicle-homing chemokine CXCL13) (17), the inducible co-stimulatory receptor ICOS (18, 19), and the inhibitory receptors PD-1 (also known as CD279) (14) and B and T lymphocyte attenuator (BTLA) (13). A previous study (13) using model antigens in adjuvant have suggested that Tfh cells are a distinct lineage of T helper cells that arises independently of Th1, Th2, and Th17 effector subsets. To date, however, no studies have examined the Tfh response during infection with a Th2-polarizing pathogen. To unambiguously identify and localize Th2 cells and IL-4 production in the reactive lymph nodes in vivo, we infected IL-4 dual reporter mice (20), in which cells that express IL-4 are marked by GFP and IL-4–producing subsets additionally display surface huCD2, with the mouse gastrointestinal helminth parasite Heligmosomoides polygyrus. We find that IL-4–producing Th2 cells localize to the B cell follicles and exhibit a Tfh cell phenotype in the draining lymph nodes of infected animals. In addition, we find that although IL-4 is dispensable for the generation of Tfh cells, it is critical for a mature B cell response. To our knowledge, this is the first report to show the location of IL-4–producing T cells in the reactive lymph nodes after helminth infection. Furthermore, our results challenge the prevailing concept that Tfh cells are a distinct T helper lineage. Rather, we suggest that Tfh cells act to support the demands of the impending immune response.     

CVID-associated TACI mutations affect autoreactive B cell selection and activation

Common variable immune deficiency (CVID) is an assorted group of primary diseases that clinically manifest with antibody deficiency, infection susceptibility, and autoimmunity. Heterozygous mutations in the gene encoding the tumor necrosis factor receptor superfamily member TACI are associated with CVID and autoimmune manifestations, whereas two mutated alleles prevent autoimmunity. To assess how the number of TACI mutations affects B cell activation and tolerance checkpoints, we analyzed healthy individuals and CVID patients carrying one or two TACI mutations. We found that TACI interacts with the cleaved, mature forms of TLR7 and TLR9 and plays an important role during B cell activation and the central removal of autoreactive B cells in healthy donors and CVID patients. However, only subjects with a single TACI mutation displayed a breached immune tolerance and secreted antinuclear antibodies (ANAs). These antibodies were associated with the presence of circulating B cell lymphoma 6–expressing T follicular helper (Tfh) cells, likely stimulating autoreactive B cells. Thus, TACI mutations may favor CVID by altering B cell activation with coincident impairment of central B cell tolerance, whereas residual B cell responsiveness in patients with one, but not two, TACI mutations enables autoimmune complications.     

Pharmacoproteomics identifies combinatorial therapy targets for diffuse large B cell lymphoma

Rationally designed combinations of targeted therapies for refractory cancers, such as activated B cell–like diffuse large B cell lymphoma (ABC DLBCL), are likely required to achieve potent, durable responses. Here, we used a pharmacoproteomics approach to map the interactome of a tumor-enriched isoform of HSP90 (teHSP90). Specifically, we chemically precipitated teHSP90-client complexes from DLBCL cell lines with the small molecule PU-H71 and found that components of the proximal B cell receptor (BCR) signalosome were enriched within teHSP90 complexes. Functional assays revealed that teHSP90 facilitates BCR signaling dynamics by enabling phosphorylation of key BCR signalosome components, including the kinases SYK and BTK. Consequently, treatment of BCR-dependent ABC DLBCL cells with PU-H71 attenuated BCR signaling, calcium flux, and NF-κB signaling, ultimately leading to growth arrest. Combined exposure of ABC DLBCL cell lines to PU-H71 and ibrutinib, a BCR pathway inhibitor, more potently suppressed BCR signaling than either drug alone. Correspondingly, PU-H71 combined with ibrutinib induced synergistic killing of lymphoma cell lines, primary human lymphoma specimens ex vivo, and lymphoma xenografts in vivo, without notable toxicity. Together, our results demonstrate that a pharmacoproteome-driven rational combination therapy has potential to provide more potent BCR-directed therapy for ABC DLCBL patients.     

P2RY8 variants in lupus patients uncover a role for the receptor in immunological tolerance

B cell self-tolerance is maintained through multiple checkpoints, including restraints on intracellular signaling and cell trafficking. P2RY8 is a receptor with established roles in germinal center (GC) B cell migration inhibition and growth regulation. Somatic P2RY8 variants are common in GC-derived B cell lymphomas. Here, we identify germline novel or rare P2RY8 missense variants in lupus kindreds or the related antiphospholipid syndrome, including a “de novo” variant in a child with severe nephritis. All variants decreased protein expression, F-actin abundance, and GPCR-RhoA signaling, and those with stronger effects increased AKT and ERK activity and cell migration. Remarkably, P2RY8 was reduced in B cell subsets from some SLE patients lacking P2RY8 gene variants. Low P2RY8 correlated with lupus nephritis and increased age-associated B cells and plasma cells. By contrast, P2RY8 overexpression in cells and mice restrained plasma cell development and reinforced negative selection of DNA-reactive developing B cells. These findings uncover a role of P2RY8 in immunological tolerance and lupus pathogenesis.     

Tumor-associated antigen PRAME exhibits dualistic functions that are targetable in diffuse large B cell lymphoma

PRAME is a prominent member of the cancer testis antigen family of proteins, which triggers autologous T cell–mediated immune responses. Integrative genomic analysis in diffuse large B cell lymphoma (DLBCL) uncovered recurrent and highly focal deletions of 22q11.22, including the PRAME gene, which were associated with poor outcome. PRAME-deleted tumors showed cytotoxic T cell immune escape and were associated with cold tumor microenvironments. In addition, PRAME downmodulation was strongly associated with somatic EZH2 Y641 mutations in DLBCL. In turn, PRC2-regulated genes were repressed in isogenic PRAME-KO lymphoma cell lines, and PRAME was found to directly interact with EZH2 as a negative regulator. EZH2 inhibition with EPZ-6438 abrogated these extrinsic and intrinsic effects, leading to PRAME expression and microenvironment restoration in vivo. Our data highlight multiple functions of PRAME during lymphomagenesis and provide a preclinical rationale for synergistic therapies combining epigenetic reprogramming with PRAME-targeted therapies.