The Epstein-Barr virus (EBV) transforming protein LMP1 appears to be a constitutively activated tumor necrosis factor receptor (TNFR) on the basis of an intrinsic ability to aggregate in the plasma membrane and an association of its cytoplasmic carboxyl terminus (CT) with TNFR-associated factors (TRAFs). We now show that in EBV-transformed B lymphocytes most of TRAF1 or TRAF3 and 5% of TRAF2 are associated with LMP1 and that most of LMP1 is associated with TRAF1 or TRAF3. TRAF1, TRAF2, and TRAF3 bind to a single site in the LMP1 CT corresponding to amino acids (aa) 199 to 214, within a domain which is important for B-lymphocyte growth transformation (aa 187 to 231). Latent-infection membrane protein 1 (LMP1) of EpsteinBarr virus (EBV) is essential for the ability of EBV to induce continuous proliferation of primary human B lymphocytes (20). LMP1 comprises an N-terminal cytoplasmic domain (amino acids [aa] 1 to 24), six markedly hydrophobic transmembrane domains separated by short reverse turns (aa 25 to 186), and a long cytoplasmic carboxyl terminus (CT) (aa 187 to 386) (6) (Fig. 1). Recombinant EBV genetic analyses indicate that the six transmembrane domains and the CT first 45 aa are sufficient for primary B-lymphocyte growth transformation in the context of a specifically mutated EBV recombinant (18,20,21,23).Several lines of evidence indicate that LMP1 is a constitutively activated tumor necrosis factor family receptor (TNFR) similar to activated CD40. First, expression of LMP1 in B lymphoblasts or in primary B lymphocytes results in NF-B activation and expression of Bcl-2, activation markers, adhesion molecules, and autocrine growth factors, all of which are induced in normal B lymphocytes after stimulation by 11,16,24,26,30,33,41,42). Second, a significant fraction of LMP1 constitutively aggregates in a patch in the plasma membrane of lymphoblastoid cell lines (LCLs) (12,26,27,29). The aggregation is dependent on the six hydrophobic transmembrane domains and is essential for transformation. Thus, LMP1 comprising only the last five transmembrane domains and the entire CT, i.e., LMP1 aa 44 to 386, diffusely distributes in the plasma membrane and is nontransforming (20, 26), whereas LMP1 comprising the last two transmembrane domains and the CT, i.e., LMP1 aa 129 to 386, diffusely distributes in all cytoplasmic membranes and has no effect on lymphoblasts (16,26,41). Third, the LMP1 CT aa 187 to 231, which are sufficient for transformation when linked to the six transmembrane domains [LMP1(1-231)] (23), interact with a protein that also interacts with the CD40 cytoplasmic domain (3,15,31,39); a CD40 cytoplasmic domain nonsignaling mutant fails to interact with this protein (15). This protein (previously called LAP1, CD40bp, CRAF1, or CAP1) is now designated TRAF3 because of its extensive C-terminal "TRAF domain" homology to TNFR-associated factor 2 (TRAF2) and TRAF1 (also called EBI6) (36, 37). The LMP1 CT can also bind to TRAF2 in vitro and associates with TRAF1, TRAF2, and TRAF3 in the plasma membrane of tran...

The Epstein-Barr virus-induced gene 3 (EBI3) is a novel soluble hematopoietin component related to the p40 subunit of interleukin 12 (IL-12). When EBI3 was expressed in cells, it accumulated in the endoplasmic reticulum and associated with the molecular chaperone calnexin, indicating that subsequent processing and secretion might be dependent on association with a second subunit. Coimmunoprecipitations from lysates and culture media of cells transfected with expression vectors for EBI3 and͞or the p35 subunit of IL-12 now reveal a specific association of EBI3 with p35. Coexpression of EBI3 and p35 mutually facilitates their secretion. Most importantly, a large fraction of p35 in extracts of the trophoblast component of a human full-term normal placenta specifically coimmunoprecipitated with EBI3, indicating that EBI3 is in a heterodimer with p35, in vivo. Because EBI3 is expressed in EBV-transformed B lymphocytes, tonsil, spleen, and placental trophoblasts, the EBI3͞p35 heterodimer is likely to be an important immunomodulator.

Latent infection membrane protein 1 (LMP1), the Epstein-Barr virus transforming protein, associates with tumor necrosis factor receptor (TNFR) associated factor 1 (TRAF1) and TRAF3. Since TRAF2 has been implicated in TNFR-mediated NF-KcB activation, we have evaluated the role of TRAF2 in LMPl-mediated NF-ucB activation. TRAF2 binds in vitro to the LMP1 carboxyl-terminal cytoplasmic domain (CT), coprecipitates with LMP1 in B lymphoblasts, and relocalizes to LMP1 plasma membrane patches. A dominant negative TRAF2 deletion mutant that lacks amino acids 6-86 (TRAF2A6-86) inhibits NF-KB activation from the LMP1 CT and competes with TRAF2 for LMP1 binding. TRAF2A6-86 inhibits NF-,cB activation mediated by the first 45 amino acids of the LMP1 CT by more than 75% but inhibits NF-ucB activation through the last 55 amino acids of the CT by less than 40%o. A TRAF interacting protein, TANK, inhibits NF-.cB activation by more than 70%16 from both LMP1 CT domains. These data implicate TRAF2 aggregation in NF-cB activation by the first 45 amino acids of the LMP1 CT and suggest that a different TRAF-related pathway may be involved in NF-KB activation by the last 55 amino acids of the LMP1 CT.

BCMA (B cell maturation) is a nonglycosylated integral membrane type I protein that is preferentially expressed in mature B lymphocytes. Previously, we reported in a human malignant myeloma cell line that BCMA is not primarily present on the cell surface but lies in a perinuclear structure that partially overlaps the Golgi apparatus. We now show that in transiently or stably transfected cells, BCMA is located on the cell surface, as well as in a perinulear Golgi-like structure. We also show that overexpression of BCMA in 293 cells activates NF-kappa B, Elk-1, the c-Jun N-terminal kinase, and the p38 mitogen-activated protein kinase. Coimmunoprecipitation experiments performed in transfected cells showed that BCMA associates with TNFR-associated factor (TRAF) 1, TRAF2, and TRAF3 adaptor proteins. Analysis of deletion mutants of the intracytoplasmic tail of BCMA showed that the 25-aa protein segment, from position 119 to 143, conserved between mouse and human BCMA, is essential for its association with the TRAFs and the activation of NF-kappa B, Elk-1, and c-Jun N-terminal kinase. BCMA belongs structurally to the TNFR family. Its unique TNFR motif corresponds to a variant motif present in the fourth repeat of the TNFRI molecule. This study confirms that BCMA is a functional member of the TNFR superfamily. Furthermore, as BCMA is lacking a "death domain" and its overexpression activates NF-kappa B and c-Jun N-terminal kinase, we can reasonably hypothesize that upon binding of its corresponding ligand BCMA transduces signals for cell survival and proliferation.

We have isolated a cDNA encoding a novel hematopoietin receptor family member related to the p40 subunit of interleukin-12 and to the ciliary neurotrophic factor receptor, whose expression is induced in B lymphocytes by Epstein-Barr virus (EBV) infection. This gene, which we have designated EBV-induced gene 3 (EBI3), encodes a 34-kDa glycoprotein which lacks a membrane-anchoring motif and is secreted. Despite the absence of a membrane-anchoring motif and of cysteines likely to mediate covalent linkage to an integral membrane protein, EBI3 is also present on the plasma membrane of EBV-transformed B lymphocytes and of transfected cells. Most newly synthesized EBI3 is retained in the endoplasmic reticulum in an endoglycosidase H-sensitive form associated with the molecular chaperone calnexin and with a novel 60-kDa protein. EBI3 is expressed in vivo by scattered cells in interfollicular zones of tonsil tissue, by cells associated with sinusoids in perifollicular areas of spleen tissue, and at very high levels by placental syncytiotrophoblasts. EBI3 expression in vitro is induced in EBV-negative cell lines by expression of the EBV latent infection membrane protein-1 and in peripheral blood mononuclear cells by pokeweed mitogen stimulation. EBI3 maps to chromosome 19p13.2/3, near genes encoding the erythropoietin receptor and the cytokine receptor-associated kinase, Tyk2. EBI3 synthesis by trophoblasts and by EBV-transformed cells and similarities to interleukin-12 p40 are compatible with a role for EBI3 in regulating cell-mediated immune responses.

SummaryTo understand the selective accumulation of memory T helper lymphocytes and of macrophages in delayed-type hypersensitivity (DTH) granulomas, we studied the in situ production of RANTES, a chemokine initially characterized on the basis of its in vitro chemotactic properties for each of these cell populations. RANTES gene expression was studied by in situ hybridization in 15 human lymph nodes presenting typical DTH lesions related to either sarcoidosis or tuberculosis. A positive signal was detected in all cases. Labeling was specific for the DTH lesions, as very few if any positive cells were detected in the normal residual lymphoid tissue surrounding them or in reactive lymph nodes involved in a B lymphocyte response. R.ANTES gene expression was associated with the production of the protein, which was detected by immunochemistry in DTH lymph nodes. The morphological characteristics and distribution of positive cells in in situ hybridization and immunochemical experiments indicated that macrophages and endothelial cells, two cell populations not previously reported to produce RANTES, contributed to its production in DTH reactions. The ability of macrophages and endothelial cells to produce R.ANTES was confirmed by in vitro studies with alveolar macrophages and umbilical vein endothelial cells. In view of the chemotactic properties of RANTES for a limited range of cell populations, these results suggest that RANTES production in DTH granulomas may play a role in the selective accumulation of macrophages and memory T helper lymphocytes characterizing this type of cell-mediated immune reaction, and that macrophages and endothelial cells are involved in this production.

EBV-induced gene 3 (EBI3) can associate with p28 to form the heterodimeric cytokine IL-27, or with the p35 subunit of IL-12 to form the EBI3/p35 heterodimer, recently named IL-35. In mice, IL-35 has been shown to be constitutively expressed by CD4+CD25+Foxp3+ regulatory T cells (Treg cells) and suggested to contribute to their suppressive activity. In this study, we investigated whether human Treg cells express IL-35. Double-staining analysis of human thymuses showed that neither Foxp3+ nor CD25+ cells coexpressed EBI3. Similarly, Foxp3+ cells present in human lymph nodes, tonsils, spleens, and intestines did not express EBI3. Consistent with these in situ observations, Treg cells purified from blood or tonsils were negative for EBI3 by immunoblotting. Other human T cell subsets, including effector T cells, naive and memory CD4+ T cells, CD8+ and γδ T cells also did not constitutively express EBI3, which contrasts with IL-35 expression observed in murine CD8+ and γδ T cells. Furthermore, although CD3/CD28 stimulation consistently induced low levels of EBI3 in various CD4+ T cell subsets, no EBI3 could be detected in CD3/CD28-stimulated Treg cells. RT-PCR analysis showed that, whereas p35 transcripts were detected in both Teff and Treg cells, EBI3 transcripts were detected only in activated Teff cells, but not in resting or activated Treg cells. Thus, in contrast to their murine counterpart, human Treg cells do not express detectable amounts of IL-35.

Interleukin (IL)-27 is a newly described member of the IL-12 family. It is a heterodimeric cytokine composed of two subunits, p28 and Epstein-Barr virus-induced gene 3 (EBI3). In vitro studies have shown that IL-27 is mainly produced by activated monocytes and dendritic cells. It induces the proliferation of naïve CD4-positive T cells and synergizes with IL-12 for interferon-gamma (IFN-gamma) production. Knock-out mice for the IL-27 receptor (WSX-1/TCCR) have impaired Th1 responses and form abnormal granulomas when injected with bacillus Calmette-Guérin. However, the expression profile of IL-27 in vivo is currently unknown. To investigate the potential role of IL-27 in the development of a Th1 response in humans in vivo, this study has analysed the in situ expression of IL-27 subunits in three types of granulomatous disease (tuberculosis, sarcoidosis, and Crohn's disease), each characterized by a Th1 response. Tissue sections from patients with tuberculosis (n = 9), sarcoidosis (n = 8), or Crohn's disease (n = 7) were analysed by immunohistochemistry with anti-EBI3 and anti-p28 antibodies, in parallel with control tissues (control reactive lymph nodes, n = 14, and control intestinal tissues, n = 11). In granulomatous tissues, EBI3 and p28 co-expression was detected in epithelioid and multinucleate giant cells in granulomas. In addition, sinus or tissue macrophages, endothelial cells, and plasma cells were found to co-express EBI3 and p28. These data support a possible role for IL-27 in human Th1 responses.