Esther J. M. Schilder-Tol

Signaling through CD44 Is Mediated by Tyrosine Kinases ASSOCIATION WITH p56lck IN T LYMPHOCYTES

Evidence from a large body of studies indicates that CD44 is involved in a number of important biological processes, including lymphocyte activation and homing, hematopoiesis, and tumor progression and metastasis. A proper understanding of the role of CD44 in these processes has been severely hampered by a lack of insight into the mode in which CD44 communicates with intracellular signal transduction pathways. In this report, we have addressed this aspect of CD44 functioning by studying CD44 signaling in T lymphocytes. We show that ligation of CD44 by monoclonal antibodies (mAbs) transduces signals to T cells which lead to tyrosine phosphorylation of ZAP-70 and other intracellular proteins. In vitro kinase assays demonstrate that cross-linking of CD44 induces an increase in the intrinsic activity of p56lck. Furthermore, immunoprecipitations show that CD44 is physically associated with p56lck. Our findings suggest that tyrosine kinases, particularly p56lck, play a central role in CD44 mediated signaling.

High prevalence of oncogenic MYD88 and CD79B mutations in primary testicular diffuse large B-cell lymphoma

High prevalence of oncogenic MYD88 and CD79B mutations in primary testicular diffuse large B-cell lymphoma

Strong expression of CD134 (OX40), a member of the TNF receptor family, in a T helper 2-type cytokine environment.

CD134 (OX40) is involved in T cell costimulation and T cell‐dependent antibody production. We show strongly increased T cell expression of CD134 in a model of T helper 2‐mediated systemic autoimmunity, induced by HgCl2. Regulation of CD134 expression on CD4+ T cells was further studied in vitro, identifying CD134 as an early marker of T cell activation. CD134 expression could be induced by interleukin‐4, but not by interferon‐γ or tumor necrosis factor‐α. Effects of interleukin‐4 and of phorbol 12‐myristate 13‐acetate on CD134 expression could be blocked by the protein kinase inhibitor staurosporin. Combination of these stimuli with ionomycin resulted in a strongly synergistic increase of CD134 expression, which was blocked by the calcineurin‐inhibitor cyclosporin A. The results demonstrate the involvement of two synergistically acting pathways in induction of CD134 expression. Furthermore, they suggest a role for interleukin‐4 in induction of CD134 expression in vivo. J. Leukoc. Biol. 64: 503–510; 1998.

Chlamydia psittaci -negative ocular adnexal marginal zone B-cell lymphomas have biased V H 4-34 immunoglobulin gene expression and proliferate in a distinct inflammatory environment

Ocular adnexal marginal zone B-cell lymphomas (OAMZLs) arise in the connective tissues of the orbit or in the mucosa-associated lymphoid tissue of the conjunctiva. Here, we present the immunological and genetic analyses of 20 primary Chlamydia psittaci (Cp)-negative OAMZLs. Analysis of the immunoglobulin variable heavy chain (IgVH) gene usage demonstrated a significant preference for VH4-34. A combined analysis across all previously published OAMZLs confirmed that this is a general feature of OAMZL, in particular of the Cp-negative group. Our series of OAMZLs did not express the characteristic rheumatoid factor VHDJH rearrangements that were previously found in salivary gland- and gastric-marginal zone B-cell lymphomas (MZBCLs). We did not detect the MZBCL-specific chromosomal translocations, t(11;18) API2-MALT1 (mucosa-associated lymphoid tissue1) and t(14;18) IgH/MALT1. Two cases contained a premature stop codon in the A20 gene (TNFAIP3) and one case harbored the activating MYD88 hotspot mutation L265P. Variable nuclear expression of BCL10, NFκB1 (p50) and NFκB2 (p52) suggests that other additional genetic abnormalities affecting the NFκB pathway exist within this group of lymphomas. OAMZL showed variable expression of the chemokine receptor CXCR3 and integrin α4β7 by the tumor B cells, and low interferon-γ and interlukin-4 mRNA levels in the tissue, indicative of an inflammatory environment with features in between those previously found in cutaneous and other extranodal MZBCL. The strongly biased usage of VH4-34 in Cp-negative OAMZLs suggests involvement of a particular stimulatory (auto-) antigen in their development.

Differential regulation of expression of the MHC class II molecules RT1.B and RT1.D on rat B lymphocytes: effects of interleukin-4, interleukin-13 and interferon-gamma

Susceptibility to induction of both T helper 1‐ (Th1) and Th2‐mediated autoimmunity is multifactorial and involves genetic linkage to the major histocompatibility complex (MHC) class II haplotype. Brown Norway (BN) rats exposed to mercuric chloride develop a Th2‐dependent systemic autoimmunity, whereas Lewis rats, which are highly susceptible to Th1‐mediated autoimmunity, develop immune suppression after mercuric chloride exposure. Exposure to mercuric chloride is known to enhance B‐lymphocyte expression of the MHC class II molecule RT1.B, predominantly in BN rats. We demonstrate that, in contrast, expression of RT1.D was unmodified on these B cells, whereas both RT1.B and RT1.D were up‐regulated on epithelial cells. Regulation of B‐cell MHC class II isotype expression was further studied in vitro, using BN rat lymph node (LN) cells. Interleukin‐4 (IL‐4) strongly enhanced B‐cell expression of RT1.B (2·8‐fold), whereas RT1.D expression was only slightly, although significantly, modified (1·2‐fold). B cells from Lewis rats showed a similar IL‐4‐induced enhancement of RT1.B expression (2·5‐fold), whereas, in contrast, RT1.D expression was unmodified. Exposure of LN cells from BN rats to interferon‐γ induced a moderate increase of B‐cell MHC class II expression, predominantly of RT1.B. Strong and rapid enhancement of B‐cell RT1.D expression was observed after stimulation by phorbol 12‐myristate 13‐acetate and ionomycin. Rat IL‐13 did not modify B‐cell MHC class II expression; however, it induced typical morphological changes in peritoneal macrophages. These experiments demonstrate isotype‐specific and strain‐dependent regulation of MHC class II expression on rat B lymphocytes, which may be of pathophysiological relevance for the strain‐dependent susceptibility for Th1‐ or Th2‐mediated autoimmunity.

Differential Expression of T‐Cell Adhesion Molecules and LFA‐1‐Dependent Intercellular Adhesion in HgCl2‐Induced Autoimmunity and Immune Suppression

Exposure of Brown Norway (BN) rats to HgCl2 induces Th2‐mediated systemic autoimmunity. In contrast, in Lewis rats, HgCl2 induces immune suppression, mediated by CD8+ T cells. HgCl2 was previously found to enhance expression of LFA‐1, ICAM‐1 and CD134 (OX40) on T cells in BN rats. In the present study, T cells from Lewis rats were studied at day 4 after injection of HgCl2. CD8+ T lymphoblasts were significantly increased, which were predominantly CD45RChi, and which showed enhanced LFA‐1 expression. Furthermore, CD4+CD45RChi T cells showed increased numbers of ICAM‐1+ cells, whereas expression of CD134 and CD26 was relatively decreased in CD4+ T lymphoblasts. Ex vivo experiments demonstrated that HgCl2‐ exposure of BN rats, but not of Lewis rats, significantly enhances PMA [phorbol 12‐myristate 13‐acetate]‐induced lymphocyte aggregation, mediated by LFA‐1 and ICAM‐1. In conclusion, HgCl2‐injected Lewis rats show early signs of T‐lymphocyte activation, predominantly on CD8+ cells. Strain‐dependent effects of HgCl2 on cell adhesion molecules and expression of CD134 may play an important role in development of either autoimmunity or immune suppression.

B-Lymphoblastic Lymphomas Evolving from Follicular Lymphomas Co-Express Surrogate Light Chains and Mutated Gamma Heavy Chains

Follicular lymphoma (FL) is an indolent B-cell non-Hodgkin lymphoma able to transform into germinal center-type diffuse large B-cell lymphoma. We describe four extraordinary cases of FL, which progressed to TdT+CD20- precursor B-lymphoblastic lymphoma (B-LBL). Fluorescence in situ hybridization analysis showed that all four B-LBLs had acquired a MYC translocation on transformation. Comparative genomic hybridization analysis of one case demonstrated that in addition to 26 numerical aberrations that were shared between the FL and B-LBL, deletion of CDKN2A/B and 17q11, 14q32 amplification, and copy-neutral loss of heterozygosity of 9p were gained in the B-LBL cells. Whole-exome sequencing revealed mutations in FMN2, NEB, and SYNE1 and a nonsense mutation in KMT2D, all shared by the FL and B-LBL, and TNFRSF14, SMARCA2, CCND3 mutations uniquely present in the B-LBL. Remarkably, all four FL-B-LBL pairs expressed IgG. In two B-LBLs, evidence was obtained for ongoing rearrangement of IG light chain variable genes and expression of the surrogate light chain. IGHV mutation analysis showed that all FL-B-LBL pairs harbored identical or near-identical somatic mutations. From the somatic gene alterations found in the IG and non-IG genes, we conclude that the FLs and B-LBLs did not develop in parallel from early t(14;18)-positive IG-unmutated precursors, but that the B-LBLs developed from preexistent FL subclones that accumulated additional genetic damage.

The immune dysregulatory compound mercuric chloride induces integrin-mediated T-lymphocyte adhesion

Exposure of Brown Norway rats to mercuric chloride induces systemic autoimmunity, involving T‐ and B‐lymphocyte activation, (auto‐)antibody production and multiorgan inflammation. Several divalent metal ions, such as Mg2+ and Mn2+, can activate binding of integrins to their ligands, thus causing lymphocyte adhesion. To test the hypothesis that Hg2+ acts in a similar way, we studied the effect of HgCl2 on integrin‐mediated T‐cell adhesion. HgCl2 induced cell–cell aggregation of human T lymphoblasts. Exposure of a human T‐cell clone to HgCl2 for 1 hr enhanced, in a dose‐dependent way, cell binding to fibronectin (FN) and to intercellular adhesion molecules (ICAM) ‐1, ‐2 and ‐3. Furthermore, HgCl2 induced strong binding of Jurkat T cells to FN. These effects of HgCl2 were of similar magnitude as the effects of phorbol 12‐myristate 13‐acetate (PMA) or MnCl2. Studies using blocking antibodies indicated the involvement of CD11a in binding to ICAMs, and of CD49d, CD49e, and CD29 in binding to FN. Adhesion to FN induced by HgCl2 or by PMA, but not by MnCl2, was dependent on temperature and on extracellular Ca2+ or Mg2+. Addition of cytochalasin B enhanced synergistically the FN adhesion induced by MnCl2, whereas the effects of PMA and HgCl2 were not modified. These results indicate that Hg2+ is a potent activator of T‐cell adhesion, mediated by several integrins and ligands. In contrast to the effect of MnCl2, HgCl2‐induced cell adhesion probably involves an intracellular pathway. Activation of integrins by HgCl2 may play an important role in activation and migration of leucocytes involved in HgCl2‐induced immune dysregulation in vivo.