Martha Hayden-Ledbetter

Characterization of Human Inducible Costimulator Ligand Expression and Function

The inducible costimulator (ICOS) is the newest member of the CD28/CD152 receptor family involved in regulating T cell activation. We constructed a soluble-Ig fusion protein of the extracellular domain of human ICOS and used it as a probe to characterize expression patterns of the ICOS ligand (ICOSL). ICOSIg did not bind to CD80- or CD86-transfected Chinese hamster ovary cell lines, demonstrating that ICOSL is distinct from those ligands identified for CD28/CD152. ICOSIg showed selective binding to monocytic and B cell lines, whereas binding was undetectable on unstimulated monocytes and peripheral blood T and B cells. Expression of ICOSL was induced on monocytes after integrin-dependent plastic adhesion. Pretreatment of monocytes with mAb to the β2-integrin subunit CD18 decreased adhesion and abolished ICOSL up-regulation but had no effect on CD80/86 (CD152 ligand (CD152L)) expression. Both ICOSL and CD152L were up-regulated on monocytes by IFN-γ but by distinct signaling pathways. Unlike CD152L expression, ICOSL expression did not change when monocytes were differentiated into dendritic cells (DCs) or after DCs were induced to mature by LPS, TNF-α, or CD40 ligation. Addition of ICOSIg to allogeneic MLRs between DCs and T cells reduced T cell proliferative responses but did so less efficiently than CTLA4Ig (CD152Ig) did. Similarly, ICOSIg also blocked Ag-specific T cell proliferation to tetanus toxoid. Thus, ICOSL, like CD80/86, is expressed on activated monocytes and dendritic cells but is regulated differently and delivers distinct signals to T cells that can be specifically inhibited by ICOSIg.

Gene therapy for cancer using single-chain Fv fragments specific for 4-1BB

Monoclonal antibodies against the T-cell activation molecule 4-1BB have been effective in the treatment of established mouse tumors. To create a vaccine that stimulates the immune system similarly to the efficacious monoclonal anti-4-1BB antibody, 1D8, we constructed a vector encoding cell-bound single-chain Fv fragments from 1D8. We transfected the vector into cells from the K1735 melanoma, selected because of its low immunogenicity and very low expression of major histocompatibility complex class I. The transfected cells induced a strong type 1 T-helper cell response, for which CD4+ but not CD8+ T lymphocytes were necessary and that involved natural killer cells. Vaccinated mice rejected established wild-type K1735 tumors growing as subcutaneous nodules or in the lung. An analogous approach may be effective against micrometastases in human patients, including tumors whose expression of major histocompatibility complex class I is very low.

Cutting Edge: CD83 Regulates the Development of Cellular Immunity

We recently found that human CD83, a marker of mature dendritic cells, is an adhesion receptor that binds to resting monocytes and a subset of activated CD8+ T cells. We injected CD83-Ig into mice transplanted with the immunogenic P815 mastocytoma and showed that it significantly enhanced the rate of tumor growth and inhibited the development of cytotoxic T cells. In contrast, mice immunized with CD83-transfected K1735 cells, a poorly immunogenic melanoma, could prevent the outgrowth of wild-type K1735 cells. Studies performed in vitro with human PBL showed that coimmobilized CD83-Ig and anti-CD3 enhanced T cell proliferation and increased the proportion of CD8+ T cells. CD83-transfected B-lymphoblastoid T51 cells stimulated T cell proliferation more effectively than untransfected T51 cells in MLR cultures and increased the generation of cytolytic T cells. We conclude that CD83 is a functionally important receptor that can regulate the development of cellular immunity by interacting with its ligand(s).

CD83 is an I-type lectin adhesion receptor that binds monocytes and a subset of activated CD8+ T cells [corrected].

To help determine CD83 function, a cDNA encoding a soluble protein containing the CD83 extracellular domain was fused with a mutated human IgG1 constant region (CD83Ig) and expressed by stable transfection of Chinese hamster ovary cells. Purified CD83Ig bound to peripheral blood monocytes and a subset of activated CD3+CD8+ lymphocytes but did not bind to FcR. Monocytes that had adhered to plastic lost their ability to bind to CD83Ig after 90 min of in vitro incubation. CD83Ig bound to two of five T cell lines tested, HPB-ALL and Jurkat. The binding to HPB-ALL cells significantly increased when they were grown at a low pH (pH 6.5), whereas binding to Jurkat cells increased after apoptosis was induced with anti-Fas mAb. B cell and monocytic lines did not bind CD83Ig and neither did CD56+ NK cells or granulocytes. Full-length CD83 expressed by a transfected carcinoma line mediated CD83-dependent adhesion to HPB-ALL cells. CD83Ig immunoprecipitated and immunoblotted a 72-kDa protein from HPB-ALL cells. Binding of CD83Ig to HPB-ALL cells was eliminated by neuraminidase treatment of the cells. We conclude that CD83 is an adhesion receptor with a counterreceptor expressed on monocytes and a subset of activated or stressed T lymphocytes, and that interaction between CD83 and its counterreceptor is dependent upon the state of glycosylation of a 72-kDa counterreceptor by sialic acid residues. In view of the selectivity of the expression of CD83 and its ligand, we postulate that the interaction between the two plays an important role in the induction and regulation of immune responses.

CD20-Directed Small Modular Immunopharmaceutical, TRU-015, Depletes Normal and Malignant B Cells

Purpose: CD20-directed therapy with rituximab is effective in many patients with malignant lymphoma or follicular lymphoma. However, relapse frequently occurs within 1 year, and patients become increasingly refractory to retreatment. Our purpose was to produce a compact, single-chain CD20-targeting immunotherapeutic that could offer therapeutic advantages in the treatment of B-cell lymphoma. Experimental Design: Rituximab is a chimeric antibody containing two heavy chains and two light chains. Here, we describe the properties of TRU-015, a small modular immunopharmaceutical specific for CD20, encoded by a single-chain construct containing a single-chain Fv specific for CD20 linked to human IgG1 hinge, CH2, and CH3 domains but devoid of CH1 and CL domains. Results: TRU-015 mediates potent direct signaling and antibody-dependent cellular cytotoxicity but has reduced size and complement-mediated cytotoxicity activity compared with rituximab. TRU-015 is a compact dimer of 104 kDa that comigrates with albumin in size exclusion chromatography and retains a long half-life in vivo. TRU-015 induced growth arrest in multiple B lymphoma cell lines in vitro and showed effective antitumor activity against large, established subcutaneous Ramos or Daudi xenograft tumors in nude mice. TRU-015 also showed rapid, dose-dependent, and durable depletion of peripheral blood B cells following single-dose administration to nonhuman primates. Conclusion: These results indicate that TRU-015 may improve CD20-directed therapy by effectively depleting embedded malignant B cells and nonmalignant pathogenic B cells and do so with reduced complement activation.

CD3-mediated activation of tumor-reactive lymphocytes from patients with advanced cancer.

Lymphocytes from blood or tumors of patients with advanced cancer did not proliferate and produced very low levels of tumor necrosis factor and IFN-γ when cultured with autologous tumor cells. Proliferation and lymphokine production dramatically increased in the presence of beads conjugated with mAbs to CD3 plus mAbs to CD28 and/or CD40, and the lymphocytes destroyed the tumor cells. Expression density of CD3 concomitantly increased from low to normal levels. Furthermore, beads providing a CD3 signal (in combination with CD28 or CD28 plus CD40) gave partial protection against the inhibitory effect of transforming growth factor type β1 on lymphocyte proliferation and production of tumor necrosis factor and IFN-γ. MHC class I-restricted cytolytic T cells lysing autologous tumor cells in a 4-h Cr51 release assay were generated when peripheral blood leukocytes were activated in the presence of autologous tumor cells and anti-CD3/CD28 or anti-CD3/CD28/CD40 beads. Experiments performed in a model system using anti-V-β1 or anti-V-β2 mAbs to activate subsets of T cells expressing restricted T cell receptor showed that lymphocytes previously activated by anti-V-β can respond to CD3 stimulation with vigorous proliferation and lymphokine production while retaining their specificity, also in the presence of transforming growth factor type β1. Our results suggest that T lymphocytes from cancer patients can proliferate and form Th1 type lymphokines in the presence of autologous tumor cell when properly activated, and that antigen released from killed tumor cells and presented by antigen-presenting cells in the cultures facilitates the selective expansion of tumor-directed, CD8+ cytolytic T cells.

T Cell Activation Inhibitors Reduce CD8+ T Cell and Pro-Inflammatory Macrophage Accumulation in Adipose Tissue of Obese Mice

Adipose tissue inflammation and specifically, pro-inflammatory macrophages are believed to contribute to insulin resistance (IR) in obesity in humans and animal models. Recent studies have invoked T cells in the recruitment of pro-inflammatory macrophages and the development of IR. To test the role of the T cell response in adipose tissue of mice fed an obesogenic diet, we used two agents (CTLA-4 Ig and anti-CD40L antibody) that block co-stimulation, which is essential for full T cell activation. C57BL/6 mice were fed an obesogenic diet for 16 weeks, and concomitantly either treated with CTLA-4 Ig, anti-CD40L antibody or an IgG control (300 µg/week). The treatments altered the immune cell composition of adipose tissue in obese mice. Treated mice demonstrated a marked reduction in pro-inflammatory adipose tissue macrophages and activated CD8+ T cells. Mice treated with anti-CD40L exhibited reduced weight gain, which was accompanied by a trend toward improved IR. CTLA-4 Ig treatment, however, was not associated with improved IR. These data suggest that the presence of pro-inflammatory T cells and macrophages can be altered with co-stimulatory inhibitors, but may not be a significant contributor to the whole body IR phenotype.

Targeting Antigens through Blood Dendritic Cell Antigen 2 on Plasmacytoid Dendritic Cells Promotes Immunologic Tolerance

The C-type lectin receptor blood dendritic cell Ag 2 (BDCA2) is expressed exclusively on human plasmacytoid DCs (pDCs) and plays a role in Ag capture, internalization, and presentation to T cells. We used transgenic mice that express human BDCA2 and anti-BDCA2 mAbs to deliver Ags directly to BDCA2 on pDCs in vivo. Targeting Ag to pDCs in this manner resulted in significant suppression of Ag-specific CD4+ T cell and Ab responses upon secondary exposure to Ag in the presence of adjuvant. Suppression of Ab responses required both a decrease in effector CD4+ T cells and preservation of Foxp3+ regulatory T cells (Tregs). Reduction in Treg numbers following Ag delivery to BDCA2 restored both CD4+ T cell activation and Ab responses, demonstrating that Tregs were required for the observed tolerance. Our results demonstrate that Ag delivery to pDCs through BDCA2 is an effective method to induce immunological tolerance, which may be useful for treating autoimmune diseases or to inhibit unwanted Ab responses.