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Biomedical subjects

R J Robb

Publications and source records attributed to R J Robb.

At least 73 records · Page 4Linked to original sources
PubMed1984

The systemic administration of purified interleukin 2 enhances the ability of sensitized murine lymphocytes to cure a disseminated syngeneic lymphoma.

Significant serum titers of interleukin 2 (IL 2) can be maintained in mice for 12 h after i.p. injection in a 15% gelatin solution. We have tested the ability of IL 2 administered systemically in this fashion to enhance the therapeutic effect of adoptively transferred specifically sensitized lymphoid cells that were expanded in IL 2. Mice with established local and disseminated FBL-3 lymphoma, induced by intrafootpad injection of 10(7) cells after 500 rad total body irradiation, were treated with a combination of i.v. injected murine splenocytes and either murine supernatants containing IL 2 or pure human IL 2 in gelatin. Splenocytes from immune mice were resensitized to irradiated tumor in vitro and were expanded for 7 days in lectin-free IL 2 supernatants. Treatment with these murine splenocytes administered with murine IL 2 supernatants prolonged mean survival to 33.6 days compared with mean survival times of 16.9 days (p less than 0.001) and 23.4 days (p = 0.007) for mice treated with IL 2 alone or splenocytes alone. Human IL 2, purified to homogeneity from the Jurkat cell line, was also capable of improving the therapeutic efficacy of transferred cells in mice. Mean survival was significantly prolonged to 32.1 days when cells and purified human IL 2 were administered, whereas mean survival times of 18.1 days (p = less than 0.001) and 21.5 (p = less than 0.001) were seen for mice treated with IL 2 alone or expanded immune cells alone. Cure rates in this model were also significantly enhanced with the combined treatment of IL 2 and expanded immune cells. Combined immunotherapy utilizing IL 2 and immune cells was thus significantly better than either component used alone. The systemic administration of IL 2 in conjunction with sensitized expanded lymphoid cells may be a useful approach to the immunotherapy of other murine and human tumors.

Animals↗
PubMed1984

In vivo administration of purified Jurkat-derived interleukin 2 in mice.

Pure human interleukin 2 (IL-2), produced by the T-cell lymphoma Jurkat, was injected in mice to study the serum half-life, toxicity, and in vivo immunological effects of IL-2. The serum half-life (t1/2) of Jurkat IL-2 in mice appeared to have two components: (a) a rapid initial phase with t1/2 of approximately 2 min during which most of the exogenous IL-2 was cleared from the serum; and (b) a second, slower component with t1/2 of about 9 min. Mice given injections i.p. or i.v. with pure Jurkat IL-2, at doses comparable on a microgram/kg basis to contemplated doses for humans, showed no signs of toxicity on the basis of serial measurements of weight, serum liver and kidney chemistries, or histology of lymphoid and vital organs. Jurkat IL-2 had no effect on the rate of growth or survival of mice with an established s.c. methylcholanthrene-induced fibrosarcoma, but Jurkat IL-2 used in conjunction with in vitro-resensitized and IL-2-expanded specific immune splenocytes prolonged survival of mice with disseminated FBL-3 tumor. This survival prolongation was highly significant when compared to treatment with Jurkat IL-2 alone (p = less than 0.001) or an equivalent number of in vitro-resensitized and expanded cells alone (p = 0.004). Treatment of mice with i.p. Jurkat IL-2 subsequent to secondary immunization with allogeneic tumor enhanced by more than 5-fold the splenocyte cytotoxicity for alloantigen measured 7 days later. Thus, purified human IL-2 derived from the Jurkat cell line has a short half-life in mice with no apparent toxicity at large doses. In vivo efficacy of human IL-2 was demonstrated in increasing alloantigen responsiveness and in increasing the efficacy of transferred expanded immune lymphocytes in the FBL-3 lymphoma model.

Animals↗
PubMed1983

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.

Amino Acid Sequence↗
PubMed1983

Direct demonstration of the identity of T cell growth factor binding protein and the Tac antigen.

Radiolabeled molecules from detergent-solubilized human T cell blasts were fractionated on affinity supports coupled with T cell growth factor (TCGF) and anti-Tac antibody. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis indicated that a glycoprotein of approximately 58,000 mol wt was bound in both cases. Sequential binding to the two affinity supports demonstrated that the molecules recognized in each instance were identical. Thus, the Tac antigen contains the cellular binding site for TCGF. Note added in proof: Preliminary binding experiments using high concentrations of [3H]leucine, lysine TCGF with a low specific radioactivity indicate the existence of a sizeable pool of receptor sites with an affinity 2,000-10,000 times lower than that of the high affinity receptors measured in Fig. 1. Such sites may explain the numerical discrepancy between early TCGF binding experiments (5) and the binding of the anti-Tac antibody. The hypothesis that the anti-Tac antibody apparently reacts with both classes of receptor would explain its effect on the physiological response (high affinity binding) and the high level of Tac antigen on the cell surface.

Antibodies, Monoclonal↗
PubMed1983

Lymphokine-activated killer cell phenomenon. III. Evidence that IL-2 is sufficient for direct activation of peripheral blood lymphocytes into lymphokine-activated killer cells.

Purified interleukin 2 (IL-2) was found to be sufficient for direct activation of peripheral blood lymphocytes into lymphokine-activated killer (LAK) cells. The LAK activation factor was directly and consistently associated with IL-2 activity using classic protein purification techniques, adsorption to IL-2-dependent cell lines, and inhibition with anti-Tac antibody. As yet, no other cytokines have been found that perform the same role.

Antibodies, Monoclonal↗
PubMed1983

Identification and initial characterization of a rat monoclonal antibody reactive with the murine interleukin 2 receptor-ligand complex.

Xenogeneic monoclonal antibodies were prepared to the murine interleukin 2 (IL-2)-dependent HT2 cell line. One rat IgM monoclonal antibody (7D4) was identified that inhibited proliferation of the HT2 cells and of IL-2-dependent CTLL cells in the presence of crude rat IL-2 as well as of purified human IL-2. The level of inhibition was dependent on both antibody and IL-2 concentration. Cell distribution studies using a fluorescence-activated cell sorter showed that the antigen identified by 7D4 is expressed at a high density on HT2 cells and on concanavalin A (Con A)-induced T-cell blasts and at a substantially lower density on lipopolysaccharide-induced B-cell blasts; 7D4 binding was not detected on greater than 95% of nonactivated thymocytes, T cells, or B cells. Competition binding studies indicated that 7D4 fails to inhibit the binding of 3H-labeled human IL-2 to CTLL cells. However, 7D4 specifically immunoprecipitated 3H-labeled human IL-2 from detergent extracts of HT2 cells or Con A-induced T-cell blasts that had been pulsed with [3H]IL-2; in contrast, 7D4 did not react with free [3H]IL-2. Initial biochemical analysis of immunoprecipitates with 7D4 of detergent extracts from surface-iodinated Con A-activated spleen cells showed a major band having apparent molecular weight of 48,000-62,000. Collectively, these results suggest that 7D4 detects an epitope on the IL-2 receptor distal to the ligand binding site or another molecule that physically associates with the receptor.

Animals↗
PubMed1983

Purification and partial sequence analysis of human T-cell growth factor.

A murine monoclonal antibody directed against human T-cell growth factor (TCGF) from the JURKAT cell line was used for affinity column purification of the factor. Bound TCGF was eluted nearly quantitatively at low pH, and the recovered factor appeared homogeneous by two-dimensional gel electrophoresis. The molecule is markedly hydrophobic, with a high content of leucine. A single NH2-terminal sequence of 36 residues was obtained by automated Edman degradation, further supporting the homogeneity of the material. Thus, significant quantities of purified TCGF have been prepared in a single step, making possible detailed analysis of its molecular structure and biological role.

Amino Acid Sequence↗
PubMed1983

Characterization of the human receptor for T-cell growth factor.

Anti-Tac monoclonal antibody has been identified as a putative antibody against the receptor for T-cell growth factor (TCGF). We now show that: (i) TCGF blocks 85% of 3H-labeled anti-Tac binding to phytohemagglutinin-activated lymphoblasts and (ii) both anti-Tac and anti-TCGF immunoprecipitate a protein band that appears to represent TCGF crosslinked to its receptor on HUT-102B2 cells. In HUT-102B2 cells, the TCGF receptor is a Mr 50,000 glycoprotein with internal disulfide bond(s) and a pI of 5.5-6.0, and it represents approximately equal to 0.05% of total cellular de novo protein synthesis. It contains a peptide of Mr 33,000 that is processed to a mature form that includes N-linked and O-linked sugars and sialic acid.

Cell Line↗
PubMed1983

Identification of a membrane antigen that is distinct from the interleukin 2 receptor and that may be required for interleukin 2-driven proliferative responses.

During the course of studies designed to raise murine monoclonal antibodies to a guinea pig alloreactive T cell line, an IgM monoclonal antibody (5C3) was identified that completely inhibited the alloantigen and IL 2-dependent proliferative response of the immunizing colony and other alloreactive T cell colonies with different antigen specificities. Tissue distribution on the FACS demonstrated that the surface antigen defined by 5C3 was preferentially expressed on T cells activated by mitogen, alloantigen, or antigen. 5C3 was also a potent inhibitor of alloantigen- and antigen-induced T cell proliferation. In addition, 5C3 substantially inhibited the proliferative response of guinea pig Con A-induced blast cells to guinea pig IL 2-containing culture fluids and to a preparation of partially purified human IL 2 but was a poor inhibitor of mitogen-induced T cell proliferation. Blast cells obtained from the co-culture of mesenteric lymph node cells with Con A in the presence of 5C3 lacked the surface molecule defined by 5C3. The 5C3- and 5C3+ blast populations, nevertheless, absorbed equivalent amounts of IL 2, which suggested that 5C3- blasts were not deficient in the expression of IL 2 receptors. However, 5C3- blast cells failed to proliferate in response to IL 2-containing culture fluids. Furthermore, 5C3 had no inhibitory activity on either the binding or subsequent degradation of radiolabeled human IL 2 by guinea pig IL 2 receptors. These data suggest that 5C3 recognizes a component of a putative IL 2 receptor-effector complex distinct from the IL 2 receptor and that this molecule plays a critical role in the processing or generation of the growth signal IL 2 transmits to T lymphocytes.

Animals↗
PubMed1983

Blockade of the interleukin-2 receptor by anti-Tac antibody: inhibition of human lymphocyte activation.

We have previously shown that monoclonal anti-Tac antibody inhibits the proliferation of interleukin-2 (IL-2) dependent human continuous T cell lines. Further, we have shown that anti-Tac specifically blocks greater than 95% of the binding of radiolabeled II-2 to a continuous T cell line. In view of these data, we suggested that anti-Tac antibody may bind to and block the human T cell receptor for IL-2. We now report the effects of anti-Tac on the activation of human peripheral blood T lymphocytes. We find that anti-Tac: 1) blocks T cell proliferation induced by soluble antigens (80-90%), autologous antigens (90%) and alloantigens (75-90%); 2) partially inhibits T cell proliferation induced by mitogenic lectins, including Concanavalin A (50-88%), pokeweed mitogen (40-87%), and phytohemagglutinin (20-80%); 3) abrogates (greater than 95%) the generation of cytolytic T lymphocytes in allogeneic cell cocultures, but does not inhibit killing by cytolytic T lymphocytes once formed; 4) and inhibits T cell dependent pokeweed mitogen activated B cell immunoglobulin production (78-95%). We further demonstrate that anti-Tac inhibition of proliferation is not secondary to diminished production of IL-2. Finally, in antigen induced T cell proliferative assays, we demonstrate that the addition of highly purified IL-2 reverses the inhibitory effects of anti-Tac. These data are consistent with the hypothesis that anti-Tac recognizes the human IL-2 receptor and illustrate ways in which this antibody can be used to modulate the human immune response.

Antibodies, Monoclonal↗
PubMed1981

T cell growth factor receptors. Quantitation, specificity, and biological relevance.

To examine directly the hypothesis that T cell growth factor (TCGF) interacts with target cells in a fashion similar to polypeptide hormones, the binding of radiolabeled TCGF to various cell populations was investigated. The results indicate that TCGF interacts with activated T cells via a receptor through which it initiates the T cell proliferative response. Internally radiolabeled TCGF, prepared from a human T leukemia cell line and purified by gel filtration and isoelectric focusing, retained biological activity and was uniform with respect to size and charge. Binding of radiolabeled TCGF to TCGF-dependent cytolytic T cells occurred rapidly (within 15 rain at 37 degrees C) and was both saturable and largely reversible. In addition, at 37 degrees C, a receptor- and lysosome-dependent degradation of TCGF occurred. Radiolabeled TCGF binding was specific for activated, TCGF-responsive T cells. Whereas unstimulated lymphocytes of human or murine origin and lipopolysaccharide-activated B cell blasts expressed few if any detectable binding sites, lectin- or alloantigen-activated cells had easily detectable binding sites. Moreover, compared with lectin- or alloantigen-activated T cells, long-term TCGF-dependent cytolytic and helper T cell lines and TCGF-dependent neo-plastic T cell lines bound TCGF with a similar affinity (dissociation constant of 5-25 pM) and expressed a similar number of receptor sites per cell (5,000-15,000). In contrast, a number of TCGF-independent cell lines of T cell, B cell, or myeloid origin did not bind detectable quantities of radiolabeled TCGF. Binding of radiolabeled TCGF to TCGF-responsive cells was specific, in that among several growth factors and polypeptide hormones tested, only TCGF competed for binding. Finally, the relative magnitude of T cell proliferation induced by a given concentration of TCGF closely paralleled the fraction of occupied receptor sites. As the extent of T cell clonal expansion depends on TCGF and on the TCGF receptor, the dissection of the molecular events surrounding the interaction of TCGF and its receptor that these studies permit, should provide new insight into the hormonelike regulation of the immune response by this lymphokine.

Animals↗
PubMed1979

Complete amino acid sequence of a papain-solubilized human histocompatibility antigen HLA-B7. 1. Isolation and amino acid composition of fragments and of tryptic and chymotryptic peptides.

As a part of the overall strategy for determining the complete covalent structure of the papain-solubilized portion of the heavy chain of the human histocompatibility antigen HLA-B7, the protein was dissected into various fragments by a combination of partial acid hydrolysis and cyanogen bromide cleavage. After purification by chromatographic procedures, these fragments have been used as a source for tryptic and chymotryptic peptides. Thirty-three major tryptic and twenty-two major chymotryptic peptides were purified in nanomole amounts and their amino acid compositions determined. These peptides account for the whole extent of the polypeptide chain with the exception of the amino-terminal CNBr pentapeptide. They provide the basis for the formal alignment of the acid cleavage and cyanogen bromide fragments of the molecule as well as the source material for the elucidation of the primary structure of the HLA-B7 heavy chain.

Amino Acid Sequence↗
PubMed1979

The heavy chain of human histocompatibility antigen HLA-B7 contains an immunoglobulin-like region.

The 88-residue fragment (ac-2) containing the second disulphide loop from HLA-B7 heavy chain is shown to have statistically significant homology with Ig constant domains, including both matches at invariant positions and conservative substitutions of structurally important residues. Thus, both the light chain (beta 2m) and a segment of the heavy chain of HLA antigens may be structurally and evolutionarily related to immunoglobulins.

Amino Acid Sequence↗
PubMed1978

Sequence of the COOH-terminal hydrophilic region of histocompatibility antigens HLA-A2 and HLA-B7.

Detergent-solubilized HLA antigens were isolated from a human lymphoblastoid cell using an anti-beta2-microglobulin immunoaffinity column. The HLA-A and HLA-B locus products were separated by thin layer isoelectric focusing. Cleavage of the p44 chain of HLA-A2 and -B7 antigens with cyanogen bromide led to the isolation of a 31-amino-acid fragment from each. The fragments were sequenced and shown to be from the COOH-terminal end of the intact chains using carboxypeptidase Y. The fragment from the HLA-B7 chain, 55% of whose amino acids were polar, contained the 2 cysteine residues not found in the papain-derived molecule. The tentative sequence of the fragment from the HLA-A2 chain was similar to that of the HLA-B7 fragment but appeared not to contain any cysteine residues. The hydrophilic COOH-terminal region of HLA antigens, which directly follows the hydrophobic, membrane-binding segment, began with a cluster of basic amino acids. This arrangement of amino acids resembles that found at the COOH terminus of the red blood cell membrane protein, glycophorin.

Amino Acid Sequence↗