Search

Evolution of apoptosis resistance in both lymphoma and leukemia cells is well documented, and induction of apoptosis in malignant cells is a major goal of cancer therapy. Up-regulation of anti-apoptotic signals is one of the mechanisms whereby resistance to apoptosis emerges. We have previously described the fusion proteins CD40·FasL and CTLA-4·FasL, which are formed from two functional membrane proteins and induce apoptosis of activated T cells. The present study explores the potential use of CD40·FasL and CTLA-4·FasL for the killing of malignant cells of lymphatic origin. Using malignant B and T cell lines that differ in surface expression of costimulatory molecules, we found that CTLA-4·FasL induces effective apoptosis of cells expressing CD95 and activates caspases 3, 8, and 9. Only B7-expressing B cells responded to CTLA-4·FasL with rapid abrogation of cFLIP expression. CD40·FasL effectively killed only the T cells that express high levels of CD40L in addition to CD95. In these cells, CD40·FasL significantly diminished cFLIP expression. Importantly, each of the fusion proteins is more potent than its respective components parts, alone or in combination. Thus, the proteins with their two functional ends deliver a pro-apoptotic signal and, in parallel, inhibit an anti-apoptotic signal, thus optimizing the wanted, death-inducing effect. Therefore, these proteins emerge as promising agents to be used for targeted and specific tumor cell killing.

Hallmarks of the pathogenesis of autoimmune encephalomyelitis include perivascular infiltration of inflammatory cells into the central nervous system, multifocal demyelination in the brain and spinal cord, and focal neuronal degeneration. Optimal treatment of this complex disease will ultimately call for agents that target the spectrum of underlying pathogenic processes. In the present study, Fn14-TRAIL is introduced as a unique immunotherapeutic fusion protein that is designed to exchange and redirect intercellular signals within inflammatory cell networks, and, in so doing, to impact multiple pathogenic events and yield a net anti-inflammatory effect. In this soluble protein product, a Fn14 receptor component (capable of blocking the pro-inflammatory TWEAK ligand) is fused to a TRAIL ligand (capable of inhibiting activated, pathogenic T cells). Sustained Fn14-TRAIL expression was obtained in vivo using a transposon-based eukaryotic expression vector. Fn14-TRAIL expression effectively prevented chronic, nonremitting, paralytic disease in myelin oligodendrocyte glycoprotein-challenged C57BL/6 mice. Disease suppression in this model was reflected by decreases in the clinical score, disease incidence, nervous tissue inflammation, and Th1, Th2, and Th17 cytokine responses. Significantly, the therapeutic efficacy of Fn14-TRAIL could not be recapitulated simply by administering its component parts (Fn14 and TRAIL) as soluble agents, either alone or in combination. Its functional pleiotropism was manifest in its additional ability to attenuate the enhanced permeability of the blood-brain barrier that typically accompanies autoimmune encephalomyelitis.

CTLA-4.Fas ligand (CTLA-4.FasL), a paradigmatic 'trans signal converter protein (TSCP)', can attach to APC (via CTLA-4 binding to B7) and direct intercellular inhibitory signals to responding T cells (via FasL binding to Fas receptor), converting an activating APC-to-T cell signal into an inhibitory one. Our previous studies established that CTLA-4.FasL inhibits human primary mixed lymphocyte reactions (MLR) and induces alloantigen-specific hyporesponsiveness ex vivo. The present study extends this to an in vivo context. Using splenocytes from MHC-mismatched C57BL/6 and Balb/c mice, we demonstrated that his(6)CTLA-4.FasL, effectively inhibits murine MLR. Moving in vivo, we demonstrated that subcutaneously administered his(6)CTLA-4.FasL modulates the in vivo response of infused allogeneic splenocytes. his(6)CTLA-4.FasL reduces the number of cells in each cell division, and increases the percentage of dead cells in each division. These findings are consistent with an antigen-induced cell death of the alloreactive cells, and bolsters recombinant TCSP promise as a therapeutic for transplantation diseases.

A successful pregnancy can only occur when the maternal immune system fails to attack the allogeneic fetus. Two plasma proteins with described immunoregulatory activities, pregnancy zone protein (PZP) and placental protein-14 (PP14; also known as glycodelin-A), increase dramatically during pregnancy, prompting us to examine their potential role in mediating fetal protection. First, we demonstrated that both native PZP and its receptor-recognized monoamine-activated form (MA-PZP) bound non-covalently and specifically to PP14, exhibiting K(d) values greater than 3 microM, as determined by surface plasmon resonance. Our evidence further suggests that PZP is potentially a more effective carrier of PP14 than its relative alpha2-macroglobulin. Second, we found that T-cell activation, as measured by increased proliferation and IL-2 production, was inhibited by either PZP or PP14 in a dose-dependent manner. However, when PZP and PP14 were combined, they acted synergistically to inhibit T cell proliferation and IL-2 production. Interestingly, the combination of PZP and PP14 had little effect on the production of T(H)2 cytokine, IL-4. Based upon these findings, we hypothesize that PZP and PP14 form a stable complex in the plasma of pregnant women and together act synergistically to selectively modulate T-cell activation. Mechanistically, this activity appears to be independent of the PZP receptor (CD91) or PZP's anti-proteinase activity.

Antigen-presenting cells (APC) can express surface ligands with both T cell activating and inhibitory capacities, prompting the question of how responding T cells integrate opposing trans signals concurrently delivered by APC. To address this question in a quantitative fashion, we turned to protein transfer as a unique experimental approach that is well-suited for addressing such questions from a quantitative standpoint. Costimulatory (either B7-1*Fc(gamma1) or Fc(gamma1)*4-1BBL) and pro-apoptotic (Fc(gamma1)*FasL) Fc fusion proteins were quantitatively "painted" in varying ratios onto surrogate APC pre-coated with palmitated-protein A, the latter serving as a surface anchor. Evaluating the signaling potential of these various painted cells in a standard in vitro T cell proliferation assay, we demonstrated that at a given level of TCR triggering, the quantitative balance between costimulator (B7-1 or 4-1BBL) and FasL dictates the magnitude of the proliferative T cell response. Furthermore, when the costimulator density is kept constant, there is also a quantitative balance between TCR-directed and FasL signals. Interesting species-specific nai;ve versus memory T cell subset differences emerged with regard to susceptibility to Fas-mediated apoptosis and costimulator:FasL opposition. Taken together, these data demonstrate for the first time a quantitative interplay between activating and pro-apoptotic trans signals that dictates the magnitude of T cell responses.

The activation of discrete T-cell responses depends on the triggering of individualized threshold numbers of T-cell receptors (TCRs). The results of this study indicate that the lipocalin placental protein 14 (PP14), a T-cell inhibitor produced by cells of the reproductive and hematopoietic systems, mediates its anti-inflammatory activity by elevating the T-cell activation threshold, thereby rendering T cells less sensitive to stimulation. Significantly, the data demonstrate hierarchical sensitivity of selected cytokine responses to PP14-mediated inhibition, with the hierarchy reflecting their respective activation thresholds. These findings suggest a novel paradigm for immunoinhibition wherein negative regulators can finely tune, rather than inactivate, T-cell responses, and thereby skew the cytokine output of immunologic responses.

Human placental protein 14 (PP14, also referred to as glycodelin and progesterone-associated endometrial protein) inhibits phytohemagglutinin (PHA)-stimulated T-cell proliferation and monokine secretion within PBMC populations. However, the mechanisms underlying these and other PP14 immunoinhibitory activities remain unclear. In the present study, we asked whether PP14's T-cell inhibitory effect is a direct one or, alternatively, an indirect consequence of accessory cell (AC) perturbation. Using either immunopurified PP14 or first-trimester amniotic fluid (AF) as a rich source of PP14, we documented inhibition of the proliferation of highly purified peripheral blood T-cells when stimulated with anti-CD3 mAbs or PHA in the presence of paraformaldehyde-fixed AC. Significantly, PP14 inhibited T-cell proliferation and IL-2 secretion induced by immobilized anti-CD3 and anti-CD28 mAbs in the absence of AC. PP14 depletion (via immunoprecipitation) abrogated AF's T-cell inhibitory activity, indicating that the PP14 within the amniotic fluid is required for this immunoregulatory effect. These findings establish that PP14 can inhibit T-cell proliferation in the absence of AC and thus add PP14 to the relatively restricted set of immunoinhibitory proteins that are known to target T-cells directly. Additional data demonstrate that PP14's inhibitory effect can be overridden by stimuli which circumvent early events during T-cell receptor (TCR) activation, namely, protein kinase C activators in combination with Ca2+ ionophores. These latter results suggest that PP14 inhibits early events in the TCR signaling pathway., (Copyright 1999 Academic Press.)

At inflammatory sites, proteoglycans are both secreted by activated mononuclear leukocytes and released as a consequence of extracellular matrix degradation. Chondroitin 4-sulfate proteoglycans constitute the predominant ones produced by activated human monocytes/macrophages. In this study, we show that two chondroitin 4-sulfate forms, CSA and CSB, can activate distinct peripheral blood mononuclear cell types. Whereas CSA activates monocytes (to secrete monokines), CSB activates B-cells (to proliferate). In contrast, the chondroitin 6-sulfate CSC and heparin do not exert these functional effects. We further show that CD44 monoclonal antibodies block CSB-induced B-cell proliferation. These findings point to glycosaminoglycans, and specifically chondroitin 4-sulfates, as a novel class of immunological mediators at inflammatory sites. Furthermore, the data link CD44 to B-cell activation, paralleling the established roles of CD44 in T-cell and monocyte activation.

Placental protein 14 (PP14), an immunosuppressive molecule previously known to be expressed in the female and male reproductive tracts only, was shown to be expressed by hematopoietic cells of the megakaryocytic lineage. Northern blot analysis confirmed the induction specificity of PP14 mRNA in phorbol ester-treated K562 cells. Potent immunosuppressive activity in conditioned medium from phorbol ester-treated K562 cells was attributed to hematopoietic PP14 by anti-PP14 antibody blocking. Immunoprecipitation with anti-PP14 antibodies from conditioned medium revealed two distinct PP14 protein isoforms, designated PP14.1 and PP14.2. Polymerase chain reaction cloning and analysis demonstrated the presence of distinct mRNA counterparts to PP14.1 and PP14.2 that had not been resolved by Northern blot analyses. Hematopoietic PP14.1 mRNA corresponds in size to endometrial PP14 mRNA, whereas the smaller hematopoietic PP14.2 mRNA displays an internal in-frame 66-nucleotide deletion that can be explained by alternative splicing and predicts a 22-amino-acid deletion in the encoded gene product. Both PP14 mRNA isoforms were additionally detected by reverse transcriptase polymerase chain reaction analyses in two human megakaryocytic cell lines and in normal human megakaryocytes and platelets. PP14 mRNA was not detected by reverse transcriptase polymerase chain reaction in a panel of nonhematopoietic, nonendometrial tissues examined. The finding of hematopoietic PP14 within the megakaryocytic lineage provides an additional regulatory link between the coagulation and immune systems in normal and pathological settings.

Bone marrow (BM) stromal cell inhibition of leukemic cell differentiation was studied in cellular coculture experiments. In coculture, a significant percentage of cells from the human myeloid leukemic cell lines HL-60, PLB-985, and K562 adhere to fibroblastic KM-102 BM stromal cells. A sensitive two-color immunofluorescence assay was developed to monitor stromal cellular effects upon leukemic cell differentiation. After chemical induction with 1 alpha,25-dihydroxyvitamin D3, strongly adherent HL-60 and PLB-985 cells were inhibited from differentiating into more mature monocytic cells, as measured by the monocytic surface marker CD14. In contrast, loosely adherent and nonadherent HL-60 and PLB-985 leukemic cells in the same cocultures, as well as both adherent and nonadherent K562 cells induced with phorbol ester, were not blocked in their capacity to differentiate. Scanning electron microscopy and intercellular dye transfer experiments correlated intimate stromal cell/leukemic cell interaction and intercellular communication with the blockade of leukemic cell differentiation. These studies indicate that there is significant variability among leukemic lines with respect to the nature of their adhesion to stromal cells. Moreover, the data implicate gap-junction formation as a potentially significant event in stromal cell-mediated leukemic cell regulation.