Ricardo García-Muñoz

IL-10 suppressor activity and ex vivo Tr1 cell function are impaired in multiple sclerosis.

T regulatory cells type 1 (Tr1 cells) are excellent candidates for cell therapy in multiple sclerosis (MS). The aim of our study was to assess the functional state of Tr1 cells and IL‐10R signaling in patients with MS. Tr1 cells were induced in vitro by activation with anti‐CD46 antibodies in controls and patients with MS. Cells were phenotyped by cytometry and suppression assays, and the expression of cytokines and transcription factors was evaluated by real‐time PCR, ELISA, cytometry and Western blotting. We found that the activity of Tr1 cells and IL‐10R signaling is impaired in MS patients since Tr1 cells isolated from MS patients produced less IL‐10 than those obtained from controls. Indeed, the supernatants from Tr1 cells from controls did not suppress the proliferation of stimulated CD4+ cells from patients with MS. Furthermore, the IL‐10R signaling pathway was not fully active in CD4+ cells from MS patients and these cells had higher baseline levels of SOCS3 transcripts than controls. Indeed, after in vitro IL‐10 stimulation, the expression levels of the STAT1, STAT3 and IL‐10RA genes were higher in MS patients than in controls. Moreover, Stat‐3 phosphorylation was lower in controls than in patients after IL‐10 stimulation. These results indicate that IL‐10 regulatory function is impaired in patients with MS.

Immunological aspects in chronic lymphocytic leukemia (CLL) development

Chronic lymphocytic leukemia (CLL) is unique among B cell malignancies in that the malignant clones can be featured either somatically mutated or unmutated IGVH genes. CLL cells that express unmutated immunoglobulin variable domains likely underwent final development prior to their entry into the germinal center, whereas those that express mutated variable domains likely transited through the germinal center and then underwent final development. Regardless, the cellular origin of CLL remains unknown. The aim of this review is to summarize immunological aspects involved in this process and to provide insights about the complex biology and pathogenesis of this disease. We propose a mechanistic hypothesis to explain the origin of B-CLL clones into our current picture of normal B cell development. In particular, we suggest that unmutated CLL arises from normal B cells with self-reactivity for apoptotic bodies that have undergone receptor editing, CD5 expression, and anergic processes in the bone marrow. Similarly, mutated CLL would arise from cells that, while acquiring self-reactivity for autoantigens—including apoptotic bodies—in germinal centers, are also still subject to tolerization mechanisms, including receptor editing and anergy. We believe that CLL is a proliferation of B lymphocytes selected during clonal expansion through multiple encounters with (auto)antigens, despite the fact that they differ in their state of activation and maturation. Autoantigens and microbial pathogens activate BCR signaling and promote tolerogenic mechanisms such as receptor editing/revision, anergy, CD5+ expression, and somatic hypermutation in CLL B cells. The result of these tolerogenic mechanisms is the survival of CLL B cell clones with similar surface markers and homogeneous gene expression signatures. We suggest that both immunophenotypic surface markers and homogenous gene expression might represent the evidence of several attempts to re-educate self-reactive B cells.

Parvovirus B19 acute infection and a reactivation of cytomegalovirus and herpesvirus 6 in a chronic myeloid leukemia patient during treatment with dasatinib (BMS-354825).

T lymphocyte activation is controlled by the T cell antigen receptor (TCR) in combination with additional signals triggered by accessory molecules present on the surface of the antigen-presenting cells (APC). Several studies have reported that treatment with imatinib can induce inhibition of the T-cell mediated immune response [1,2]. Imatinib inhibits T cell receptor-mediated T cell proliferation and activation in a dose-dependent manner and reduces tyrosine phosphorylation of ZAP-70 and LAT in response to activation through TCR [1]. Studies conducted in a mouse model indicate that imatinib treatment also leads to a selective inhibition of memory CTLs without affecting primary T or B cells responses [2]. Recent reports describing the development of severe viral infections such as varicella-zoster [3] and fatal hepatitis B virus reactivation in patients with CML treated with imatinib along with in vitro data suggest that the potential for immune suppression and viral reactivation in patients treated with abl and src kinase inhibitors should be considered [4]. We describe the development of a cytomegalovirus and herpes 6 virus associated with treatment with dasatinib (BMS354825) in a patient with diagnosis of CML. A 53-year-old woman was diagnosed with CML in November 2003. Treatment with imatinib was initiated at doses of 400 mg/day and further increased to 800 mg/day due to the persistence of BCR-ABL chromosome after 1 year. Treatment with imatinib was discontinued 2 years after diagnosis (September 2005) due to the development of grade III/IV cutaneous and gastrointestinal complications. In March 2006 treatment with dasatinib (BMS354825) was started at doses of 140 mg/day. One month after initiation of treatment with dasatinib, the patient was admitted to the hospital with atypical pneumonia (bilateral infiltrates in chest radiography, fever, dry cough, dyspnea and pleuritic pain). Despite extensive work-up, only three positive citomegalovirus cells were identified by the rapid viral culture (shell vial method) from peripheral blood. The patient was treated with antiviral (Ganciclovir) and antibiotic therapy (TeicoplaninMeropenem) with good response and was discharged after 15 days. In May 2006, the patient presented with a generalized cutaneous rash with positive serological test for parvovirus B19 (IgG titer 512 and IgM titer 40) and herpesvirus 6 (IgG titer 80). This pattern is compatible with an acute infection from parvovirus B19 because detectable low levels of B19 specific IgM can be found within 7 – 10 days of the exposure. We excluded the possibility to herpesvirus 6 infection because the patient had low levels of specific herpesvirus 6 IgG and a percentage of healthy adults are IgM positive at any time, making

Autoimmunity and lymphoma: Is mantle cell lymphoma a mistake of the receptor editing mechanism?

Mantle cell lymphoma (MCL) is an aggressive B cell malignancy, which is believed to originate from naïve B cells in the mantle zone of lymph nodes. We speculate that a possible mechanistic hypothesis for the generation of MCL is one in which receptor editing and germinal centre exclusion could be involved in the molecular development and in the display of clinical characteristics of this rare, aggressive and scarcely understood lymphoma. The hypothesis is supported by a preferential autoimmune related IGVH gene utilization in MCL, where VH3-21, VH4-34 and VH5-51 genes are predominant, and by the fact that MCL expresses immunoglobulin light chain (IgL) lambda more frequently than other non-Hodgkins lymphomas and that IgL lambda-producing B cells usually delete one or both their IgL kappa genes.

Splenic marginal zone lymphoma with Evans’ syndrome, autoimmunity, and peripheral gamma/delta T cells

Dear Editor, Several theories have tried to explain the occurrence of autoantibodies in lymphoid malignancies. Autoimmune phenomena like Evans’ syndrome, lupus anticoagulants, autoimmune thrombocytopenia, and autoimmune hemolytic anemias are frequently associated with lymphomas [4, 5]. Recently, systemic lupus erythematosus (SLE) was associated with an increased risk of diffuse large B cell and marginal zone lymphomas [3]. Splenic marginal zone lymphoma (SMZL) is an indolent B cell lymphoma, which generally presents splenomegaly and involvement of both bone marrow and peripheral blood. In this report, we describe a patient with autoimmunity and bone marrow infiltration that probably broke the mechanisms of central tolerance in bone marrow producing the emergency of new auto-reactive clones and autoantibodies. A 60-year-old man had a 12-month history of B symptoms. Physical examination was unremarkable, except for the presence of massive splenomegaly. A computerized tomograhpy (CT) scan of the abdomen confirmed splenomegaly and visualized enlarged lymph nodes in retroperitoneum (Fig. 1). Blood smear and peripheral blood immunophenotype suggest SMZL with villous lymphocytes. The patient underwent laparotomy for splenectomy, lymph node, and bone marrow biopsies. Histology corresponds to SMZL. Follow-up studies detected the persistence of lymphoid neoplastic cells (1.4%) and a γδ T cell population (TCR γδ CD2+, CD3+, CD5 low+, CD7+, CD8 low+/−, CD16 −/+ CD56+, CD57 +/−) that represented 12% of the cells in peripheral blood. Five months after diagnosis, the patient presented with abdominal discomfort. On a CT scan, abdominal adenopatic progression was observed and the two populations (3.4% of SMZL and 4.9% of γδ T cells) persisted in peripheral blood. The patient received eight cycles of chemotherapy with cyclophosphamide–doxorubicin–vincristin–prednisone regime, resulting in radiological remission. At this moment, flow cytometry of peripheral blood revealed 0.0026% of tumor cells and 13.75% of γδ T cells. Four months after chemotherapy, the patient was admitted to the hospital with a recurrence of SZML. He showed, besides, respiratory insufficiency and Evans’ syndrome, associated with high titers of antiphospholipid antibodies, lupus anticoagulant and prolonged partial tromboplastin time, high levels of immune complexes, and low levels of both immunoglobulins and C3 and C4 fractions of complement. Antinuclear, antineutrophil cytoplasmic, anticardiolipin, antiplatelets, anti-HLA I, and antiEPCR antibodies were also detected. CT scan ascertained the presence of pulmonary emphysema and hepatomegaly, without associated enlarged lymph nodes. Peripheral blood flow-cytometry indicated an increase in the neoplastic B cell population (50%) and a decrease of γδ T cells (5%). The bone marrow biopsy confirmed lymphoid infiltration (SMZL 35% and γδ T cells 4%) by immunophenotype. Ann Hematol (2009) 88:177–178 DOI 10.1007/s00277-008-0555-z

Homeobox NKX2-3 promotes marginal-zone lymphomagenesis by activating B-cell receptor signalling and shaping lymphocyte dynamics

NKX2 homeobox family proteins have a role in cancer development. Here we show that NKX2-3 is overexpressed in tumour cells from a subset of patients with marginal-zone lymphomas, but not with other B-cell malignancies. While Nkx2-3-deficient mice exhibit the absence of marginal-zone B cells, transgenic mice with expression of NKX2-3 in B cells show marginal-zone expansion that leads to the development of tumours, faithfully recapitulating the principal clinical and biological features of human marginal-zone lymphomas. NKX2-3 induces B-cell receptor signalling by phosphorylating Lyn/Syk kinases, which in turn activate multiple integrins (LFA-1, VLA-4), adhesion molecules (ICAM-1, MadCAM-1) and the chemokine receptor CXCR4. These molecules enhance migration, polarization and homing of B cells to splenic and extranodal tissues, eventually driving malignant transformation through triggering NF-κB and PI3K-AKT pathways. This study implicates oncogenic NKX2-3 in lymphomagenesis, and provides a valid experimental mouse model for studying the biology and therapy of human marginal-zone B-cell lymphomas.

Chronic lymphocytic leukaemia: could immunological tolerance mechanisms be the origin of lymphoid neoplasms?

Immunological tolerance theory in chronic lymphocytic leukaemia (CLL): we suggest that B cells that express B‐cell receptors (BCR) that recognize their own BCR epitopes are viewed by immune system as ‘dangerous cells’. BCR autonomous signalling may induce constant receptor editing and mistakes in allelic exclusion. The fact that whole BCR recognizes a self‐antigen or foreing antigen may be irrelevant in early B cell development. In early B cells, autonomous signalling induced by recognition of the BCR’s own epitopes simulates an antigen‐antibody engagement. In the bone marrow this interaction is viewed as recognition of self‐molecules and induces receptor editing. In mature B cells autonomous signalling by the BCR may promote ‘reversible anergy’ and also may correct self‐reactivity induced by the somatic hypermutation mechanisms in mutated CLL B cells. However, in unmutated CLL B cells, BCR autonomous signalling in addition to self‐antigen recognition augments B cell activation, proliferation and genomic instability. We suggest that CLL originates from a coordinated normal immunologic tolerance mechanism to destroy self‐reactive B cells. Additional genetic damage induced by tolerance mechanisms may immortalize self‐reactive B cells and transform them into a leukemia.

Common variable immunodeficiency and Evans syndrome complicated by autoimmune hemolysis due to anti-Jka auto-antibodies

Common variable immunodeficiency (CVID) is a syndrome characterised by generalised defective antibody production and recurrent infections. A marked decrease in serum IgG associated with significant reduction of IgM and/or IgA levels is the hallmark of the disease [1]. Patients with CVID are at increased risk of developing autoimmune disorders and malignancy, as a consequence of abnormal immune regulation as well as immunodeficiency [2]. Treatment options include corticosteroids and intravenous immunoglobulin [3]. Twenty percent of patients with CVID develop clinical features suggestive of autoimmune disease. Our report is the first case of CVID and Evans syndrome, complicated with autoimmune hemolysis due to anti-JKa auto-antibodies. A 54-year-old man with a diagnosis of CVID was admitted to our hospital in August 2007 with a severe Coombs-positive autoimmune hemolytic anemia (AIHA). He reported jaundice, fatigue and symptoms and signs of anemia in the days preceding admission. Laboratory studies showed normal platelet and white blood cell counts with a normal differentiation, but severe anemia (Hb of 6.5 g/dL) with reticulocytosis was found. As bilirubin (5.32 mg/dL) and LDH (480 UI/L) were all elevated, hemolysis was suspected. The patient had a positive direct anti-globulin test (DAT) with IgG (7), IgA (7), IgM (7) C3d(þ) and a serum auto-antibody with anti-JKa specificity in the eluate prepared from his red blood cells. Indirect anti-globulin test (IAT) also was positive. Serum immunoglobulin levels were IgG 441 mg/ dL, IgA 22 mg/dL, IgM 22 mg/dL and IgD 4 UI/L. Low serum levels of IgG1 262 mg/dL, IgG2 93 mg/ dL and IgG4 0.36 mg/dL were also found. Serum protein electrophoresis showed hypogammaglobulinemia with no evidence of monoclonal gammopathy. C4 10 mg/dL and C3 74 mg/dL fractions of complement were low. Anti-nuclear antibodies, Mycoplasma and viral serologies were negative but anti-platelet antibodies against GP IIb/IIIa, Ib/IX and Ia/IIa were found. Normal levels of CD4, CD8 T cells and B cells were detected; however significant low levels of NK cells were observed (Table I). When dissecting the B cell compartment that comprises 3.2% (195 cells/mL) of peripheral blood cells (PBLs) according to surface Ig and CD27 expression, we detected a significant decrease in the class switched IgD-CD27þ memory B cells containing only 0.18% (3 cells/mL). The remaining memory B cells 1.62% (99 cells/mL) of PBLs corresponded to non class switched CD27þIgDþ B cells. Within this last memory subpopulation the majority of cells expressed CD27þIgMþIgDþ phenotype (48.81%), suggesting an expansion of splenic marginal zone memory cells B (Table II). The patient had a history of CVID diagnosed 18 years prior to admission and complicated by splenomegaly, pyogenic skin infections, respiratory infections, neutropenia, immune thrombocytopenic purpura (ITP) and autoimmune hemolytic anemia (Evans syndrome), that had been treated with IVIG

Acute lymphoblastic leukemia developing during maintenance therapy with lenalidomide in a patient with multiple myeloma

In an attempt to improve the duration of response to therapy in multiple myeloma, maintenance strategies using interferon, thalidomide and corticosteroids have been developed. Unfortunately, data relating to the use of interferon-a as maintenance therapy did not show significant benefit in terms of both prolonged disease response and overall survival [1]. High-dose corticosteroid maintenance (50 mg oral prednisone given on alternate days) resulted in the prolongation of disease response and overall survival in patients; however, no data are available regarding its efficacy post-high-dose therapy/autologous stem cell transplant (HDT/ASCT) [2]. Newer approaches such as those based on thalidomide are often limited by the development of side effects, including peripheral neuropathy and thrombo embolic disease, and the majority of patients discontinue therapy because of this [3]. Recently, three high-profile randomized placebo-controlled trials employing lenalidomide as maintenance therapy in patients with multiple myeloma have been published [4–6]. Importantly, a marked prolongation in progression-free survival (PFS) in all three trials was reported, and one study also demonstrated a survival benefit with maintenance lenalidomide [4–6]. This notwithstanding, some experts consider that as of today, there are insufficient data to recommend routine lenalidomide maintenance to all patients with multiple myeloma, given the lack of survival benefit in two of the three trials and the risk of secondary malignancies [7]. Herein, we report a case of a patient with multiple myeloma who developed acute lymphoblastic leukemia (ALL) during maintenance treatment with lenalidomide. A 62-year-old female patient was diagnosed with multiple myeloma, immunoglobulin G (IgG) lambda, stage IIIB (creatinine 2.03 mg/dL) in January 2009; she received treatment with four cycles of bortezomib–dexamethasone (February 2009) with a partial response and renal function improvement. However, she developed peripheral neuropathy and hypotension, which forced withdrawal of treatment with bortezomib. She continued treatment with high dexamethasone doses for two cycles (June and July), with no response. In order to reach an intense response prior to HDT/ASCT, she received two alternating chemotherapy cycles of vincristine, BCNU, melphalan, cyclophosphamide, prednisone/vincristine, BCNU, adriamycin and dexamethasone (VBMCP/VBAD) (September 2009–January 2010), without response. The patient was thus treated with three cycles of the combination of lenalidomide plus dexamethasone (February– April 2010) with a very good, albeit partial, response. The patient was consolidated with ASCT conditioned with melphalan 200 mg/m2 in June 2010. Six months after ASCT the patient remained positive on immunofixation assay. She started maintenance with lenalidomide (10 mg  21-day cycles every 28 days) in November 2010. In the 20th maintenance cycle in July 2012, the patient required a lower dose of lenalidomide (5 mg) because of thrombocytopenia and neutropenia. Two weeks later the patient was observed to have noticeable cytopenias, but without blasts in the peripheral blood smear. The patient started granulocyte-colony stimulating factor (G-CSF) therapy and withdrew lenalidomide. One week later, pancytopenia as well as asthenia, gait instability and dizziness were observed. A bone marrow aspiration was performed that demonstrated 47% of blast infiltration with irregular nuclei and variable numbers of distinct nucleoli. Vacuoles were seen occasionally. Multiparametric flow cytometry demonstrated CD19 CD34 CD10  CD20 CD22, cytoplasmic Cd79a, terminal deoxynucleotidyl transferase (TdT) and IgMc blasts, compatible with pre-B cell ALL. Cytogenetics showed a normal karyotype. Computed tomography of the brain was normal. A lumbar puncture was performed to obtain cerebrospinal fluid for cytological and flow cytometric analyses, which were negative for leukemic infiltration or myelomatosis. The patient received treatment with induction therapy for adult ALL; unfortunately she died of septicemia. B cell ALL is a clonal hematopoietic stem cell disorder with evidence of early B cell differentiation. The disease is characterized by the presence of a rapidly proliferating population of immature blasts, with minimal morphologic evidence of differentiation. Distinguishing these tumors generally requires immunophenotypic demonstration of B L eu k L ym ph om a D ow nl oa de d fr om in fo rm ah ea lth ca re .c om b y U ni ve rs ity o f C on ne ct ic ut o n 10 /2 8/ 14

First-line use of rituximab correlates with increased overall survival in late post-transplant lymphoproliferative disorders: retrospective, single-centre study

This retrospective study evaluates the impact of rituximab on PTLD response and survival in a single‐centre cohort. PTLD cases between 1984 and 2009, including heart, kidney, liver and lung transplant recipients, were included. Survival was analysed taking into account the type of PTLD (monomorphic vs. polymorphic), EBV infection status, IPI score, Ann Arbor stage and use of rituximab. Among 1335 transplanted patients, 24 developed PTLD. Median age was 54 yr (range 29–69), median time to diagnosis 50 months (range 0–100). PTLD type was predominantly late/monomorphic (79% and 75%), mostly diffuse large B‐cell type. Overall response rate (ORR) was 62% (66% rituximab vs. 50% non‐rituximab; P = 0.5). R‐CHOP‐like regimens were used most frequently (72% of patients treated with rituximab). Median overall survival was 64 months (CI 95% 31–96). OS was significantly increased in patients treated with rituximab (P = 0.01; CI 95% rituximab 58–79 months; non‐rituximab 1–30 months). Post‐transplant immunosuppression regimen had no effect on survival or time to PTLD, except for cyclosporine A (CyA), which associated with increased time to PTLD (P = 0.02). Rituximab was associated with increased survival in our single‐centre series, and it should be considered as first‐line therapy for PTLD patients. The possible protective effect of CyA for development of PTLD should be prospectively evaluated.