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CD82 is a tetraspan transmembrane protein on NK/LAK-susceptible targets. A single highly glycosylated protein of heterogeneous molecular mass (50-90 kDa) was immunoprecipitated by anti-CD82 from Nonidet P-40 lysates of various B cell lines, Raji, Daudi, 721, and 721.134. Using the milder detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), additional proteins were coprecipitated with CD82 from surface iodinated B cell lines, including a major band at 45 kDa, identified as the HLA class I heavy chain by sequential immunoprecipitations and sequential immunoprecipitation-Western blot analysis. Cocapping experiments confirmed the molecular association of CD82 and HLA class I at the cell surface of these B cell lines. CD82 could be coprecipitated with both mature and beta 2-microglobulin (beta 2m)-free heavy chains of MHC-I from CHAPS extracts. No association between MHC-I and CD82 was found in the beta 2m-deficient Daudi cell line or after co-in vitro translation of CD82, MHC heavy chain, and beta 2m mRNA. The most likely source of free class I heavy chains at the cell surface is by dissociation of beta 2m-associated class I molecules. These results suggest that association of CD82-MHC-I takes place at the cell surface and could interfere with the capacity of the MHC-I complex to protect targets from NK-mediated cytotoxicity.

In vitro and in vivo experiments indicate that the production of interleukin 2 (IL 2) by T lymphocytes is critical for the development of the effector phase of immunity. Complex cellular interactions are involved for the induction of IL 2 production. We have shown in a previous study that in humans monocytes can transmit opposite signals to the IL 2-producing cells. In addition to the positive signal delivered through the release of interleukin 1, human monocytes can deliver a negative signal through the release of prostaglandin E2 (PGE2). This monokine, known to activate suppressor mechanisms in several systems, was shown to inhibit IL 2 production. The data presented in this paper show that this PGE2-dependent inhibition is strictly dependent upon the presence of radiosensitive T cells in the culture, suggesting that PGE2 induces the activation of suppressor T cells modulating IL 2 production. Kinetics experiments indicate that these suppressor cells are radiosensitive during their induction phase but become radioresistant after 18 hr of incubation in the presence of PGE2. Successful in vitro induction of suppressor cells by incubation of enriched T cells with PGE2 was decisive for the analysis of the phenomenon. The induced suppressors were capable of inhibiting IL 2 production by fresh autologous T cells as well as inhibiting PHA proliferative response by these cells. A quantitative evaluation of IL 2 receptors on PGE2-treated cells has indicated that this absorption capacity was similar to the capacity of PBL known to express a low number of IL 2 receptors, thus excluding a suppression via absorption or competition for IL 2. No detectable killing of IL 2-producing cells by PGE2-induced suppressors was observed. The OKT4 and OKT8 phenotype of suppressor cells was examined. T cells were purified at two stages of differentiation before or after induction by PGE2 in vitro treatment. We conclude from these experiments that PGE2 activates suppressor cells among precursors segregating predominantly with the OKT8 subset and fewer cells with the OKT4 subset. After differentiation, however, the suppressor cells segregate with the OKT8 subset only. Such results were obtained by using positive selection (cellular affinity columns) and negative selection (monoclonal antibodies plus complement).

Cytokines may exert anti-inflammatory properties, allowing the hypothesis of their potential therapeutic use. Targeting the cytokines balance may modify the course of subsequent inflammatory events in an autoimmune disease. Many data are available. Interferon-gamma can be blocked by an anti-interferon-gamma monoclonal antibody: if the treatment is administered in the early phase of an autoimmune disease such as collagen-induced arthritis, the course of the disease is worsened; if the treatment is given later, the disease can be improved; these results are mirrored by treatment with high doses of the cytokine itself. Pharmacologic effects of antiinflammatory cytokines such as interleukin (IL)-4, IL-10 or IL-13 are a protection against several experimental autoimmune diseases. The very short half-life of cytokines makes them difficult and expensive to use directly; in such occurrence, high quantities have to be frequently injected. In this context, gene therapy appears as an effective alternative solution: the transfection of cells with cytokines genes (e.g. either IL-4 or IL-13) then the engraftment of these vectors in animals, permit the in vivo secretion of high levels of cytokines, and result in the protection of the animals from the development of the disease.

Gene therapy is rheumatoid arthritis is presently in an experimental phase. Genes encoding for antiinflammatory proteins can be transfected in joint cells. Therefore gene encoding for interleukin-1 receptor antagonist has been transfected into synovial cells or into chondrocytes. Gene expression can be maintained as long as 8 weeks after cell transfer. We have reported that clinical and histopathological parameters of collagen-induced arthritis (an animal model of rheumatoid arthritis) can be reduced by engraftment of CHO cells transfected with genes encoding for antiinflammatory cytokines such as interleukine-4 or interleukine-13. Some of the questions which need to be assessed before planning clinical trial are the choice of vectors and the most efficient target cells; and the genes to be transfected have to be chosen among many candidates.

The role of MHC-encoded class I molecules in allogeneic activation and proliferation of human T lymphocytes was investigated. The study was performed by using primary mixed culture of lymphocytes from MHC recombinant siblings identical for MHC class II Ag (DR, DP, DQ) and displaying MHC class I disparity. The results indicate that such allogeneic combination is sufficient to trigger early activation steps within responder T cells without promoting a significant proliferation. After MHC class I allosensitization, a significant proportion of cells entered the cell cycle (G0----G1). The stimulatory potential of MHC class I Ag was further stressed by the specific induction on responder cells of IL-2R (22% T cell activation Ag positive). Under the same experimental conditions, transferrin receptor expression and IL-2 activity were not detectable. This is consistent with the low T cell proliferation. Exogenous rIL-1 did not improve IL-2 production and the subsequent T cell proliferation indicating that these two events were not associated with a defective accessory cell function involving IL-1 release. MHC class I disparity can also prime precursor CTL to differentiate into IL-2-dependent functional MHC restricted cytotoxic T cells. Conversely IFN-gamma had no effect. Addition to the culture of W6/32, a mAb specifically directed against a monomorphic determinant on human class I HLA-A, -B, and -C Ag was able to block all these activation events. These data clearly indicate a role of HLA class I Ag involvement in the early events triggering allogeneic T cell activation.

Patients with active Hodgkin's disease demonstrate a significant depression of cellular immunity. The present study, performed with lymphocytes from 16 untreated patients with active Hodgkin's disease and 13 healthy control volunteers, demonstrate an equivalent IL 2 production in vitro in both groups. Our results, however, demonstrate an abnormal regulation of IL 2 production in patients. A negative control of IL 2 production involving monocytes producing PGE 2 able to induce radiosensitive suppressor T lymphocytes, has been identified previously with cells from healthy donors. In the present study we demonstrate that this negative control is hyper functioning with cells from Hodgkin's disease patients.

Monocyte chemotactic protein-3 (MCP-3) is a C-C chemokine that interacts with the CCR1, CCR2, and CCR3 receptors and has a spectrum of action encompassing T cells, NK cells, eosinophils, and dendritic cells (DC), in addition to mononuclear phagocytes. This broad spectrum of action prompted the present study aimed at assessing the antitumor activity of MCP-3 in a gene transfer approach and at providing information as to the actual in vivo leukocyte recruiting capacity of MCP-3. P815 mastocytoma cells transfected with the gene coding MCP-3 (P815/MCP-3) grew in syngeneic hosts and underwent rejection. Rejection was associated with profound alterations of leukocyte infiltration and resistance to subsequent challenge with P815 cells. Tumor-associated macrophages, already present in copious numbers, T cells, eosinophils, and neutrophils, increased in tumor tissues after gene transfer. DC, identified as DEC205+, high MHC class II+, CD11c+ cells, did not increase substantially in the tumor mass. However, in peritumoral tissues, DC accumulated in perivascular areas. P815/MCP-3-transfected tumor cells grew normally in nude mice. Increased accumulation of macrophages and polymorphonuclear neutrophils was evident also in nude mice. mAb against CD4, CD8, and IFN-gamma, but not against IL-4, inhibited rejection of MCP-3-producing cells. An anti-polymorphonuclear mAb caused only a retardation of MCP-3-elicited tumor rejection. Thus, MCP-3 gene transfer elicits tumor rejection by activating type I T cell-dependent immunity. It is tempting to speculate that altered trafficking of APCs, which express receptors and respond to MCP-3, together with recruitment of activated T cells, underlies activation of specific immunity by MCP-3-transfected cells.