Functional consequences of anti-sense RNA-mediated inhibition of CD8 surface expression in a human T cell clone.

An experimental approach for defining the function of CD8 has been developed by linking anti-sense RNA mutagenesis and T cell cloning technologies. We have transfected an anti-sense CD8 episomal expression vector into a CD8+ nontransformed human T cell clone that is specific for the human class I alloantigen HLA-B35. Expression of CD8 on this T cell clone, JH.ARL.1, was selectively and efficiently inhibited. Stimulation of this CD8- variant with specific alloantigen resulted in a marked loss of a number of functional responses, including cytotoxicity, proliferation, IL-2 secretion, and IL-2-R expression. However, these same functional responses could be elicited with stimuli that do not require antigen recognition to activate the T cell (anti-CD3 mAbs, PHA). The results of our study support the hypothesis that CD8 is required for recognition of class I MHC alloantigens that results in activation of T cell functional responses.

The gene encoding decay-accelerating factor (DAF) is located in the complement-regulatory locus on the long arm of chromosome 1.

Delay-accelerating factor (DAF) protects host cells from complement-mediated damage by regulating the activation of C3 convertases on host cell surfaces. Using a panel of hamster-human somatic cell hybrids, the DAF gene was mapped to human chromosome 1. In situ hybridization studies using human metaphase cells further localized the gene to bands 1q31-41, with the largest cluster of grains at 1q32. This establishes the close linkage of the DAF gene to genes for four other proteins (C3b/C4b receptor or complement receptor 1, C3d receptor or complement receptor 2, factor H, and C4-binding protein) that share 60-amino-acid homologous repeats as well as complement-regulatory or -receptor activity, thereby enlarging the complement-regulatory gene family on the long arm of human chromosome 1.

Treatment and prevention of rat glioblastoma by immunogenic C6 cells expressing antisense insulin-like growth factor I RNA.

Rat C6 glioma cells express insulin-like growth factor I (IGF-I) and form rapidly growing tumors in syngeneic animals. When transfected with an episome-based vector encoding antisense IGF-I complementary DNA, these cells lost tumorigenicity. Subcutaneous injection of IGF-I antisense-transfected C6 cells into rats prevented formation of both subcutaneous tumors and brain tumors induced by nontransfected C6 cells. The antisense-transfected cells also caused regression of established brain glioblastomas when injected at a point distal to the tumor. These antitumor effects result from a glioma-specific immune response involving CD8+ lymphocytes. Antisense blocking of IGF-I expression may reverse a phenotype that allows C6 glioma cells to evade the immune system.

Decay-accelerating factor protects human tumor cells from complement-mediated cytotoxicity in vitro.

The disialoganglioside GD2 is expressed on a wide spectrum of human tumor types, including neuroblastomas and melanomas. Upon binding of 3F8, a murine monoclonal antibody (MAb) specific for GD2, neuroblastomas and some melanomas are sensitive to killing by human complement, whereas some melanomas are not. To investigate the mechanism underlying these differences in complement mediated cytotoxicity, complement-insensitive melanoma cell lines were compared with respect to expression of the decay-accelerating factor (DAF), a membrane regulatory protein that protects blood cells from autologous complement attack. While DAF was undetectable among neuroblastomas, it was present in complement-insensitive melanomas. When the function of DAF was blocked by anti-DAF MAb, C3 uptake and complement-mediated lysis of the insensitive melanoma lines were markedly enhanced. F(ab')2 fragments were as effective in enhancing lysis as intact anti-DAF MAb. The DAF-negative and DAF-positive melanoma cell lines were comparably resistant to passive lysis by cobra venom factor-treated serum. The data suggest that in some tumors, DAF activity accounts for their resistance to complement-mediated killing. The ability to render these cells complement-sensitive by blocking DAF function may have implications for immunotherapy.

Addition of lipid substituents of mammalian protein glycosylphosphoinositol anchors.

A single metabolic path leading to synthesis of ether lipids is known in animal cells, the major products of which are plasmalogens. To learn whether this peroxisomal path is also responsible for the synthesis of base-resistant lipid components of glycosylphosphoinositol (GPI)-anchored membrane proteins, we have investigated the structure of anchor precursor mannolipids both in wild-type cells (CHO-K1 and a macrophage-like line, RAW 264.7) and in two corresponding mutant cells in which ether lipid biosynthesis is severely impaired. We observe that the precursor mannolipids of both the wild-type and mutant cells do not include alkylglycerol. Nevertheless, both wild-type and mutant cells express cell surface GPI-anchored placental alkaline phosphatase (AP) which includes alkali-resistant hydrophobic chains in its anchor moiety. Thus, (i) in normal AP GPI anchor synthesis, any ether-linked substituents must be added either immediately before, during, or after anchor addition to AP, and (ii) the classical peroxisomal path for ether lipid synthesis appears not to contribute to the synthesis of GPI anchors.

Induction of antitumor immunity via intratumoral tetra-costimulator protein transfer.

Our group recently described a novel two-step Fc(gamma1) fusion protein transfer method, which entails the docking of Fc(gamma1) fusion proteins onto cells precoated with chemically palmitated protein A (pal-prot A). In the present study, we have adapted this protein transfer method, originally used in an ex vivo context, for in situ tumor cell engineering, and in so doing, we have evaluated its utility for the induction of antitumor immunity via combinatorial costimulator protein transfer on to tumor cell surfaces. The feasibility of "painting" cells with preformed conjugates of a murine B7-1 costimulator derivative, B7-1.Fc(gamma1), and pal-prot A in a single step was first established ex vivo. Next, B7-1.Fc(gamma1):pal-prot A transfer was accomplished in vivo by directly injecting the preformed conjugates into highly aggressive L5178Y-R lymphomas grown intradermally in syngeneic mice. The presence of cell surface-associated B7-1 epitopes on cells of the injected tumors was documented by flow cytometric analysis of cells recovered subsequently from the injected tumors. B7-1.Fc(gamma1), along with Fc(gamma1) fusion protein derivatives of three additional costimulators (Fc(gamma1).4-1BBL, CD48.Fc(gamma1), and Fc(gamma1).CD40L) geared toward a variety of immune effectors, were together preconjugated with pal-prot A and injected directly into tumor beds. Significantly, this "tetra-costimulator" combination, delivered intratumorally, induced complete tumor regression in approximately 45% of treated mice, whereas control injections of pal-prot A alone had no therapeutic effect. Furthermore, there was evidence for systemic antitumor immunity in that tumor-specific CTLs were detected in spleens recovered from cured mice, and these mice were uniformly protected against tumor rechallenge at distant tumor sites. Hence, combinatorial costimulator transfer, coupled to intratumoral delivery, may have special advantages for the induction of antitumor immunity.

Alloantigenic recognition of artificial glycosyl phosphatidylinositol-anchored HLA-A2.1.

Alloantigen presentation by GPI-reanchored variants of the human class I MHC molecule HLA-A2.1 was studied in human cellular systems. To this end, we generated chimeric coding sequences for two GPI-modified HLA-A2.1 heavy chain derivatives. In these chimeras, the coding sequence for the HLA-A2.1 heavy chain was fused in-frame to alternative overlapping sequences from the 3'-end of human DAF containing the GPI-modification signal sequence. The encoded polypeptides HLA-A2.1:DAF-S and HLA-A2.1:DAF-L differed by 53 amino acids of additional DAF sequence in the latter. Both were detected on stably transfected C1R cell surfaces by HLA-A2.1-specific mAb, and their GPI-modification was confirmed by PI-PLC enzymatic cleavage. Immunoprecipitation analysis of surface-biotinylated C1R transfectants revealed heterodimeric association for both HLA-A2.1:DAF-L and HLA-A2.1:DAF-S heavy chains with beta 2m. Alloantigenic stimulation by, and cytotoxic recognition of, both HLA-A2.1:DAF-S/C1R and HLA-A2.1/CIR cells was observed; however, HLA-A2.1:DAF-L/C1R cells could not serve as allostimulators or allotargets. These findings establish that polymorphic human class I MHC molecules can function, when artificially GPI-reanchored, as alloantigenic targets. Moreover, the data suggest that the sequence bridging the HLA-A2 extracellular domain and the membrane can influence alloantigenic presentation.

Rabbit MHC antigens: occurrence of non-beta-2-microglobulin-associated class I molecules.

Two monoclonal antibodies, L12/36 and L13/112, prepared from spleens of BALB/c mice immunized with extracts of the rabbit lymphoid cell line RL-5, precipitate a 42,000 mol. wt molecule from detergent lysates of this cell line. This molecule is not associated with beta-2-microglobulin (beta 2m) and has been shown by sequential precipitation experiments to be antigenically distinct from the population of class I rabbit MHC (RLA) molecules that is associated with beta 2m. In spite of this antigenic difference, amino acid sequence analyses indicate that the RLA heavy chain precipitated with anti-beta 2m and that precipitated with the monoclonal antibodies, have identical N-terminal sequences. The sequence determined to position 36 is GSHSMRYFYTSVSRPGLGXPRFIIVGYVXXTXFVRF. The isoleucine assigned to position 24 is the first species-specific residue found for RLA class I molecules. Analysis of the beta 2m associated and non-associated molecules by two-dimensional gel electrophoresis revealed no differences between the RLA heavy chains. Differences in the subcellular locations of the determinants were indicated by fluorescence microscopy and confirmed by immunoelectron microscopy. It was shown that the specificity recognized by the monoclonal antibodies is principally located on the cytoplasmic face of the plasma membrane whereas the majority of anti-beta 2m reactive specificities are on the extracellular side of the cell membrane.

Directional antisense and sense cDNA cloning using Epstein-Barr virus episomal expression vectors.

A set of Epstein-Barr virus (EBV) episomal expression vectors, incorporating either the Rous sarcoma virus 3' long terminal repeat or the human metallothionein IIA gene promoter, were constructed. The transcriptional cassettes encompassed by these vectors were designed to permit both antisense and sense RNA transcription. A novel methodology was developed for directional cDNA cloning using an oligodeoxyribonucleotide adapter; the EBV episomal vectors alternatively enabled the insertion of cDNA segments in antisense or sense orientations. We propose a strategy for random antisense RNA mutagenesis exploiting this vector system and a method for episome-based directional antisense cDNA cloning and expression, permitting the rapid identification of genes mediating selectable cellular functions.

Non-glycosylated human B7-1(CD80) retains the capacity to bind its counter-receptors.

Though the cell surface-associated costimulator B7-1(CD80) is known to be highly N-glycosylated, the functional significance of this N-glycosylation has not been evaluated. Two experimental approaches were taken to assess the influence of N-glycosylation on human B7-1 function. First, stable K562 transfectants expressing human B7-1 were treated with the N-glycosylation inhibitor tunicamycin. This treatment reduced the levels of B7-1 at the cell surface as judged by both indirect immunofluorescence/flow cytometry and immunoprecipitation analyses. Significantly, the non-glycosylated cell surface-associated B7-1 on tunicamycin-treated cells retained the capacity to bind CTLA-4 x Ig, a soluble derivative of the CTLA-4(CD152) counter-receptor. Second, experiments were performed with bacterially-produced non-glycosylated derivatives of human B7-1, comprising either the complete B7-1 extracellular domain (hB7-1 x ed) or the membrane-proximal IgC-homologue domain of B7-1 in isolation (hB7-1 x IgC). While the hB7-1 x IgC derivative failed to bind to CTLA-4, the larger hB7-1 x ed derivative associated with CTLA-4 x Ig in cell-free binding assays. Futhermore, recombinant hB7-1 x ed effectively blocked B7-1-mediated costimulation in an in vitro T cell proliferation assay, suggesting that this soluble non-glycosylated B7-1 derivative is capable of engaging CD28, the B7 counter-receptor implicated in T cell activation. Taken together, these data indicate that the N-glycosylation of B7-1 is not required for its association with counter-receptors. Moreover, the findings pave the way for the therapeutic use of recombinant bacterial B7-1 derivatives as competitive inhibitors of B7-mediated signals.

A receptor for the lipocalin placental protein 14 on human monocytes.

Human placental protein 14 (PP14), a member of the lipocalin structural superfamily, is an abundant amniotic fluid glycoprotein with documented immunoinhibitory activities. While receptors have been characterized for several other lipocalins, none have been reported to date for PP14. In the present study, two-color immunofluorescence and flow cytometry was used to screen peripheral blood mononuclear cell subpopulations for their capacity to engage fluoresceinated recombinant PP14. The tagged PP14 bound strongly in a specific and saturable fashion to CD14+ (monocyte lineage) cells, but not to CD20+ (B cell lineage) or CD3+ (T cell lineage) cells. This binding was both pH- and temperature-sensitive, and was reduced by proteolytic pre-digestion of the cells with trypsin or proteinase K. Scatchard analysis demonstrated a single class of receptors on CD14+ cells, with a K(D) of approximately 1 x 10(-8) and approximately 10-35,000 receptors per cell. These findings constitute the first report of a cell surface-associated binding protein for PP14 and set the stage for exploring the molecular mechanisms of PP14-mediated signaling and immunomodulation.

Prospects for anti-rejection therapies based upon CD8-dependent immunoregulation.

In this article we propose that anti-rejection immunotherapies for countering acute allograft rejection can be designed around manipulation of the cell surface phenotype of alloantigen-presenting cells (allo-APCs) in ways that convert them from T cell activators to inhibitors. It is further suggested that a class of molecules, termed "coinhibitors," can be defined that carry out this APC conversion process. Data are summarized indicating that the human lymphoid cell surface molecule CD8 has such a trans-coinhibitor function which is in addition to the cis-coreceptor and adhesin functions traditionally ascribed to it. Antisense and sense gene transfer studies indicate that CD8 on the surface of allo-APCs leads to inhibition of allospecific T cell responders. We have explored the possibility of using protein, rather than gene, transfer as a therapeutic strategy for delivering CD8 to APC surfaces. Two membrane-binding variants of CD8 have been assembled to show retention of the coinhibitor function of native CD8. Immunotherapeutic possibilities associated with these chimeric CD8 polypeptides in the clinical context of renal and other organ transplantation are considered.

Expression of the imprinted H19 oncofetal RNA in epithelial ovarian cancer.

STUDY: To examine the expression of the imprinted maternally expressed H19 gene in benign, low malignant potential (borderline) and malignant surface epithelial ovarian tumors. DESIGN: In situ hybridization for H19 RNA using S-labeled and digoxigenin-labeled probes was performed on paraffin sections of ovarian surface epithelial tumors. The serous tumors included nine section cystadenomas, twelve serous tumors of low malignant potential and twenty serous carcinomas, grade I-IIII (FIGO classification). A smaller group included two mucinous cystadenomas, four mucinous tumors of low malignant potential and two mucinous cystadenocarcinomas. RESULTS: H19 expression was found to be positive in 6/9 (67%) serous cystadenomas, 9/12 (75%) of serous tumors of low malignant potential and 13/20 (65%) of invasive serous carcinomas. Expression in mucinous tumors was confined to the stroma beneath the epithelial lining. CONCLUSION: H19 is expressed in the majority of serous epithelial tumors. Taking into consideration the high percentage of H19 expressing serous ovarian neoplasms we suggest that H19 RNA may be used as an adjuvant tumor marker for the diagnosis and mainly for staging and follow-up of patients with serous ovarian carcinoma.

CD40.FasL inhibits human T cells: evidence for an auto-inhibitory loop-back mechanism.

A chimeric CD40.FasL (CD40-CD95L) protein was designed with the combined capacities to bind to two surface receptors on activated T cells, CD40 ligand (CD40L; CD154) and Fas receptor (CD95). CD40.FasL, once tethered to the cell surface via one of its ends, can transmit a signal via its other end. In principle, simultaneous triggering from both ends is possible, and thus there is the intriguing potential for 'auto-inhibition' if such dual triggering occurs on the same cell itself. Several lines of evidence support this mechanism: (i) CD40.FasL is cytotoxic to Fas receptor-positive cell lines of different cell lineages, (ii) CD40.FasL's function is potentiated when there is enforced expression of CD40L on target cells, (iii) CD40.FasL inhibition does not require intercellular contact, as demonstrated by soft agar clone formation and cell dilution analysis and (iv) introduction of exogenous CD40 into the system interferes with CD40.FasL inhibition. Taken together, these data are consistent with a 'loop-back' inhibitory mechanism within individual activated (CD40L and Fas receptor expressing) T cells causing suicide of these T cells. Significantly, this type of fusion protein provides a unique way to confine immunoinhibition to activated T cells.

CTLA-4.FasL inhibits allogeneic responses in vivo.

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.

Bone marrow stromal cell blockade of human leukemic cell differentiation.

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.

The expansion of human gammadelta T cells in response to Daudi cells requires the participation of CD4+ T cells.

The Burkitt's lymphoma cell line Daudi is a potent inducer of human gammadelta T-cell expansion. Using an in vitro culture system comprised of irradiated Daudi cells as stimulators and normal human lymphocytes as responders, the cellular determinants of this response were investigated. Three of four monoclonal antibodies (mAbs 1-1C4, L243, and 9.3F10) directed against disparate epitopes of human major histocompatibility complex (MHC) class II, as well as a mAb with specificity for CD4 (OKT4), inhibited the expansion of gammadelta T cells in response to Daudi cell stimulators. mAbs with a specificity for CD74 and CD8 were non-inhibitory. Lymphocyte depletion experiments demonstrated a critical role for the CD4+ T-cell subset in the expansion of gammadelta T cells. Other data pointed towards requirements for direct cell contact in this system, and the addition of exogenous recombinant interleukin (IL)-2, IL-4, and IL-12 failed to reconstitute gammadelta T-cell expansion in CD4+ lymphocyte-depleted cultures. These results complement previous findings in murine infectious disease and mycobacterial systems, providing a direct demonstration that CD4+ T cells play a role in gammadelta T-cell expansion through an interaction with human leucocyte antigen (HLA) class II on Daudi cells. The data point towards important functional links between the acquired and natural immune systems.

CG dinucleotide clusters in MHC genes and in 5' demethylated genes.

In the DNA of higher vertebrates the dinucleotide CG is unique in two respects: it occurs far less frequently than would be expected on the basis of the content of cytosine and guanine in a given DNA segment ("CG suppression") and it contains predominantly 5-methyl-cytosine, the only modified nucleotide common in vertebrate DNA. Here we point out the existence of CG clusters, i.e. localized lapses in the usual CG suppression, in two categories of DNA segments from vertebrates: around the polymorphic exons of major histocompatibility complex (MHC) genes and in the 5' regions of certain other genes. These observations contradict the recent suggestion that CG frequency is uniform over long contiguous segments of DNA containing several genes. A model for the origin of these CG clusters as a consequence of regional demethylation of germline DNA is supported by analysis of other sequence features of these regions as well as by previously published data on the methylation status in sperm DNA of two of these CG-rich regions.