EBV‐associated but HHV8‐unrelated double‐hit effusion‐based lymphoma

Effusion‐based lymphoma is a rare and unique type of large B‐cell lymphoma presenting in effusion without a mass lesion. It shares many clinicopathological features with primary effusion lymphoma (PEL), but is distinct from PEL by the absence of HHV8 association. Double hit lymphoma (DHL) is an aggressive B‐cell lymphoma, defined by concurrent rearrangement of MYC and BCL2 or BCL6 . DHL often presents as lymphadenopathy or an extranodal mass, but rarely occurs in effusion. Here we report a 61‐year‐old male with alcoholic cirrhosis presenting as massive ascites and left pleural effusion. He has no HIV, HBV or HCV infection and no mass lesion by CT scans. Cytology of both pleural effusion and ascites show large lymphoma cells with plasmablastic morphology characterized by pleomorphic and eccentric nuclei, prominent nucleoli and frequent mitoses. Immunohistochemical study with cell block shows that the lymphoma cells express plasma cell‐related markers (CD138, MUM‐1 and EMA), but not CD3, CD30, CD45, B‐cell markers (CD19, CD20, CD79a, and PAX5), HHV8, ALK or cytokeratin. EBER is positive in most lymphoma cells. Fluorescence in situ hybridization reveals rearrangement at the IGH, BCL2, and MYC loci, but not at BCL6 . It is diagnosed as an EBV‐associated but HHV8‐unrelated double hit effusion‐based lymphoma with plasmablastic features. The patient passed away soon after diagnosis without chemotherapy. This is the first reported case of double‐hit effusion‐based lymphoma with MYC and BCL2 rearrangement. This case illustrates the importance of integrating clinical, cytological, immunophenotypical, and molecular findings to reach a correct diagnosis. Diagn. Cytopathol. 2017;45:257–261. © 2016 Wiley Periodicals, Inc.     

Enteroantigen (eAg)‐binding B lymphocytes in the mouse – phenotype,distribution, function and eAg‐specific antibody secretion

Studies reporting beneficial effects of B lymphocytes in autoimmune diseases have been accumulating and a regulatory role for certain B cell subsets is hence getting more and more recognition. Recently, B cells were shown to exhibit a regulatory effect in a T cell transfer model of colitis. Here, B cells exposed to enteroantigen (eAg) ex vivo abrogated the colitogenic effect if co‐transplanted with Treg‐depleted (CD4+CD25?) T cells into severe combined immune deficiency (SCID) mice. These data may imply a role for B cells that bind eAg (eAg+ B cells) in the immunopathology of colitis. Here, we report the detection of a subset of eAg+ B cells, including both B2 and B1 lineages, and show that these cells are present in all peripheral lymphoid organs of the mouse including the peritoneal cavity. eAg+ B cells are far more efficient as eAg‐presenting cells than unfractionated splenocytes or eAg? B cells in causing proliferation of eAg‐specific T cells. In comparison with eAg? B cells, eAg+ B cells secrete a significant amount of IL‐10 in vitro, suggesting an anti‐inflammatory potential. Compared with wild‐type B cells, B cell receptor (BCR) transgenic, hen egg lysozyme‐specific B cells show inferior eAg binding and T cell stimulatory activity suggesting involvement of the BCR in eAg binding and processing. After activation of CD19⁺ B cells by eAg and hybridization with hypoxanthine‐aminopterin‐thymidine (HAT) sensitive ×63 lymphoma cells followed by cloning at limiting dilution conditions, around 10% of the hybridoma cells secrete eAg‐specific antibodies.     

Expansion of regulatory T cells via IL‐2/anti‐IL‐2 mAb complexes suppresses experimental myasthenia

Human autoimmune diseases are often characterized by a relative deficiency in CD4⁺CD25⁺ regulatory T cells (Treg). We therefore hypothesized that expansion of Treg can ameliorate autoimmune pathology. We tested this hypothesis in an experimental model for autoimmune myasthenia gravis (MG), a B‐cell‐mediated disease characterized by auto‐Ab directed against the acetylcholine receptor within neuromuscular junctions. We showed that injection of immune complexes composed of the cytokine IL‐2 and anti‐IL‐2 mAb (JES6‐1A12) induced an effective and sustained expansion of Treg, via peripheral proliferation of CD4⁺CD25⁺Foxp3⁺ cells and peripheral conversion of CD4⁺CD25^?Foxp3^? cells. The expanded Treg potently suppressed autoreactive T‐ and B‐cell responses to acetylcholine receptor and attenuated the muscular weakness that is characteristic of MG. Thus, IL‐2/anti‐IL‐2 mAb complexes can expand functional Treg in vivo, providing a potential clinical application of this modality for treatment of MG and other autoimmune disorders.     

Human CD22 cannot fully substitute murine CD22 functions in vivo,as shown in a new knockin mouse model

CD22, an inhibitory co‐receptor of the B‐cell receptor, shows a B‐cell‐specific expression pattern and is expressed on most B‐cell lymphomas. The anti‐CD22 antibody Epratuzumab is in clinical trials for B‐cell non‐Hodgkin lymphoma and systemic lupus erythematosus, but shows a mostly unknown mode of action. We generated a new mouse model that expresses human CD22 instead of murine CD22 (Huki CD22 mice), in which human CD22 can be targeted. Expression of human CD22 on the B cells of Huki CD22 mice does not generally interfere with B‐cell development. However, Huki CD22 mice show a reduction of the population of mature recirculating B cells in the bone marrow and reduced transitional and marginal zone B cells in the spleen, phenotypes resembling that of CD22‐deficient mice. Similarly, enhanced BCR‐induced Ca²⁺ signalling is observed in Huki CD22 mice, which also mount normal immune responses toward different classes of antigens. Huki CD22 B cells show a normal anti‐hCD22 antibody‐mediated endocytosis. In conclusion, human CD22 cannot fully substitute for murine CD22 functions, possibly due to the changed intracellular tail of the protein or due to lower expression levels. Huki CD22 mice are a valuable new model for both antibody‐ and immunotoxin‐mediated targeting of human CD22.     

Cellular‐FLIP,Raji isoform (c‐FLIPR) modulates cell death induction upon T‐cell activation and infection

Dysregulation of apoptosis caused by an imbalance of pro‐ and anti‐apoptotic protein expression can lead to cancer, neurodegenerative, and autoimmune diseases. Cellular‐FLIP (c‐FLIP) proteins inhibit apoptosis directly at the death‐inducing signaling complex of death receptors, such as CD95, and have been linked to apoptosis regulation during immune responses. While the isoforms c‐FLIP_L and c‐FLIP_S are well characterized, the function of c‐FLIP_R remains poorly understood. Here, we demonstrate the induction of endogenous murine c‐FLIP_R in activated lymphocytes for the first time. To analyze c‐FLIP_R function in vivo, we generated transgenic mice expressing murine c‐FLIP_R specifically in hematopoietic cells. As expected, lymphocytes from c‐FLIP_R transgenic mice were protected against CD95‐induced apoptosis in vitro. In the steady state, transgenic mice had normal cell numbers and unaltered frequencies of B cells and T‐cell subsets in lymphoid organs. However, when challenged with Listeria monocytogenes, c‐FLIP_R transgenic mice showed less liver necrosis and better bacterial clearance compared with infected wild‐type mice. We conclude that c‐FLIP_R expression in hematopoietic cells supports an efficient immune response against bacterial infections.     

Memory IL‐22‐producing CD4+ T cells specific for Candida albicans are present in humans

Co‐expression of IL‐22 and IL‐17 has been identified and demonstrated to be involved in the immunopathogenesis of some autoimmune diseases as well as the defense against pathogenic infections in animal studies. However, the properties of IL‐22‐producing cells in humans remain largely unclear. In the present study, we showed that IL‐22 could be induced from human PBMC following various polyclonal stimulations. The majority of IL‐22‐producing cells in PBMC were CD4⁺ T cells with a memory cell phenotype. In addition, we found that a subset of IL‐22⁺ T cells produced IL‐22 alone, whereas other IL‐22⁺ T cells co‐expressed cytokines typical of Th1, Th2 and Th17 cells. Importantly, stimulation of PBMC from healthy adults with heat‐inactivated Candida albicans (C. albicans) yeast or hyphae resulted in IL‐22 production by central and effector memory CD4⁺ T cells. Moreover, CD4⁺CCR6⁺ but not CD4⁺CCR6^? T cells produced IL‐22 when stimulated with either C. albicans or PMA and ionomycin. In addition, PBMC from the individuals infected with C. albicans produced a significantly higher amount of IL‐22 compared with healthy controls following stimulation with C. albicans. These data demonstrate that IL‐22‐producing T cells in humans may play an important role in the defense against fungal infections such as C. albicans.     

Boswellic acids reduce Th17 differentiation via blockade of IL‐1β‐mediated IRAK1 signaling

Interferon‐gamma producing CD4⁺ T (Th1) cells and IL‐17‐producing CD4⁺ T (Th17) cells are involved in the pathogenesis of several autoimmune diseases including multiple sclerosis. Therefore, the development of treatment strategies controlling the generation and expansion of these effector cells is of high interest. Frankincense, the resin from trees of the genus Boswellia, and particularly its prominent bioactive compound acetyl‐11‐keto‐β‐boswellic acid (AKBA), have potent anti‐inflammatory properties. Here, we demonstrate that AKBA is able to reduce the differentiation of human CD4⁺ T cells to Th17 cells, while slightly increasing Th2‐ and Treg‐cell differentiation. Furthermore, AKBA reduces the IL‐1β‐triggered IL‐17A release of memory Th17 cells. AKBA may affect IL‐1β signaling by preventing IL‐1 receptor‐associated kinase 1 phosphorylation and subsequently decreasing STAT3 phosphorylation at Ser727, which is required for Th17‐cell differentiation. The effects of AKBA on Th17 differentiation and IL‐17A release make the compound a good candidate for potential treatment of Th17‐driven diseases.     

Impaired B‐cell tolerance checkpoints promote the development of autoimmune diseases and pathogenic autoantibodies

A role for B cells in autoimmune diseases is now clearly established both in mouse models and humans by successful treatment of multiple sclerosis and rheumatoid arthritis with anti‐CD20 monoclonal antibodies that eliminate B cells. However, the underlying mechanisms by which B cells promote the development of autoimmune diseases remain poorly understood. Here, we review evidence that patients with autoimmune disease suffer from defects in early B‐cell tolerance checkpoints and therefore fail to counterselect developing autoreactive B cells. These B‐cell tolerance defects are primary to autoimmune diseases and may result from altered B‐cell receptor signaling and dysregulated T‐cell/regulatory T‐cell compartment. As a consequence, large numbers of autoreactive naive B cells accumulate in the blood of patients with autoimmune diseases and may promote autoimmunity through the presentation of self‐antigen to T cells. In addition, new evidence suggests that this reservoir of autoreactive naive B cells contains clones that may develop into CD27^?CD21^?/lo B cells associated with increased disease severity and plasma cells secreting potentially pathogenic autoantibodies after the acquisition of somatic hypermutations that improve affinity for self‐antigens.     

Translational Mini‐Review Series on B Cell‐Directed Therapies: Recent advances in B cell‐directed biological therapies for autoimmune disorders

B cell‐directed therapies are promising treatments for autoimmune disorders. Besides targeting CD20, newer B cell‐directed therapies are in development that target other B cell surface molecules and differentiation factors. An increasing number of B cell‐directed therapies are in development for the treatment of autoimmune disorders. Like rituximab, which is approved as a treatment for rheumatoid arthritis (RA), many of these newer agents deplete B cells or target pathways essential for B cell development and function; however, many questions remain about their optimal use in the clinic and about the role of B cells in disease pathogenesis. Other therapies besides rituximab that target CD20 are the furthest along in development. Besides targeting CD20, the newer B cell‐directed therapies target CD22, CD19, CD40–CD40L, B cell activating factor belonging to the TNF family (BAFF) and A proliferation‐inducing ligand (APRIL). Rituximab is being tested in an ever‐increasing number of autoimmune disorders and clinical studies of rituximab combined with other biological therapies are being pursued for the treatment of rheumatoid arthritis (RA). B cell‐directed therapies are being tested in clinical trials for a variety of autoimmune disorders including RA, systemic lupus erythematosus (SLE), Sjögren's syndrome, vasculitis, multiple sclerosis (MS), Graves' disease, idiopathic thrombocytopenia (ITP), the inflammatory myopathies (dermatomyositis and polymyositis) and the blistering skin diseases pemphigus and bullous pemphigoid. Despite the plethora of clinical studies related to B cell‐directed therapies and wealth of new information from these trials, much still remains to be discovered about the pathophysiological role of B cells in autoimmune disorders.     

Signaling pathways involved in the T‐cell‐mediated immunity against Epstein‐Barr virus: Lessons from genetic diseases

Primary immunodeficiencies (PIDs) provide researchers with unique models to understand in vivo immune responses in general and immunity to infections in particular. In humans, impaired immune control of Epstein‐Barr virus (EBV) infection is associated with the occurrence of several different immunopathologic conditions; these include non‐malignant and malignant B‐cell lymphoproliferative disorders, hemophagocytic lymphohistiocytosis (HLH), a severe inflammatory condition, and a chronic acute EBV infection of T cells. Studies of PIDs associated with a predisposition to develop severe, chronic EBV infections have led to the identification of key components of immunity to EBV – notably the central role of T‐cell expansion and its regulation in the pathophysiology of EBV‐associated diseases. On one hand, the defective expansion of EBV‐specific CD8 T cells results from mutations in genes involved in T‐cell activation (such as RASGRP1, MAGT1, and ITK), DNA metabolism (CTPS1) or co‐stimulatory pathways (CD70, CD27, and TNFSFR9 (also known as CD137/4‐1BB)) leads to impaired elimination of proliferating EBV‐infected B cells and the occurrence of lymphoma. On the other hand, protracted T‐cell expansion and activation after the defective killing of EBV‐infected B cells is caused by genetic defects in the components of the lytic granule exocytosis pathway or in the small adapter protein SH2D1A (also known as SAP), a key activator of T‐ and NK cell‐cytotoxicity. In this setting, the persistence of EBV‐infected cells results in HLH, a condition characterized by unleashed T‐cell and macrophage activation. Moreover, genetic defects causing selective vulnerability to EBV infection have highlighted the role of co‐receptor molecules (CD27, CD137, and SLAM‐R) selectively involved in immune responses against infected B cells via specific T‐B cell interactions.     

Translational Mini‐Review Series on B Cell‐Directed Therapies: B cell‐directed therapy for autoimmune diseases

B cells play an important role in the pathogenesis of both systemic and organ‐specific autoimmune diseases. Autoreactive B cells not only produce autoantibodies, but are also specialized to present specific autoantigens efficiently to T cells. Furthermore, these B cells can secrete proinflammatory cytokines and can amplify the vicious cycle of self‐destruction. Thus, B cell‐directed therapies are potentially an important approach for treating autoimmune diseases. On the other hand, like T cells, there are subsets of B cells that produce anti‐inflammatory cytokines and are immunosuppressive. These regulatory B cell subsets can protect against and ameliorate autoimmune diseases. Thus targeting B cells therapeutically will require this balance to be considered. Here we summarize the roles of pathogenic and regulatory B cells and current applications of B cell‐directed therapy in autoimmune diseases. Considerations for future development of B cell‐directed therapy for autoimmune diseases have also been discussed.     

New assay to detect low‐affinity interactions and characterization of leukocyte receptors for collagen including leukocyte‐associated Ig‐like receptor‐1 (LAIR‐1)

Leukocyte activity is controlled by numerous interactions between membrane receptors and ligands on the cell surface. These interactions are of low affinity making detection difficult. We developed a sensitive assay that could readily detect extremely weak interactions such as that between CD200 and the activating receptor CD200RLa (K_d>500 μM) at the protein level. We used the new technology to screen for interactions of inhibitory receptors for collagens. We confirmed that both human and mouse leukocyte‐associated Ig‐like receptor‐1, and in addition the related inhibitory leukocyte Ig‐like receptor subfamily B member 4 (CD85K, Gp49B), bound collagen specifically, whereas other cell surface proteins gave no binding. The monomeric affinities of the interactions were then determined to allow comparison with other leukocyte interactions and indicate conditions when these interactions might lead to inhibitory signals.     

Of self‐lipids,CD1‐restricted T cells,and contact sensitization

Contact hypersensitivity (CHS) in rodents and contact dermatitis in humans are long‐known pathological conditions caused by MHC‐restricted T‐cell responses. These responses are triggered upon T‐cell recognition of neo‐antigenic determinants, which are generated by a variety of environmental contact sensitizer (CS) chemicals associating with self‐proteins to comprise these neo‐antigens. In this issue of the European Journal of Immunology, Betts et al. [Eur. J. Immunol. 2017. 47: 1171–1180] provide intriguing data implying that common small molecule CSs such as dinitrochlorobenzene can also recruit and activate autoreactive CD1‐restricted T cells specific for cell‐endogenous lipids, which are enriched in human skin. The effects of dinitrochlorobenzene on CD1 T‐cell recruitment and function were dependent on newly synthesized CD1 molecules and the presence of endogenous lipids. These findings shed new light on unanticipated mechanisms that have potential clinical relevance on a common and highly distressing disease state.     

Interleukin‐2 treatment reverses effects of cAMP‐responsive element modulator α‐over‐expressing T cells in autoimmune‐prone mice

Systemic autoimmune diseases, such as systemic lupus erythematosus (SLE), are often characterized by a failure of self‐tolerance and result in an uncontrolled activation of B cells and effector T cells. Interleukin (IL)‐2 critically maintains homeostasis of regulatory T cells (T_reg) and effector T cells in the periphery. Previously, we identified the cAMP‐responsive element modulator α (CREMα) as a major factor responsible for decreased IL‐2 production in T cells from SLE patients. Additionally, using a transgenic mouse that specifically over‐expresses CREMα in T cells (CD2CREMαtg), we provided in‐vivo evidence that CREMα indeed suppresses IL‐2 production. To analyse the effects of CREMα in an autoimmune prone mouse model we introduced a Fas mutation in the CD2CREMαtg mice (FVB/Fas^–/–CD2CREMαtg). Overexpression of CREMα strongly accelerated the lymphadenopathy and splenomegaly in the FVB/Fas^–/– mice. This was accompanied by a massive expansion of double‐negative (DN) T cells, enhanced numbers of interferon (IFN)‐γ‐producing T cells and reduced percentages of T_regs. Treatment of FVB/Fas^–/–CD2CREMαtg mice with IL‐2 restored the percentage of T_regs and reversed increased IFN‐γ production, but did not affect the number of DNTs. Our data indicate that CREMα contributes to the failure of tolerance in SLE by favouring effector T cells and decreasing regulatory T cells, partially mediated by repression of IL‐2 in vivo .     

CD38 protein deficiency induces autoimmune characteristics and its activation enhances IL‐10 production by regulatory B cells

CD38 is a transmembrane protein expressed in B lymphocytes, and is able to induce responses as proliferation, differentiation or apoptosis. Several reports propose that CD38 deficiency accelerates autoimmune processes in murine models of autoimmune diabetes, lymphoproliferation and rheumatoid arthritis. Other reports have shown elevated CD38 expression in B and T cells from patients with autoimmunity; however, the role of CD38 is still unclear in the development of autoimmunity. Recently, it has been characterized as CD1d^hi CD5⁺ regulatory B cell subpopulation able to produce IL‐10, and the loss of these cells exacerbates the autoimmunity in murine models. Here, we report that CD38 ^{−/ −} mice exhibited elevated titres of ANAS, anti‐dsDNA autoantibodies from 12 months of age and were higher by 16 months of age and mice presented kidney damage. Interestingly, there is a reduction in the survival of CD38 ^{−/ −} mice compared to the WT. Furthermore, CD38 is highly expressed by CD1d^high CD5⁺ regulatory B cells, and the agonistic anti‐CD38 stimulus plus LPS was able to increase the percentage of this cell subset and its ability to induce IL‐10 production. Together, these results suggest that CD38 could play a role in the control of autoimmune diseases through their expression on regulatory B cells.     

Steady‐state antigen‐expressing dendritic cells terminate CD4+ memory T‐cell responses

CD4⁺ T cells are important effectors of inflammation and tissue destruction in many diseases of immune dysregulation. As memory T cells develop early during the preclinical stages of autoimmune and inflammatory diseases, immunotherapeutic approaches to treatment of these diseases, once established, must include the means to terminate memory T‐cell responses. Traditionally, it has been considered that, due to their terminally differentiated nature, memory T cells are resistant to tolerance induction, although emerging evidence indicates that some immunotherapeutic approaches can terminate memory T‐cell responses. Here, we demonstrate that CD4⁺ memory T‐cell responses can be terminated when cognate antigen is transgenically expressed in steady‐state DC. Transfer of in‐vitro‐generated CD4⁺ memory T cells establishes, in nontransgenic recipients, a stable and readily recalled memory response to cognate antigen. In contrast, upon transfer to mice expressing cognate antigen targeted to DC, memory CD4⁺ T cells undergo a phase of limited proliferation followed by substantial deletion, and recall responses are effectively silenced. This finding is important in understanding how to effectively apply immunotherapy to ongoing T‐cell‐mediated autoimmune and inflammatory diseases.     

B7‐H1 and CD8+ Treg: The enigmatic role of B7‐H1 in peripheral tolerance

The interaction between B7‐H1 (PD‐L1) expressed on APC with PD‐1 expressed by T cells was shown previously to result in inhibition of T‐cell activation and autoimmune diseases. A paper in this issue of the European Journal of Immunology demonstrates that DC B7‐H1 expression can in fact enhance autoimmunity, rather than suppress it. Using a model of direct injection of self antigen‐loaded DC into the CNS, the authors demonstrate that DC with intact B7‐H1 expression exacerbate CNS autoimmune disease. Importantly, the improved disease outcome in animals treated with B7‐H1^?/? DC is a result of a population of CD8⁺ Treg cells that expand at the site of autoimmune inflammation.     

B‐cell inhibition by cross‐linking CD79b is superior to B‐cell depletion with anti‐CD20 antibodies in treating murine collagen‐induced arthritis

Depletion of B cells with the anti‐CD20 antibody rituximab is an established therapy for rheumatoid arthritis. However, rituximab has only moderate efficacy, most likely due to insufficient depletion of B cells in lymphoid organs and expansion of pathogenic B cells. We found that an antibody against mouse CD79b profoundly blocks B‐cell proliferation induced via the B‐cell receptor, CD40, CD180, and chondroitin sulfate, but not via TLR4 or TLR9. Treatment with anti‐CD79b also induces death in resting and activated B cells. B‐cell inhibition is mediated by cross‐linkage of CD79b, but independent of Fc‐receptor engagement. In the model of collagen‐induced arthritis, an antibody against mouse CD20 depletes B cells very efficiently but fails to suppress the humoral immune response against collagen and the development of arthritis. In contrast, the antibody against CD79b, and a deglycosylated variant of this antibody, almost completely inhibits the increase in anti‐collagen antibodies and the development of arthritis. In mice with established arthritis only the fully glycosylated antibody against CD79b is effective. Our data show that targeting B cells via CD79b is much more effective than B‐cell depletion with anti‐CD20 antibodies for therapy of arthritis. These findings may have important implications for treatment of B‐cell–mediated autoimmune diseases.