Begomovirus coat protein interacts with a small heat-shock protein of its transmission vector (Bemisia tabaci).

Tomato yellow leaf curl Sardinia virus (TYLCSV) is transmitted from plant to plant by the whitefly Bemisia tabaci in a persistent-circulative manner. The coat protein (CP) plays an important role in this transmission cycle. In this study, the CP was used to screen a Bemisia tabaci cDNA library using the yeast two-hybrid system, in a search for interacting partners. A member of the small heat-shock protein family (termed BtHSP16) was identified and its interaction with the CP was verified by an in vitro pull-down assay. The binding domain was located at the variable N-terminal part of the CP, while full-length BtHSP16 is required for the interaction. The putative role for this interaction in the transmission cycle by the whitefly is discussed.

The Tomato mosaic virus 30 kDa movement protein interacts differentially with the resistance genes Tm-2 and Tm-2(2).

In tomato plants ( Lycopersicon esculentum Mill.), the genes Tm-2 and Tm-2(2) confer resistance to Tomato mosaic virus (ToMV). Sequence analysis of ToMV strains able to break the Tm-2 or Tm-2(2) resistance revealed distinct amino acid exchanges in the viral 30 kDa protein, suggesting that the movement protein is recognized by both resistance genes to induce the plant defense reaction. To analyze the interactions between the ToMV movement protein and the Tm-2 and Tm-2(2) genes in detail, we generated transgenic tomato lines expressing various movement protein gene constructs. Crosses of the transgenic tomato lines with cultivars containing either the Tm-2 or the Tm-2(2) gene demonstrated that both genes are able to elicit a hypersensitive reaction in response to movement proteins from resistance inducing ToMV strains. However, the domains and the structural requirements for induction of the necrotic response by the ToMV movement protein are completely different for either resistance gene. In the context of the Tm-2 gene, the resistance determinant lies within the N-terminal 188 amino acids of the ToMV movement protein. Interaction of the 30 kDa protein with the Tm-2(2) gene requires two distinct domains localized at the C-terminus and in a different region of the protein, respectively.

Immunotherapy of human tumors with T-cell-activating bispecific antibodies: stimulation of cytotoxic pathways in vivo.

Bispecific monoclonal antibodies (Bi-mAbs) specific for a tumor-associated antigen and the CD3 or CD28 antigen on T lymphocytes represent one of the most successful experimental strategies for the immunotherapy of cancer. We report that the in vivo administration of both alpha-CD3/CD30 and alpha-CD28/CD30 Bi-mAbs results in the specific activation of xenotransplanted, resting human T cells infiltrating the CD30-positive Hodgkin's tumor. Bi-mAb treatment resulted in enhanced expression of cytokines such as interleukin 1beta, interleukin 2, tumor necrosis factor type alpha, and activation markers including Ki-67, CD25, and CD45RO in tumor-infiltrating lymphocytes. This antigen-dependent, local T-cell stimulation led to the activation of the cytolytic machinery in T lymphocytes, determined by the up-regulation of mRNA-encoding perforin and the cytotoxic serine-esterases granzymes A and B. The Bi-mAb-induced generation of CTLs depended on the presence of the CD30 antigen and the combined application of both Bi-mAbs. Our findings suggest that the combined application of T-cell-activating Bi-mAbs is able to achieve a tumor site-specific activation of the T-cell cytolytic machinery in vivo. The fact that these cytotoxic cells do not home in tumor-associated antigen-negative tissue and do not enter circulation might explain our previous observations (C. Renner et al., Blood, 87: 2930-2937, 1996) of a high cure rate in preclinical models even at an advanced stage of disease.

RP1, a new member of the adenomatous polyposis coli-binding EB1-like gene family, is differentially expressed in activated T cells.

Cross-linking of the CD3 and CD28 molecules on T lymphocytes represents one of the most effective signals for T lymphocyte activation and triggering of their cytotoxic effector function. To identify genes that are expressed in T cells after stimulation, mRNA from T lymphocytes that had been activated by the simultaneous stimulation of the CD3 and CD28 trigger molecules was transcribed for a differential mRNA display analysis into cDNA and was compared with cDNA from CD28- or CD3-activated or resting lymphocytes. Differential expression was confirmed subsequently by Northern blot analysis. One of the cDNA fragments expressed specifically in CD3- and CD28-activated T cells was designated RP1. The predictive protein-coding region of RP1 had a significant homology to members of the recently found adenomatous polyposis coli (APC) protein-binding EB1 gene family, which codes for yet unknown protein(s). Bacterially expressed RP1 protein revealed specific binding to wild-type but not to mutated APC protein. The rapid up-regulation of RP1 mRNA in properly activated T cells suggests that this gene might belong to the immediate/early gene family, which controls the signal transduction cascade downstream of the TCR. As the expression level of the RP1 gene in activated T cells and a spectrum of tumor-derived cell lines correlates with the proliferative status of the cells, members of the EB1-like gene family may not only be involved in the tumorigenesis of colorectal cancers but may also play a role in the proliferative control of normal cells.

Role of naive and memory T cells in tumor cell lysis mediated by bi-specific antibodies.

Bispecific monoclonal antibodies (Bi-mAb) with specificity for a tumor associated antigen and the CD3 or CD28 antigen on T lymphocytes, respectively, induce activation of resting T lymphocytes and target-specific tumor cell lysis. Former studies had confirmed that T cells expressing the CD45RO "memory" antigen at high levels were the most potent effectors of Bi-mAb-mediated cytotoxicity when compared to their "naive" counterparts expressing the CD45RA antigen. Further analysis of the T cell subpopulations revealed that within the memory T cell pool, CD8+ T cells were the effector cell, population with strongest cytolytic activity. The cytolytic activity was correlated with the expression level of perforin and granzymes B mRNA. Ca2+ complexing agents, which abrogate perforin activity, reduced necrosis, while inhibition of granzyme activity in effector or target-cells had a similar effect on apoptosis. These results confirm the crucial role perforin and granzymes play in target-cell lysis and explain why CD8+CD45RO+ T cells activated by combined CD3 and CD28 antigen triggering represent the T cell pool with highest cytolytic potential.

Role of perforin, granzymes and the proliferative state of the target cells in apoptosis and necrosis mediated by bispecific-antibody-activated cytotoxic T cells.

Bispecific monoclonal antibodies (bi-mAb), directed against a tumor-associated antigen and the CD3 or CD28 antigen on T lymphocytes, induce activation of resting T lymphocytes and target-specific tumor cell lysis. We now show that both necrosis and apoptosis contribute to T-cell-mediated tumor cell destruction. Even though T cells up-regulate FAS/APO-1 expression upon bi-mAb stimulation, FAS/APO-1-mediated apoptosis does not contribute to bi-mAb-mediated destruction of Hodgkin's cells. CD8+ lymphocytes were the most potent effectors of bi-mAb-mediated cytotoxicity and had the highest levels of mRNA coding for perforin and granzyme A and B. Ca2+-complexing agents, which abrogate perforin activity, led to decreased levels of necrosis, while inhibition of granzyme activity in effector or target cells had a similar effect on apoptosis. Granzyme-mediated apoptosis critically dependent on the proliferative state of the target cells, while perforin-induced necrosis was not cell-cycle-dependent. Our results underline the importance of the expression levels of perforin and granzymes in the effector T cells and of the proliferative state of the target cells in bi-mAb-mediated apoptosis and necrosis of tumor cells.

T cells from patients with Hodgkin's disease have a defective T-cell receptor zeta chain expression that is reversible by T-cell stimulation with CD3 and CD28.

To investigate the mechanisms underlying the deficiency of T lymphocytes from patients with Hodgkin's disease, we investigated the expression of the T-cell receptor (TCR) zeta chain in patients with Hodgkin's disease. By flow cytometry using an anti-zeta chain monoclonal antibody, peripheral blood T lymphocytes from patients with untreated Hodgkin's disease were shown to express decreased levels of the TCR zeta chain. After stimulation by combined CD3 and CD28 cross-linking, T cells from Hodgkin's disease patients upregulated zeta chain protein expression to normal values within 48 hours and achieved a cytolytic potential and levels of interleukin (IL)-2 secretion that were not different from T cells obtained from healthy controls. These results show that downregulation of the TCR zeta chain in Hodgkin's T lymphocytes is a reversible event. Costimulation of CD3 and CD28 is a novel approach for overcoming the T-cell deficiency in Hodgkin's disease and might be exploited clinically. As upregulation of the zeta chain can also be achieved using bispecific monoclonal antibodies (BI-MoAbs) with specificity for tumor antigens and CD3 and CD28, respectively, an immunotherapy with CD3/CD30 and CD28/CD30 Bi-MoAbs may overcome and should therefore, not be jeopardized by the inherent T-cell deficiency in patients with Hodgkin's disease.

Interleukin-12 increases bispecific-antibody-mediated natural killer cell cytotoxicity against human tumors.

The combination of CD16/CD30 bispecific monoclonal antibodies (bi-mAb) and unstimulated human resting natural killer (NK) cells can cure about 50% of mice with severe combined immunodeficiency (SCID) bearing subcutaneously growing established Hodgkin's lymphoma. As interleukin-2 (IL-2) and IL-12 have been shown to increase NK cell activity, we tested the capacity of these cytokines to increase bi-mAb-mediated NK cell cytotoxicity against two types of human tumors (Hodgkin's disease and colorectal carcinoma). Unstimulated NK cells needed a three- to five-times higher antibody concentration than cytokine-stimulated NK cells to exert similar levels of bi-mAb-mediated cytotoxicity. The augmented tumor cell lysis was achieved with IL-12 at considerably lower concentrations than with IL-2 and was associated with a significantly increased bi-mAb-mediated intracellular Ca2+ mobilization. The efficiency of IL-12 in this setting together with its low toxicity make it the ideal candidate for a combination therapy with NK-cell-activating bi-mAb in human tumors that are resistant to standard treatment.