Conjugates of folate and anti-T-cell-receptor antibodies specifically target folate-receptor-positive tumor cells for lysis.

High-affinity folate receptors (FRs) are expressed at elevated levels on many human tumors. Bispecific antibodies that bind the FR and the T-cell receptor (TCR) mediate lysis of these tumor cells by cytotoxic T lymphocytes. In this report, conjugates that consist of folate covalently linked to anti-TCR antibodies are shown to be potent in mediating lysis of tumor cells that express either the alpha or beta isoform of the FR. Intact antibodies with an average of five folate per molecule exhibited high affinity for FR+ tumor cells but did not bind to FR- tumor cells. Lysis of FR+ cell lines could be detected at concentrations as low as 1 pM (approximately 0.1 ng/ml), which was 1/1000th the concentration required to detect binding to the FR+ cells. Various FR+ mouse tumor cell lines could be targeted with each of three different anti-TCR antibodies that were tested as conjugates. The antibodies included 1B2, a clonotypic antibody specific for the cytotoxic T cell clone 2C; KJ16, an anti-V beta 8 antibody; and 2C11, an anti-CD3 antibody. These antibodies differ in affinities by up to 100-fold, yet the cytolytic capabilities of the folate/antibody conjugates differed by no more than 10-fold. The reduced size (in comparison with bispecific antibodies) and high affinity of folate conjugates suggest that they may be useful as immunotherapeutic agents in targeting tumors that express folate receptors.

De novo formation of caveolae in lymphocytes by expression of VIP21-caveolin.

Caveolae are plasma membrane invaginations, which have been implicated in endothelial transcytosis, endocytosis, potocytosis, and signal transduction. In addition to their well-defined morphology, caveolae are characterized by the presence of an integral membrane protein termed VIP21-caveolin. We have recently observed that lymphocytes have no detectable VIP21-caveolin and lack plasma membrane invaginations resembling caveolae. Here we transiently express VIP21-caveolin in a lymphocyte cell line using the Semliki Forest virus expression system and show de novo formation of plasma membrane invaginations containing VIP21-caveolin. These invaginations appear homogeneous in size and morphologically indistinguishable from caveolae of nonlymphoid cells. Moreover, the glycosylphosphatidylinositol-anchored protein. Thy1, patched by antibodies, redistributes to the newly formed caveolae. Our results show that VIP21-caveolin is a key structural component required for caveolar biogenesis.

Amino-terminal alteration of the HLA-A*0201-restricted human immunodeficiency virus pol peptide increases complex stability and in vitro immunogenicity.

Initial studies suggested that major histocompatibility complex class I-restricted viral epitopes could be predicted by the presence of particular residues termed anchors. However, recent studies showed that nonanchor positions of the epitopes are also significant for class I binding and recognition by cytotoxic T lymphocytes (CTLs). We investigated if changing nonanchor amino acids could increase class I affinity, complex stability, and T-cell recognition of a natural viral epitope. This concept was tested by using the HLA-A 0201-restricted human immunodeficiency virus type 1 epitope from reverse transcriptase (pol). Position 1 (P1) amino acid substitutions were emphasized because P1 alterations may not alter the T-cell receptor interaction. The peptide with the P1 substitution of tyrosine for isoleucine (I1Y) showed a binding affinity for HLA-A 0201 similar to that of the wild-type pol peptide in a cell lysate assembly assay. Surprisingly, I1Y significantly increased the HLA-A 0201-peptide complex stability at the cell surface. I1Y sensitized HLA-A 0201-expressing target cells for wild-type pol-specific CTL lysis as well as wild-type pol. Peripheral blood lymphocytes from three HLA-A2 HIV-seropositive individuals were stimulated in vitro with I1Y and wild-type pol. I1Y stimulated a higher wild-type pol-specific CTL response than wild-type pol in all three donors. Thus, I1Y may be an "improved" epitope for use as a CTL-based human immunodeficiency virus vaccine component. The design of improved epitopes has important ramifications for prophylaxis and therapeutic vaccine development.

High-efficiency retroviral-mediated gene transfer into human and nonhuman primate peripheral blood lymphocytes.

Peripheral blood lymphocytes (PBLs) are primary targets for gene therapy of inherited and acquired disorders of the immune system. We describe the development of an optimized transduction system that provides for high-efficiency retrovirus-mediated gene transfer into primary PBLs. This optimized transduction protocol combines centrifugation of the lymphocytes (1000 x g) at the inception of transduction with phosphate depletion, low-temperature incubation (32 degrees C), and the use of the packaging cell line PG13. Gene marking studies of human and primate PBLs using these optimized transduction conditions demonstrated that the transduction efficiency exceeded 50% of the total lymphocyte population. The optimized transduction efficiency of PBLs with amphotropic retroviral vectors was in excess of 25%. The transduction procedure does not alter phenotype, viability, or expansion of the transduced cells. Our data indicate that this optimized transduction system leads to high-efficiency gene transfer into primary human lymphocytes, which obviates the requirement for selection of transduced cells prior to gene-therapy procedures. Thus, large quantities of healthy retrovirally transduced lymphocytes containing a broad immunological repertoire can be generated for use in clinical protocols. Our results represent a significant improvement in the methodology for the transduction of lymphocytes for gene therapy.

Apoptosis induced by a human milk protein.

To the breast-fed infant, human milk is more than a source of nutrients; it furnishes a wide array of molecules that restrict microbes, such as antibodies, bactericidins, and inhibitors of bacterial adherence. However, it has rarely been considered that human milk may also contain substances bioactive toward host cells. While investigating the effect of human milk on bacterial adherence to a human lung cancer cell line, we were surprised to discover that the milk killed the cells. Analysis of this effect revealed that a component of milk in a particular physical state--multimeric alpha-lact-albumin--is a potent Ca(2+)-elevating and apoptosis-inducing agent with broad, yet selective, cytotoxic activity. Multimeric alpha-lactalbumin killed all transformed, embryonic, and lymphoid cells tested but spared mature epithelial elements. These findings raise the possibility that milk contributes to mucosal immunity not only by furnishing antimicrobial molecules but also by policing the function of lymphocytes and epithelium. Finally, analysis of the mechanism by which multimeric alpha-lactalbumin induces apoptosis in transformed epithelial cells could lead to the design of antitumor agents.

Upregulation of class I major histocompatibility complex antigens by interferon gamma is necessary for T-cell-mediated elimination of recombinant adenovirus-infected hepatocytes in vivo.

Recombinant adenoviruses are attractive vehicles for liver-directed gene therapy because of the high efficiency with which they transfer genes to hepatocytes in vivo. First generation recombinant adenoviruses deleted of E1 sequences also express recombinant and early and late viral genes, which lead to development of destructive cellular immune responses. Previous studies indicated that class I major histocompatibility complex (MHC)-restricted cytotoxic T lymphocytes (CTLs) play a major role in eliminating virus-infected cells. The present studies utilize mouse models to evaluate the role of T-helper cells in the primary response to adenovirus-mediated gene transfer to the liver. In vivo ablation of CD4+ cells or interferon gamma (IFN-gamma) was sufficient to prevent the elimination of adenovirus-transduced hepatocytes, despite the induction of a measurable CTL response. Mobilization of an effective TH1 response as measured by in vitro proliferation assays was associated with substantial upregulation of MHC class I expression, an effect that was prevented in IFN-gamma-deficient animals. These results suggest that elimination of virus-infected hepatocytes in a primary exposure to recombinant adenovirus requires both induction of antigen-specific CTLs as well as sensitization of the target cell by TH1-mediated activation of MHC class I expression.

Resistance to human immunodeficiency virus type 1 infection conferred by transduction of human peripheral blood lymphocytes with ribozyme, antisense, or polymeric trans-activation response element constructs.

Human peripheral blood lymphocytes (PBLs) were transduced with a number of recombinant retroviruses including RRz2, an LNL6-based virus with a ribozyme targeted to the human immunodeficiency virus (HIV) tat gene transcript inserted within the 3' region of the neomycin-resistance gene; RASH5, and LNHL-based virus containing an antisense sequence to the 5' leader region of HIV-1 downstream of the human cytomegalovirus promoter; and R20TAR, an LXSN-based virus with 20 tandem copies of the HIV-1 trans-activation response element sequence driven by the Moloney murine leukemia virus long terminal repeat. After G418 selection, transduced PBLs were challenged with the HIV-1 laboratory strain IIIB and a primary clinical isolate of HIV-1, 82H. Results showed that PBLs from different donors could be transduced and that this conferred resistance to HIV-1 infection. For each of the constructs, a reduction of approximately 70% in p24 antigen level relative to the corresponding control-vector-transduced PBLs was observed. Molecular analyses showed constitutive expression of all the transduced genes from the retroviral long terminal repeat, but no detectable transcript was seen from the internal human cytomegalovirus transcript was seen from the internal human cytomegalovirus promoter for the antisense construct. Transduction of, and consequent transgene expression in, PBLs did not impact on the surface expression of either CD4+/CD8+ (measured by flow cytometry) or on cell doubling time (examined by [3H]thymidine uptake). These results indicate the potential utility of these anti-HIV-1 gene therapeutic agents and show the preclinical value of this PBL assay system.

Interleukin 12 induces tyrosine phosphorylation and activation of STAT4 in human lymphocytes.

Interleukin 12 (IL-12) is an important immunoregulatory cytokine whose receptor is a member of the hematopoietin receptor superfamily. We have recently demonstrated that stimulation of human T and natural killer cells with IL-12 induces tyrosine phosphorylation of the Janus family tyrosine kinase JAK2 and Tyk2, implicating these kinases in the immediate biochemical response to IL-12. Recently, transcription factors known as STATs (signal transducers and activators of transcription) have been shown to be tyrosine phosphorylated and activated in response to a number of cytokines that bind hematopoietin receptors and activate JAK kinases. In this report we demonstrate that IL-12 induces tyrosine phosphorylation of a recently identified STAT family member, STAT4, and show that STAT4 expression is regulated by T-cell activation. Furthermore, we show that IL-12 stimulates formation of a DNA-binding complex that recognizes a DNA sequence previously shown to bind STAT proteins and that this complex contains STAT4. These data, and the recent demonstration of JAK phosphorylation by IL-12, identify a rapid signal-transduction pathway likely to mediate IL-12-induced gene expression.

CD4+ blood lymphocytes are rapidly killed in vitro by contact with autologous human immunodeficiency virus-infected cells.

We have investigated the ability of human immunodeficiency virus (HIV)-infected cells to kill uninfected CD4+ lymphocytes. Infected peripheral blood mononuclear cells were cocultured with autologous 51Cr-labeled uninfected cells. Rapid death of the normal CD4-expressing target population was observed following a brief incubation. Death of blood CD4+ lymphocytes occurred before syncytium formation could be detected or productive viral infection established in the normal target cells. Cytolysis could not be induced by free virus, was dependent on gp120-CD4 binding, and occurred in resting, as well as activated, lymphocytes. CD8+ cells were not involved in this phenomenon, since HIV-infected CEMT4 cells (CD4+, CD8- cells) mediated the cytolysis of uninfected targets. Reciprocal isotope-labeling experiments demonstrated that infected CEMT4 cells did not die in parallel with their targets. The uninfected target cells manifested DNA fragmentation, followed by the release of the 51Cr label. Thus, in HIV patients, infected lymphocytes may cause the depletion of the much larger population of uninfected CD4+ cells without actually infecting them, by triggering an apoptotic death.

Lymphocytes selected in allogeneic thymic epithelium mediate dominant tolerance toward tissue grafts of the thymic epithelium haplotype.

Athymic mice grafted at birth with allogeneic thymic epithelium (TE) from day 10 embryos before hematopoietic cell colonization reconstitute normal numbers of T cells and exhibit full life-long tolerance to skin grafts of the TE haplotype. Intravenous transfers of splenic cells, from these animals to adult syngeneic athymic recipients, reconstitute T-cell compartments and the ability to reject third-party skin grafts. The transfer of specific tolerance to skin grafts of the TE donor strain, however, is not observed in all reconstituted recipients, and the fraction of nontolerant recipients increases with decreasing numbers of cells transferred. Furthermore, transfers of high numbers of total or CD4+ T cells from TE chimeras to T-cell receptor-anti-H-Y antigen transgenic immunocompetent syngeneic hosts specifically hinder the rejection of skin grafts of the TE haplotype that normally occurs in such recipients. These observations demonstrate (i) that mice tolerized by allogeneic TE and bearing healthy skin grafts harbor peripheral immunocompetent T cells capable of rejecting this very same graft; and (ii) that TE selects for regulatory T cells that can inhibit effector activities of graft-reactive cells.

Identification of human granzyme B promoter regulatory elements interacting with activated T-cell-specific proteins: implication of Ikaros and CBF binding sites in promoter activation.

Granzyme B serine protease is found in the granules of activated cytotoxic T cells and in natural and lymphokine-activated killer cells. This protease plays a critical role in the rapid induction of target cell DNA fragmentation. The DNA regulatory elements that are responsible for the specificity of granzyme B gene transcription in activated T-cells reside between nt -148 and +60 (relative to the transcription start point at +1) of the human granzyme B gene promoter. This region contains binding sites for the transcription factors Ikaros, CBF, Ets, and AP-1. Mutational analysis of the human granzyme B promoter reveals that the Ikaros binding site (-143 to -114) and the AP-1/CBF binding site (-103 to -77) are essential for the activation of transcription in phytohemagglutinin-activated peripheral blood lymphocytes, whereas mutation of the Ets binding site does not affect promoter activity in these cells.

Prolonged survival of pancreatic islet allografts mediated by adenovirus immunoregulatory transgenes.

The adenovirus (Ad) early region 3 (E3) genes code for at least four proteins that inhibit the host immune responses mediated by cytotoxic T lymphocytes and tumor necrosis factor alpha. To evaluate the potential use of these immunoregulatory viral functions in facilitating allogeneic cell transplantation, the Ad E3 genes were expressed in pancreatic beta cells in transgenic mice under control of the rat insulin II promoter. Transgenic H-2b/d (C57BL/6 x BALB/c) islets, expressing the Ad E3 genes, remained viable for at least 94 days after transplantation under the kidney capsule of BALB/c (H-2d) recipients. Nontransgenic H-2b/d control islets were rejected as anticipated between 14 and 28 days. Histological analysis of the transplanted transgenic islets revealed normal architecture. Immunohistochemical studies with antisera to islet hormones revealed the presence of both beta and non-beta islet cells, suggesting a propagation of the immunosuppressive effect of Ad proteins from beta cells to other islet cells. The use of viral genes, which have evolved to regulate virus-host interactions, to immunosupress the anti-genicity of donor transplant tissue suggests additional ways for prolonging allograft survival. In addition, these findings have implications for designing Ad vectors for gene therapy.

Preferential expansion and survival of B lymphocytes based on VH framework 1 and framework 3 expression: "positive" selection in appendix of normal and VH-mutant rabbits.

B cells with a rearranged heavy-chain variable region VHa allotype-encoding VH1 gene segment predominate throughout the life of normal rabbits and appear to be the source of the majority of serum immunoglobulins, which thus bear VHa allotypes. The functional role(s) of these VH framework region (FR) allotypic structures has not been defined. We show here that B cells expressing surface immunoglobulin with VHa2 allotypic specificities are preferentially expanded and positively selected in the appendix of young rabbits. By flow cytometry, a higher proportion of a2+ B cells were progressing through the cell cycle (S/G2/M) compared to a2- B cells, most of which were in the G1/G0 phase of the cell cycle. The majority of appendix B cells in dark zones of germinal centers of normal 6-week-old rabbits were proliferating and very little apoptosis were observed. In contrast, in 6-week-old VH-mutant ali/ali rabbits, little cell proliferation and extensive apoptosis were observed. Nonetheless even in the absence of VH1, B cells with a2-like surface immunoglobulin had developed and expanded in the appendix of 11-week-old mutants. The numbers and tissue localization of B cells undergoing apoptosis then appeared similar to those found in 6-week-old normal appendix. Thus, B cells with immunoglobulin receptors lacking the VHa2 allotypic structures were less likely to undergo clonal expansion and maturation. These data suggest that "positive" selection of B lymphocytes through FR1 and FR3 VHa allotypic structures occurs during their development in the appendix.

A small bispecific antibody construct expressed as a functional single-chain molecule with high tumor cell cytotoxicity.

Construction of a bispecific single-chain antibody derivative is described that consists of two different single-chain Fv fragments joined through a Gly-Ser linker. One specificity of the two Fv fragments is directed against the CD3 antigen of human T cells and the other is directed against the epithelial 17-1A antigen; the latter had been found in a clinical trial to be a suitable target for antibody therapy of minimal residual colorectal cancer. The construct could be expressed in CHO cells as a fully functional protein, while its periplasmic expression in Escherichia coli resulted in a nonfunctional protein only. The antigen-binding properties of the bispecific single-chain antibody are indistinguishable from those of the corresponding univalent single-chain Fv fragments. By redirecting human peripheral T lymphocytes against 17-1A-positive tumor cells, the bispecific antibody proved to be highly cytotoxic at nanomolar concentrations as demonstrated by 51Cr release assay on various cell lines. The described bispecific construct has a molecular mass of 60 kDa and can be easily purified by its C-terminal histidine tail on a Ni-NTA chromatography column. As bispecific antibodies have already been shown to be effective in vivo in experimental tumor systems as well as in phase-one clinical trials, the small CD3/17-1A-bispecific antibody may be more efficacious than intact antibodies against minimal residual cancer cells.

T lymphocytes that infiltrate tumors and atherosclerotic plaques produce heparin-binding epidermal growth factor-like growth factor and basic fibroblast growth factor: a potential pathologic role.

Despite significant infiltration into tumors and atherosclerotic plaques, the role of T lymphocytes in these pathological conditions is still unclear. We have demonstrated that tumor-infiltrating lymphocytes (TILs) and plaque-infiltrating lymphocytes (PILs) produce heparin-binding epidermal growth factor-like growth factor (HB-EGF) and basic fibroblast growth factor (bFGF) in vitro under nonspecific conditions and in vivo in tumors by immunohistochemical staining. HB-EGF and bFGF derived from TILs and PILs directly stimulated tumor cells and vascular smooth muscle cells (SMCs) in vitro, respectively, while bFGF displayed angiogenic properties. Therefore, T cells may play a critical role in the SMC hyperplasia of atherosclerosis and support tumor progression by direct stimulation and angiogenesis.