J M Depper

Qualitative analysis of immune function in patients with the acquired immunodeficiency syndrome: evidence for a selective defect in soluble antigen recognition

We studied purified subpopulations of lymphocytes from patients with the acquired immunodeficiency syndrome (AIDS) in order to determine whether intrinsic defects in lymphocyte function, aside from those due to alterations in lymphocyte numbers, were present. Mitogen-stimulated DNA synthesis, production of gamma interferon, production of interleukin-2, and expression of interleukin-2 receptors, although variably decreased in unseparated cell populations, were normal in populations of purified T-cell subsets. In contrast, DNA synthesis in response to the soluble protein antigen tetanus toxoid was decreased in both unseparated and purified T-cell subpopulations. Cell-mixing experiments demonstrated that the hyporesponsiveness of the unfractionated lymphocytes from patients with AIDS was not due to active suppression. We conclude that the lymphocytes of patients with AIDS, although capable of undergoing a normal degree of blast transformation and lymphokine production after mitogenic stimulation, have an intrinsic defect in their ability to recognize and respond to soluble antigen.

Functional and phenotypic comparison of human T cell leukemia/lymphoma virus positive adult T cell leukemia with human T cell leukemia/lymphoma virus negative Sézary leukemia, and their distinction using anti-Tac. Monoclonal antibody identifying the human receptor for T cell growth factor.

Adult T cell leukemia (ATL) and Sézary leukemia are malignant proliferations of T lymphocytes that share similar cell morphology and clinical features. ATL is associated with HTLV (human T cell leukemia/lymphoma virus), a unique human type C retrovirus, whereas most patients with the Sézary syndrome do not have antibodies to this virus. Leukemic cells of both groups were of the T3, T4-positive, T8-negative phenotype. Despite the similar phenotype, HTLV-negative Sézary leukemic cells frequently functioned as helper cells, whereas some HTLV-positive ATL and HTLV-positive Sézary cells appeared to function as suppressors of immunoglobulin synthesis. One can distinguish the HTLV-positive from the HTLV-negative leukemias using a monoclonal antibody (anti-Tac) that appears to identify the human receptor for T cell growth factor (TCGF). Resting normal T cells and most HTLV-negative Sézary cells were Tac-negative, whereas all ATL cell populations were Tac-positive. The observation that ATL cells manifest TCGF receptors suggests the possibility that an abnormality of the TCGF-TCGF receptor system may partially explain the uncontrolled growth of these cells.

The Human Interleukin-2 Receptor: Normal and Abnormal Expression in T Cells and in Leukemias Induced by the Human T-Lymphotropic Retroviruses

The human receptor for interleukin-2 (T-cell growth factor) plays a critical role in the growth of T cells and is required for full expression of the normal immune response. Through hybridoma and recombinant DNA techniques, the interleukin-2 receptor protein has been biochemically characterized and purified; full-length copies of its complementary DNA have been molecularly cloned, sequenced, and expressed in eukaryotic cells; and the receptor gene has been characterized. Transient expression of the interleukin-2 receptor gene occurs during normal T-cell activation, and high- and low-affinity forms of the membrane receptor exist. A naturally occurring, soluble receptor has also been isolated, and its levels in serum correlate with the activity of various diseases. Deregulation of interleukin-2 receptor expression occurs in T-cell leukemias produced by the human T-lymphotropic retroviruses types I and II (HTLV-I and -II) and has been causally linked to the action of the trans-activator (taf) gene of these viruses. Monoclonal antibodies specific for the interleukin-2 receptor are being evaluated in the treatment of HTLV-I-induced leukemias and other conditions involving the inappropriate function of activated T cells.

The Human Interleukin-2 Receptor: Analysis of Structure and Function

Considerable information presently exists regarding the molecular, biochemical, and biological features of the human IL-2 receptor. The IL-2 receptor protein, multiple receptor mRNAs, and a single structural gene have now been identified. The important role of this receptor in normal T-cell growth is well established and its potential participation in B-cell growth and differentiation appreciated. The availability of cloned gene products for both the IL-2 receptor and IL-2 may permit the future development of novel biological agents capable of either augmenting or blunting the T-cell immune response. The intriguing interrelationship of HTLV-I and -II infection and altered IL-2 receptor expression is now being unraveled. However, the structural difference in high and low affinity receptors as well as the mechanism by which signals for T-cell growth are propagated through the high affinity receptor remain dominant, unanswered questions in the field.

A Monoclonal Antibody to the Human Receptor for T Cell Growth Factor

T cell growth factor (TCGF or interleukin-2) is a 14800-dalton glycoprotein hormone which is essential for a normal immune response. It has been extensively reviewed previously (Smith, 1980; Ruscetti and Gallo, 1981). Morgan et al. (1976) were the first to report that conditioned media from lectin-stimulated mononuclear cells contained a factor capable of supporting the exponential growth of lectin-activated human T cells. That factor became known as TCGF, and is now known to be essential for the expansion and continued proliferation of cytotoxic (Gillis et al., 1978a), suppressor (Coutinho et al., 1979), and some helper T cells (Watson, 1979). TCGF has permitted the long-term maintenance of T cell lines in vitro (Morgan et al., 1976; Ruscetti et al., 1977). It is important to recognize that the specificity of the immune response is determined by the antigen, while TCGF provides the mitogenic stimulus in an antigen nonspecific and non-restricted manner (Smith et al., 1979). Utilizing a sensitive bioassay (Gillis et al., 1978b), this lymphokine was biochemically characterized (Mier and Gallo, 1980; Gillis et al., 1980), purified to homogeneity (Robb, 1982; Robb et al., 1983), and its N-terminal protein sequence determined (Robb et al., 1983). Recently, the gene encoding TCGF has been cloned and expressed using a eukaryotic vector (Taniguchi et al., 1983; Cheroutre et al., 1983; Clark et al., 1983; Lin et al., 1983).

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).

The Human Interleukin-2 Receptor

SUMMARY Complementary DNAs corresponding to the human receptor for interleukin-2 (IL-2) have been molecularly cloned, sequenced, and expressed in both COS-1 and L cells. The human genome appears to contain a single structural gene for this receptor located on the short arm of chromosome 10 (band 14–15). However, when transcribed, at least two families of mRNAs are produced, which vary in length due to the use of at least three different polyadenylation signals. Sequence analysis of the cloned cDNAs and S1 nuclease protection assays indicate an alternative pathway of mRNA processing for this receptor whereby a 216 base-pair segment contained within the protein coding region is spliced, resulting in an mRNA unable to encode a functional IL-2 receptor. In contrast, cDNAs corresponding to mRNA retaining this 216 base-pair region code membrane receptors that bind both IL-2 and anti-Tac (monoclonal anti-IL-2 receptor antibody). Analysis of the deduced amino acid sequence reveals that the receptor is composed of 272 amino acids including a signal peptide 21 amino acids in length. Hydrophobicity analysis suggests a single, 19 amino acid transmembrane domain. A short intracytoplasmic domain composed of 13 amino acids is present and contains two potential phosphate acceptor sites (serine and threonine but not tyrosine) as well as positively charged residues presumably involved in cytoplasmic anchoring. Two sites for N-linked glycosylation sites and numerous extracytoplasmic O-linked glycosylation sites are present.

Interleukin-2 Receptors

Interleukin-2 (IL-2) is a lymphokine synthesized by some T-cells following activation. Resting T-cells do not express IL-2 receptors but receptors are rapidly expressed on T-cells following the interaction of antigens, mitogens, or monoclonal antibodies with the antigen specific T-cell receptor complex. Using anti-Tac a monoclonal antibody that recognizes the IL-2 receptor, the receptor has been purified. The receptor is a 33kd peptide that is posttranslationally glycosylated to a 55kd mature form. Mature receptors contain both N-linked and O-linked sugars and are both sulfated and phosphorylated. Using an oligonucleotide probe, based on the N-terminal amino acid sequence, cDNAs encoding this receptor have been cloned, sequenced and expressed. The addition of anti-Tac to in vitro culture systems blocks the IL-2 induced DNA synthesis of IL-2 dependent T-cell lines and inhibits soluble auto- and alloantigen induced T-cell proliferation. Furthermore, it prevents the generation of cytotoxic and suppressor effector T cells. The anti-receptor antibody also inhibits lectin stimulated immunoglobulin synthesis and the sequential expression of late appearing activation antigens on T-cells. Normal resting T-cells and most leukemic T-cell populations do not express IL2 receptors however the leukemic cells of all patients with human T-cell leukemia/lymphoma virus (HTLV-1) associated, adult T-cell leukemia (ATL) examined expressed the Tac antigen. In HTLV-I infected cells the 42kd LOR protein encoded in part, by the pX region of HTLV-I may act as a transacting transcriptional activator that induces transcription of the IL-2 receptor gene thus providing an explanation for the constant association of HTLV-I infection of lymphoid cells and IL-2 receptor expression. The constant display of large numbers of IL-2 receptors which may be aberrant in the ATL cells may play a role in the uncontrolled growth of these leukemic T-cells. Patients with the Tac positive adult T-cell leukemia are being treated with the anti-Tac monoclonal antibody directed towards this growth factor receptor.

Molecular Analysis of the Human Interleukin-2 Receptor

Complete expression of the human immune response requires the generation of activated T cells1. This activation sequence is initiated by an interaction of antigen with specific receptors present on the membrane of resting T cells. This receptor-ligand interaction, in the presence of macrophage derived interleukin-1 (IL-1), then triggers the production of interleukin-2 (IL-2, previously designated T cell growth factor)2,3. IL-2 is a well-characterized 14,500 dalton glycoprotein which promotes T cell proliferation following binding to specific high affinity IL-2 membrane receptors4–6. However, unlike receptors for antigen, IL-2 receptors are not expressed by resting T cells, but like IL-2, are synthesized following antigen activation. The interaction of IL-2 with its inducible receptor results in T cell proliferation and expansion of the antigen reactive T cell clone and culminates in the emergence of T cells mediating helper, suppressor, and cytotoxic T cell function. Thus, both the specificity and magnitude of the T cell immune response is in large measure controlled at the level of IL-2 receptor expression.

Molecular Cloning of cDNA for the Human Interleukin-2 Receptor

Interleukin-2 (IL-2 or T cell growth factor) is a 14,500 daltons glycoprotein critically involved in the development of a normal human immune response [1, 2]. Recently, cDNA for this lymphokine has been isolated and expressed in both prokaryotic and eukaryotic cells [3–5]. Further, the human IL-2 gene has been cloned, sequenced [6], and localized to chromosome 4. As with other polypeptide hormones, IL-2 exerts its biologic effects through binding to specific high affinity membrane receptors [7]. However, neither IL-2 nor IL-2 receptors are produced by resting T cells [7, 8]. Following exposure to antigen, T cells binding antigen enter a program of cellular activation leading to de novo synthesis and secretion of IL-2 and expression of IL-2 receptors. The interaction of IL-2 with its cellular receptor then triggers cellular proliferation, resulting in the growth and development of helper, suppressor, and cytotoxic T cells.