Mitogen-activated protein kinases are involved in cucurbitacin D-induced antitumor effects on adult T-cell leukemia cells.

Adult T cell leukemia (ATL) is an aggressive and malignant blood disease. We previously reported that steroid-structured cucurbitacin D (CuD) induces apoptosis in ATL cells. In this study, we investigated the effects of mitogen-activated protein kinase (MAPK) signaling inhibitors on CuD-induced cell death in peripheral blood lymphocytes (PBLs) isolated from ATL/acute lymphoblastic leukemia (ALL) patients and two human leukemia cell lines (MT-1 and MT-4). PBLs were isolated from an ATL/ALL patient as well as from a healthy donor. Cell surface markers were examined using flow cytometry. Serum cytokine levels were estimated using LEGENDplex or analyzed at the Center for Clinical and Translational Research of Kyushu University Hospital. Cell proliferation was assessed using the Cell Titer-Glo luminescent cell viability assay. Protein expression was determined by western blotting. PBLs from patients highly expressed CD4 and CD5. Serum from the patient contained high levels of interleukin (IL)-8, IL-10, IL-18, and interferon-γ compared to the healthy donor. CuD-induced cell death was enhanced by the mitogen-activated protein kinase kinase (MEK)1/2 inhibitor U0126. However, a c-Jun N-terminal kinase (JNK) inhibitor prevented CuD-induced cell death. Immunoblot analyses revealed that CuD reduced the phosphorylation of extracellular signal-regulated kinase (ERK), p38, and JNK, and co-treatment with CuD and U0126 did not affect the phosphorylation of ERK. MEK1/2 and p38 inhibitors enhanced CuD-induced cell death, and U0126 enhanced the CuD-induced de-phosphorylation of ERK in MT-1 and MT-4 cells. We conclude that CuD reduces ERK activation, resulting in enhanced antitumor effects on leukemic cells.

Endogenous co-expression of two T cell receptors promotes lymphopenia-induced proliferation via increased affinity for self-antigen.

Approximately 10% of peripheral T cells express 2 functional TCR αß heterodimers. Receptor co-expression changes the repertoire of TCRs produced during thymic development, enabling generation of T cells bearing TCRs not capable of mediating positive selection or that would normally be negatively selected. The effect of receptor co-expression on the composition and functionality of the peripheral TCR repertoire is not well defined, though evidence demonstrates dual TCR cells pose an increased risk for unwanted immune responses such as autoimmunity and alloreactivity. Based on our previous finding that dual TCR expression promotes positive selection, we hypothesized that dual TCR expression may enhance T cell homeostasis via increased reactivity against self-peptide:MHC (pMHC) ligands. To examine the effect of dual TCR expression on T cell homeostasis, we performed cotransfer experiments comparing T cells genetically deficient for dual TCR expression (TCRα+/- ) with wild-type T cells in models of acute and chronic lymphopenia-induced proliferation (LIP). Lack of dual TCR expression resulted in reduced LIP. The effect of dual TCR expression on LIP was most pronounced in acute lymphopenia, which is driven by recognition of low-affinity self-pMHC ligands. Differences in homeostatic proliferation were not attributable to differences in total TCR expression or signaling, but were dependent on interaction with MHC and associated with increased affinity for positively selecting self-pMHC as evidenced by higher expression of CD5 by dual TCR cells from wild-type mice. These results represent an unappreciated novel mechanism driving homeostasis and shaping the T cell repertoire, potentially promoting autoreactive or heterologous immune responses.

Prognostic impact of CD5 expression in diffuse large B-cell lymphoma in patients treated with rituximab-EPOCH.

OBJECTIVES: CD5-positive (CD5+) diffuse large B-cell lymphoma (DLBCL) represents 5%-10% of all DLBCL cases, which has been associated with a poorer prognosis in patients treated with R-CHOP. Prognostic impact of CD5 expression in patients with DLBCL treated with R-EPOCH has not been evaluated. METHODS: We studied 130 patients with de novo DLBCL who received frontline R-EPOCH therapy. The clinicopathologic features and overall survival (OS) were compared between patients with CD5+ and CD5- DLBCL. MYC, BCL2, and BCL6 rearrangements were examined by fluorescent in situ hybridization. RESULTS: Sixteen (12.3%) of 130 DLBCLs were CD5+. Most clinicopathologic features including cell of origin and frequency of MYC, BCL2, and BCL6 rearrangements were similar between CD5+ and CD5- groups. Patients with CD5+ DLBCL, however, showed higher rate of central nervous system relapse (33.3% vs 15.6%; P<.01) and a higher Ki67 proliferative index compared with CD5- patients. The median OS was significantly worse in CD5+ than CD5- patients (28.13 months vs not reached, P=.006). CD5 expression was an independent prognostic factor for OS in multivariate analysis. CONCLUSIONS: R-EPOCH therapy does not seem to impact the known poorer prognosis of patients with de novo CD5+ DLBCL, and CD5 expression was still an independent prognostic factor in R-EPOCH-treated patients with DLBCL.

Developmental expression of B cell molecules in equine lymphoid tissues.

Identification and classification of B cell subpopulations has been shown to be challenging and inconsistent among different species. Our study tested aspects of ontogeny, phenotype, tissue distribution, and function of equine CD5hi B cells, which represented a greater proportion of B cells early in development and in the peritoneal cavity. CD5hi and CD5lo B cells differentially expressed B cell markers (CD2, CD21, IgM) measured using flow cytometry, but similar mRNA expression of signature genes (DGKA, FGL2, PAX5, IGHM, IL10) measured using quantitative RT-PCR. Sequencing lambda light chain segments revealed that CD5hi B cells generated diverse immunoglobulin repertoires, and more frequently bound to fluorescence-labeled phosphorylcholine. This study shows developmental characteristics and tissue distribution of a newly described subpopulation of B cells in the horse.

Retinal pigment epithelium and microglia express the CD5 antigen-like protein, a novel autoantigen in age-related macular degeneration.

We report on a novel autoantigen expressed in human macular tissues, identified following an initial Western blot (WB)-based screening of sera from subjects with age-related macular degeneration (AMD) for circulating auto-antibodies (AAbs) recognizing macular antigens. Immunoprecipitation, 2D-gel electrophoresis (2D-GE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS), direct enzyme-linked immunosorbent assays (ELISA), WBs, immunohistochemistry (IHC), human primary and ARPE-19 immortalized cell cultures were used to characterize this novel antigen. An approximately 40-kDa autoantigen in AMD was identified as the scavenger receptor CD5 antigen-like protein (CD5L), also known as apoptosis inhibitor of macrophage (AIM). CD5L/AIM was localized to human RPE by IHC and WB methods and to retinal microglial cells by IHC. ELISAs with recombinant CD5L/AIM on a subset of AMD sera showed a nearly 2-fold higher anti-CD5L/AIM reactivity in AMD vs. Control sera (p = 0.000007). Reactivity ≥0.4 was associated with 18-fold higher odds of having AMD (χ2 = 21.42, p = 0.00063). Circulating CD5L/AIM levels were also nearly 2-fold higher in AMD sera compared to controls (p = 0.0052). The discovery of CD5L/AIM expression in the RPE and in retinal microglial cells adds to the known immunomodulatory roles of these cells in the retina. The discovery of AAbs recognizing CD5L/AIM identifies a possible novel disease biomarker and suggest a potential role for CD5L/AIM in the pathogenesis of AMD in situ. The possible mechanisms via which anti-CD5L/AIM AAbs may contribute to AMD pathogenesis are discussed. In particular, since CD5L is known to stimulate autophagy and to participate in oxidized LDL uptake in macrophages, we propose that anti-CD5L/AIM auto-antibodies may play a role in drusen biogenesis and inflammatory RPE damage in AMD.

CD5 Binds to Interleukin-6 and Induces a Feed-Forward Loop with the Transcription Factor STAT3 in B Cells to Promote Cancer.

The participation of a specific subset of B cells and how they are regulated in cancer is unclear. Here, we demonstrate that the proportion of CD5(+) relative to interleukin-6 receptor α (IL-6Rα)-expressing B cells was greatly increased in tumors. CD5(+) B cells responded to IL-6 in the absence of IL-6Rα. IL-6 directly bound to CD5, leading to activation of the transcription factor STAT3 via gp130 and its downstream kinase JAK2. STAT3 upregulated CD5 expression, thereby forming a feed-forward loop in the B cells. In mouse tumor models, CD5(+) but not CD5(-) B cells promoted tumor growth. CD5(+) B cells also showed activation of STAT3 in multiple types of human tumor tissues. Thus, our findings demonstrate a critical role of CD5(+) B cells in promoting cancer.

[A rare CD5-positive subgroup of diffuse large B-cell lymphoma - clinical, morphological and immunophenotypic features in Polish patients].

The aim of the study was to assess the incidence of CD5-positive diffuse large B-cell lymphoma (DLBCL) in the Polish population and to describe its morphologic and clinical characteristics. The study included 36 patients with CD5-positive DLBCL, diagnosed and treated in the Maria Sklodowska-Curie Institute and Oncology Centre, Warsaw, Poland and the Medical University of Warsaw, Poland in the years 2002-2013. The control group consisted of 28 patients with CD5-negative DLBCL. CD5-positive DLBCL accounted for 6.26% of all DLBCL cases diagnosed in the Maria Sklodowska-Curie Institute and Oncology Centre in the years 2008-2012. The incidence is comparable to other European countries, lower than noted in Japan and higher than in the US. Patients with CD5-positive DLBCL, in comparison to the CD5-negative group, were characterized by: (1) older age (≥ 60 vs. younger) and worse general status (ECOG ≥ 2 vs. < 2), (2) lower frequency of complete remission (CR), (3) higher expression of unfavorable prognostic factors (BCL2, FOXP1, CD44) and MMP-9, and (4) lower expression of favorable prognostic factors (CD30, cyclin D1, cyclin D3) and TIMP-2.

CD5-positive follicular lymphoma: clinicopathologic correlations and outcome in 88 cases.

Follicular lymphoma is a low-grade B-cell lymphoma of germinal center B-cell origin that typically lacks CD5 expression. We describe the clinicopathologic features of 88 cases of CD5+ follicular lymphoma (53 men, 35 women; median age, 60 years; range, 31-86). Follicular lymphoma was diagnosed initially in lymph nodes in 66 and extranodal sites in 22 patients. Eighty-one patients had lymphadenopathy, 66 had more than one involved site, 46 had bone marrow involvement, and 7 had splenomegaly. Staging information was available for 84 patients: 52 stage IV, 18 stage III, 12 stage II, and 2 stage I. Sixty-one cases were grade 1 or 2 and 27 were grade 3. The median proliferation index (Ki-67) was 30%. CD5 expression was detected by flow cytometry in 69, immunohistochemistry in 8, and both methods in 11 cases. The presence of t(14;18)(q32;q21)/IGH-BCL2 or other BCL2 translocation was detected in 28/44 (64%) cases. A total of 38 (43%) patients also had diffuse large B-cell lymphoma, concurrent with (n=20), subsequent to (n=13), or developing before CD5+ follicular lymphoma (n=5). All patients received chemotherapy; 12 also received stem-cell transplantation. With a median follow-up of 55 months (range, 0.5-207), 15 patients died, 46 were alive with disease, and 20 were in clinical remission. Compared with a matched group of patients with CD5- follicular lymphoma, patients with CD5+ follicular lymphoma more commonly had an International Prognostic Index >2 (35/80 vs 10/99, P<0.001), more often developed diffuse large B-cell lymphoma (38/88 vs 17/99; P<0.001), and had a shorter median progression-free survival (44 vs 89 months, P=0.0042). Higher Ki-67 and International Prognostic Index were identified as poor prognostic factors in both the groups. We conclude that CD5 expression in follicular lymphoma is associated with a higher International Prognostic Index, higher rate of transformation to diffuse large B-cell lymphoma, and shorter progression-free survival.

Clinical and biological significance of de novo CD5+ diffuse large B-cell lymphoma in Western countries.

CD5 is a pan-T-cell surface marker and is rarely expressed in diffuse large B-cell lymphoma (DLBCL). Large-scale studies of de novo CD5+ DLBCL are lacking in Western countries. In this study by the DLBCL Rituximab-CHOP Consortium, CD5 was expressed in 5.5% of 879 DLBCL patients from Western countries. CD5+ DLBCL was associated with higher frequencies of >1 ECOG performance status, bone marrow involvement, central nervous system relapse, activated B-cell-like subtype, Bcl-2 overexpression, and STAT3 and NF-κB activation, whereas rarely expressed single-stranded DNA-binding protein 2 (SSBP2), CD30 or had MYC mutations. With standard R-CHOP chemotherapy, CD5+ DLBCL patients had significantly worse overall survival (median, 25.3 months vs. not reached, P< .0001) and progression-free survival (median, 21.3 vs. 85.8 months, P< .0001) than CD5- DLBCL patients, which was independent of Bcl-2, STAT3, NF-κB and the International Prognostic Index. Interestingly, SSBP2 expression abolished the prognostic significance of CD5 expression, suggesting a tumor-suppressor role of SSBP2 for CD5 signaling. Gene-expression profiling demonstrated that B-cell receptor signaling dysfunction and microenvironment alterations are the important mechanisms underlying the clinical impact of CD5 expression. This study shows the distinctive clinical and biological features of CD5+ DLBCL patients in Western countries and underscores important pathways with therapeutic implications.

Splenic B cells from Hymenolepis diminuta-infected mice ameliorate colitis independent of T cells and via cooperation with macrophages.

Helminth parasites provoke multicellular immune responses in their hosts that can suppress concomitant disease. The gut lumen-dwelling tapeworm Hymenolepis diminuta, unlike other parasites assessed as helminth therapy, causes no host tissue damage while potently suppressing murine colitis. With the goal of harnessing the immunomodulatory capacity of infection with H. diminuta, we assessed the putative generation of anti-colitic regulatory B cells following H. diminuta infection. Splenic CD19(+) B cells isolated from mice infected 7 [HdBc(7(d))] and 14 d (but not 3 d) previously with H. diminuta and transferred to naive mice significantly reduced the severity of dinitrobenzene sulfonic acid (DNBS)-, oxazolone-, and dextran-sodium sulfate-induced colitis. Mechanistic studies with the DNBS model, revealed the anti-colitic HdBc(7(d)) was within the follicular B cell population and its phenotype was not dependent on IL-4 or IL-10. The HdBc(7(d)) were not characterized by increased expression of CD1d, CD5, CD23, or IL-10 production, but did spontaneously, and upon LPS plus anti-CD40 stimulation, produce more TGF-ß than CD19(+) B cells from controls. DNBS-induced colitis in RAG1(-/-) mice was inhibited by administration of HdBc(7(d)), indicating a lack of a requirement for T and B cells in the recipient; however, depletion of macrophages in recipient mice abrogated the anti-colitic effect of HdBc(7(d)). Thus, in response to H. diminuta, a putatively unique splenic CD19(+) B cell with a functional immunoregulatory program is generated that promotes the suppression of colitis dominated by TH1, TH2, or TH1-plus-TH2 events, and may do so via the synthesis of TGF-ß and the generation of, or cooperation with, a regulatory macrophage.

Egress of CD19(+)CD5(+) cells into peripheral blood following treatment with the Bruton tyrosine kinase inhibitor ibrutinib in mantle cell lymphoma patients.

Ibrutinib (PCI-32765) is a highly potent oral Bruton tyrosine kinase (BTK) inhibitor in clinical development for treating B-cell lymphoproliferative diseases. Patients with chronic lymphocytic leukemia (CLL) often show marked, transient increases of circulating CLL cells following ibrutinib treatments, as seen with other inhibitors of the B-cell receptor (BCR) pathway. In a phase 1 study of ibrutinib, we noted similar effects in patients with mantle cell lymphoma (MCL). Here, we characterize the patterns and phenotypes of cells mobilized among patients with MCL and further investigate the mechanism of this effect. Peripheral blood CD19(+)CD5(+) cells from MCL patients were found to have significant reduction in the expression of CXCR4, CD38, and Ki67 after 7 days of treatment. In addition, plasma chemokines such as CCL22, CCL4, and CXCL13 were reduced 40% to 60% after treatment. Mechanistically, ibrutinib inhibited BCR- and chemokine-mediated adhesion and chemotaxis of MCL cell lines and dose-dependently inhibited BCR, stromal cell, and CXCL12/CXCL13 stimulations of pBTK, pPLCγ2, pERK, or pAKT. Importantly, ibrutinib inhibited migration of MCL cells beneath stromal cells in coculture. We propose that BTK is essential for the homing of MCL cells into lymphoid tissues, and its inhibition results in an egress of malignant cells into peripheral blood. This trial was registered at www.clinicaltrials.gov as #NCT00114738.

Primary hepatosplenic CD5-positive diffuse large B-cell lymphoma: a case report with literature review.

De novo CD5-positive diffuse large B-cell lymphoma (CD5(+) DLBCL) accounts for approximately 10% of DLBCL, and is usually associated with aggressive clinical course. We report a case of CD5(+) DLBCL with primary involvement of the spleen and liver, and no distinct mass lesions or lymphadenopathy. The patient had stage IV disease with bone marrow involvement by lymphoma. The lymphoma cells showed characteristic portal and intrasinusoidal pattern of infiltrate in the liver. The literature was reviewed and the clinicopathologic features of 7 similar reported cases were summarized. All cases share the common features of hepatosplenomegaly without mass lesions, exclusive red pulp infiltrate with a diffuse and cordal pattern in the spleen, portal and intrasinusoidal pattern of infiltrate in the liver, and stage IV disease with poor response to conventional chemotherapy. This may represent a distinct subgroup of CD5(+) DLBCL and the diagnosis is important for prompt clinical treatment.

Recombinant murine calreticulin fragment 39-272 expands CD1d(hi)CD5+ IL-10-secreting B cells that modulate experimental autoimmune encephalomyelitis in C57BL/6 mice.

Calreticulin (CRT) is a Ca²âº binding molecular chaperone in the endoplasmic reticulum, but can also accumulate in soluble form in serum and/or synovial fluid of patients with rheumatic disorders. We have recently shown that a prokaryotically expressed recombinant CRT fragment 39-272 (rCRT/39-272) exhibited potent stimulatory activities against macrophages and B cells. However, intraperitoneal (i.p.) administration of rCRT/39-272 effectively suppressed delayed-type hypersensitivity in mice, attributable to production of anti-CRT Abs favoring Th2 cell differentiation. In this study, we further demonstrate that i.p. injection of rCRT/39-272 reduced disease severity in mouse model of experimental autoimmune encephalomyelitis (EAE), by inhibiting autoantigen-specific Th1 differentiation in vivo. Interestingly, the EAE-modulating effect of rCRT/39-272 was attributed to activation/expansion of CD1d(hi)CD5⁺ IL-10-secreting B (B10) cells rather than induction of CRT-specific antibodies in mice. In vitro, rCRT/39-272 could activate and expand murine splenic B10 cells through a Toll like receptor 4 (TLR4)-dependent pathway. The rCRT-activated B10 cells were able to not only enhance Th2 cell differentiation in vitro but also reduce EAE scores of recipient animals in passive transfer experiments. These results revealed soluble CRT, likely released by tissue cells under stress conditions, as a potentially important multi-faced player in immunoregulation and immunopathological responses.

B cell: T cell interactions occur within hepatic granulomas during experimental visceral leishmaniasis.

Hepatic resistance to Leishmania donovani infection in mice is associated with the development of granulomas, in which a variety of lymphoid and non-lymphoid populations accumulate. Although previous studies have identified B cells in hepatic granulomas and functional studies in B cell-deficient mice have suggested a role for B cells in the control of experimental visceral leishmaniasis, little is known about the behaviour of B cells in the granuloma microenvironment. Here, we first compared the hepatic B cell population in infected mice, where ≈60% of B cells are located within granulomas, with that of naïve mice. In infected mice, there was a small increase in mIgM(lo)mIgD(+) mature B2 cells, but no enrichment of B cells with regulatory phenotype or function compared to the naïve hepatic B cell population, as assessed by CD1d and CD5 expression and by IL-10 production. Using 2-photon microscopy to quantify the entire intra-granuloma B cell population, in conjunction with the adoptive transfer of polyclonal and HEL-specific BCR-transgenic B cells isolated from L. donovani-infected mice, we demonstrated that B cells accumulate in granulomas over time in an antigen-independent manner. Intra-vital dynamic imaging was used to demonstrate that within the polyclonal B cell population obtained from L. donovani-infected mice, the frequency of B cells that made multiple long contacts with endogenous T cells was greater than that observed using HEL-specific B cells obtained from the same inflammatory environment. These data indicate, therefore, that a subset of this polyclonal B cell population is capable of making cognate interactions with T cells within this unique environment, and provide the first insights into the dynamics of B cells within an inflammatory site.

Immunologic and cytogenetic markers expressed in non-Hodgkin lymphoma of head and neck.

Very often, the first doctor who examines a patient with malignant lymphoma is an ENT specialist, because non-Hodgkin lymphoma is five times more frequent than Hodgkin disease in the head and neck region. Approximately 25% of extranodal lymphoma occurs in the head and neck and extranodal presentation is twice as frequent as nodal presentation. This paper present a study of the patients from ENT, Head & Neck Surgery Clinic of Coltea Clinical Hospital, Bucharest, Romania, diagnosed with malignant lymphoma. We developed a specific scheme for collecting data about patients, together with pathology details, immunology and cytogenetic markers. We tried to establish a relation between immunologic and cytogenetic markers and the clinical evolution of non-Hodgkin lymphoma. For this study, we analyzed data regarding CD10, CD5, CD20, Bcl-2, Bcl-6, Ki67 expression obtained from 58 patients with follicular lymphoma. An attempt was made to correlate the presence of certain immunohistochemical and cytogenetic markers with the evolution and aggressiveness of the disease, and we can say that Bcl-2 is positive in all tumor subtypes, being associated relatively frequently with CD5 expression, and is a marker of poor prognosis, while Bcl-6 is positive especially in the tumor forms associated with the predominance of small cells and is a marker of favorable prognosis.