Richard J. Robb

Interleukin 2: the molecule and its function

IL2 plays an essential role in triggering proliferation of activated T cells, a response mediated by interaction of the factor with a high-affinity membrane receptor. IL2 also influences a number of other cellular responses, including natural killer cell activity and antibody secretion. Thus, directly or indirectly, IL2 has the properties of both a growth factor and a differentiation signal. Both IL2 and its receptor have been purified to homogeneity and extensively characterized at the molecular level. In this review, Richard Robb discusses the current biochemical understanding of IL2 and its receptor and new information relating to its role in cellular responses.

Human Autologous Tumor-Specific T-Cell Responses Induced by Liposomal Delivery of a Lymphoma Antigen

Purpose: The idiotype (Id) of the immunoglobulin on a given B-cell malignancy is a clonal marker that can serve as a tumor-specific antigen. We developed a novel vaccine formulation by incorporating Id protein with liposomal lymphokine that was more potent than a prototype, carrier-conjugated Id protein vaccine in preclinical studies. In the present study, we evaluated the safety and immunogenicity of this vaccine in follicular lymphoma patients. Experimental Design: Ten patients with advanced-stage follicular lymphoma were treated with five doses of this second generation vaccine after chemotherapy-induced clinical remission. All patients were evaluated for cellular and humoral immune responses. Results: Autologous tumor and Id-specific type I cytokine responses were induced by vaccination in 10 and 9 patients, respectively. Antitumor immune responses were mediated by both CD4+ and CD8+ T cells, were human lymphocyte antigen class I and II associated, and persisted 18 months beyond the completion of vaccination. Specific anti-Id antibody responses were detected in four patients. After a median follow-up of 50 months, 6 of the 10 patients remain in continuous first complete remission. Conclusions: This first clinical report of a liposomal cancer vaccine demonstrates that liposomal delivery is safe, induces sustained tumor-specific CD4+ and CD8+ T-cell responses in lymphoma patients, and may serve as a model for vaccine development against other human cancers and infectious pathogens.

Retention of biological activity following radioiodination of human interleukin 2: Comparison with biosynthetically labeled growth factor in receptor binding assays

Human interleukin 2 (IL-2) was radioiodinated by a modified form of the chloramine-T reaction. Comparison with biosynthetically radiolabeled IL-2 demonstrated that the iodinated molecule retained similar receptor binding characteristics and proliferation-inducing ability. The iodinated molecule also possessed the distinct advantages of a higher specific radioactivity and a reduced processing time for the assay samples. The majority of the iodine was incorporated at the tyrosine in position 45 of the polypeptide chain. Evidently, this residue is unimportant for the molecules association with its receptor. The development of active radioiodinated IL-2 should facilitate routine measurement of the high-affinity IL-2 binding sites which mediate the physiological response to this lymphokine.

An ELISA-based assay for quantitation of human interleukin 2

An ELISA-based method for the quantitation of human interleukin 2 is described. A murine monoclonal antibody and rabbit polyclonal antibody, both of which recognize the various glycosylated forms of human IL2, were used in a sandwich technique together with alkaline phosphatase-conjugated goat anti-rabbit immunoglobulin. The specificity of the reaction was dependent upon the monoclonal reagent. By generating a standard curve with a defined preparation of IL2, it was possible to quantitate accurately the concentration of the factor in crude lymphokine preparations and in serum samples from IL2-treated patients. Substances in human serum and mitogens such as phytohemagglutinin and phorbol myristic acetate did not interfere in the measurement. This assay provides a rapid, automated alternative to the biological assay generally used to quantitate IL2.

HepG2 cells predominantly express the type II interleukin 1 receptor (biochemical and molecular characterization of the IL-1 receptor)

In this study we have characterized the cell surface interleukin 1 (IL-1) receptor in HepG2 hepatoma cells. We found that HepG2 cells bind both IL-1 alpha and beta with high affinity, KDs of 136 and 180 pM and receptor densities of 16,000 and 8500 binding sites/cell respectively. The binding sites appeared to be predominantly type II since phorbol ester treatment of the cells, which selectively downregulates type II IL-1 receptors, reduced binding by 68% while treatment of the cells with an inhibitory monoclonal antibody specific for the type I receptor had no significant effect on IL-1 binding. Competition studies with a modified IL-1 beta analog (Glu4) also revealed binding kinetics more consistent with binding to type II receptors than to type I. Crosslinking and ligand blotting with human 125I-IL-1 demonstrated the presence of two bands, a 78 kDa band typical of crosslinking to type II (p60) receptor, and a 98 kDa band, typical of crosslinking to the type I (p80) receptor. Low level expression of the type I receptor was consistent with molecular biological studies employing polymerase chain reaction (PCR) amplification which indicated that mRNA for the type I receptor was produced by the HepG2 cells. Functional receptors were demonstrated by the induction of IL-8 by IL-1 stimulated cells.

Posttranslational modification of human T-cell growth factor

Amino-terminal sequence analysis of human T-cell growth factor indicated that the amino acid in position 3 of the polypeptide chain was modified. Examination of the N-terminal octapeptide using the amino acid analyzer and mass spectrometry demonstrated that position 3 was a threonine which was linked to N-acetyl-D-galactosamine. This site of glycosylation is of practical significance since it appears to play a role in the selectivity of a monoclonal antibody for the factor.

Receptors for T-Cell Growth Factor: Structure, Function and Expression on Normal and Neoplastic Cells

“During the years since its discovery (Morgan et al., 1976), the preception of T-cell growth factor (TCGF), also called Interleukin-2 (IL-2),” an in vitro novelty has given way to an appreciation of its crucial role both in the immune response and in the etiology and potential therapy of a number of diseases. In addition to playing a pivotal role in the culturing and cloning of T cells (Gillis and Smith, 1977; Schreier et al., 1980), TCGF potentiates the release of a number of other important lymphokines, including γ-interferon (γ-IFN) (Farrar et al., 1981; Kasahara et al., 1983), B-cell growth factor (BCGF) (Howard et al., 1983), and B-cell differentiation factor (BCDF) (Inaba et al., 1983). Moreover, TCGF or defects in its production or function have been implicated in such pathological states as congenital and acquired immunodeficiency (Palladino et al, 1984; Flomenberg et al, 1983; Harel-Bellan et al., 1983; Stotter et al, 1980; Thoman and Weigle, 1982), autoimmunity (Altman et al, 1981; Dauphinee et al., 1981; Linker-Israeli et al., 1983), and cancer (Gootenberg et al., 1981; Rey et al, 1983). Indeed, the receptor for this growth factor is expressed on the cells of several types of neoplasms (see Section VII), raising the question of whether its presence in such instances is fortuitous or physiologically significant. From the viewpoint of clinical therapy, several reports have appeared demonstrating the efficacy of TCGF in promoting in vitro and in vivo activity of alloreactive (Kern et al,1981) and tumor-reactive T-cells as well as natural and lymphokine-activated killer populations (Lotze et al, 1980, 1981; Wagner et al., 1980; Gillis and Watson, 1981; Cheever et al., 1982; Hefeneider et al., 1983; Eberlein et al., 1982; Rosenberg et al., 1983; Paetkau et al., 1982; Merluzzi et al., 1983; Henney et al., 1981, Donohue et al., 1984; Grimm et al., 1983).

The effect of cholesterol in a liposomal Muc1 vaccine.

A liposomal Muc1 mucin vaccine for treatment of adenocarcinomas was formulated by incorporating a synthetic Muc1 mucin-based lipopeptide and Lipid A into a DPPC/cholesterol bilayer. Vaccination of mice with the liposomal formulation produced a peptide-specific immune response dependent on the cholesterol content. The response occurred at a threshold of 20-23 mol% cholesterol, and was optimal at cholesterol levels of > or =30 mol%. To understand this cholesterol dependency, we studied the effect of cholesterol on the liposomal bilayer and surface properties. Freeze-fracture electron microscopy showed a unique surface texture that was codependent upon cholesterol (> or =20 mol%) and lipopeptide content. Fluorescence anisotropy measurements exhibited a significant decrease in the rotational motion of 1,6-diphenyl-1,3,5-hexatriene in formulations containing >20 mol% cholesterol and only in the presence of the lipopeptide. At 20 mol% cholesterol and with lipopeptide, DSC showed a significant increase in the main phase transition of the DPPC bilayers, while Raman spectroscopy indicated a more ordered arrangement of DPPC molecules compared to control liposomes containing DPPC/cholesterol alone. Taken together, the data suggest the presence of lipopeptide-rich microdomains at and above a threshold of 20 mol% cholesterol that may play a role in the induction of a peptide-specific immunological response.

Interleukin-2-induced small unilamellar vesicle coalescence

Recombinant human interleukin-2 (rhIL-2) was incorporated in liposomes for potential therapeutic applications using a novel process. In this process, rhIL-2 caused the formation of large, unique multilamellar vesicles (MLVs) from small unilamellar vesicles (SUVs) of dimyristoylphosphatidylcholine (DMPC). Vesicle coalescence occurred most rapidly at 19 degrees C, between the pre- and main phase transition temperatures of DMPC, and showed a dependence upon pH (pH <5.5), ionic strength (>50 mM) and the initial size of the unilamellar vesicles (<or=25 nm). Intermediates (partially coalesced vesicles) within the forming multilamellar structures were identified by freeze-fracture electron microscopy and their presence was corroborated by differential scanning calorimetry. Several distinct steps were identified in the coalescence process. In the initial step, rhIL-2 rapidly bound to the DMPC SUVs. This was followed by a pH-dependent conformational change in the protein, as evidenced by an increase in tryptophan fluorescence intensity. The SUVs then aggregated in large clusters that eventually annealed to form closed MLVs. In this process over 90% of the rhIL-2 was bound to and incorporated within the multilamellar structures.

Interleukin-2 Receptors on Activated Malignant and Normal B-Cells

A battery of antigen non-specific, genetically unrestricted factors derived from T cells have been shown to play a role in the regulation of B-cell responses (Howard and Paul 1983). One such factor designated B-cell growth factor (BCGF) appears to be required for the proliferation of a subset of B cells following their interaction with antigen or with anti-immunoglobulin molecules. A second set of factors termed B-cell differentiation or T-cell replacing factors (TRF) is involved in the terminal maturation of such proliferating B-cells into immunoglobulin secreting cells. There has been controversy concerning the possible involvement of T-cell growth factor or interleukin-2 (IL-2) in B-cell responses and concerning the ability of this growth factor to act directly on B lymphocytes. The proponents of a direct action of IL-2 on B-cells have shown that depletion of IL-2 from cofactor rich supernatants by absorption on IL-2 dependent T-cell lines also removes a factor required for B-cell differentiation (Parker 1982; Leibson et al. 1981). The view that IL-2 acts directly on B-cells has been challenged since the IL-2 containing supernatants generally used in the previous studies also contained BCGF and one or more TRF’s (Howard and Paul 1983). Furthermore, IL-2 was not absorbed by resting B-cells, LPS stimulated splenic lymphoblasts or by either of the two Burkitt’s lymphoma B-cell lines examined suggesting that B-cells do not manifest receptors for IL-2 (Robb et al. 1981).