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Biomedical subjects

H R MacDonald

Publications and source records attributed to H R MacDonald.

At least 217 records · Page 12Linked to original sources
PubMed1987

Phenotypic identification of memory cytolytic T lymphocytes in a subset of Lyt-2+ cells.

Murine peripheral Lyt-2+ T cells could be subdivided according to surface expression of the Pgp-1 glycoprotein into major (71%) Pgp-1- and minor (29%) Pgp-1+ subsets. A striking correlation was observed between Pgp-1 expression and enrichment for antigen-specific memory cytolytic T lymphocyte precursors (CTLp). After immunization with the male minor transplantation antigen H-Y, virtually all the H-Y-specific CTLp were found in the minor Pgp-1+ subset of Lyt-2+ cells. In addition, after alloimmunization the frequency of allospecific CTLp resistant to inhibition by anti-Lyt-2 antibody was markedly enriched within the Pgp-1+ cells, suggesting an enrichment for CTLp bearing high avidity antigen receptors. Taken together, these data suggest that surface Pgp-1 expression is stably acquired at the time of primary antigenic stimulation by virgin T cells. As such, Pgp-1 represents an important marker for identifying a subset of Lyt-2+ T cells with the quantitative and qualitative properties of memory CTLp.

Animals↗
PubMed1987

Granzyme A secretion by normal activated Lyt-2+ and L3T4+ T cells in response to antigenic stimulation.

Granzyme A is a serine esterase initially isolated from cytoplasmic granules of cytolytic T cell lines (Masson, D. et al., EMBO J. 1986. 5: 1595). Among normal T lymphocytes activated by allogeneic stimulation it is found in both Lyt-2+ and L3T4+ subsets in comparable amounts. Here we show that normal alloantigen-activated T cells secrete the enzyme specifically when mixed with appropriate stimulator cells. Specific enzyme release is dependent on external calcium and removal of calcium blocks further secretion within a few minutes. Both Lyt-2+ and L3T4+ cells specifically secrete the enzyme with similar rates, and anti-L3T4 and anti-Lyt-2 antibodies block secretion by the responder cells expressing these markers. Anti-LFA-1 antibodies, on the other hand, block secretion by either subset.

Animals↗
PubMed1987

Anti-Thy-1-induced proliferation of immature thymocytes expressing the CD3-associated gamma/delta heterodimer.

A series of rat monoclonal antibodies (mAb) directed against the Thy-1 molecule have been evaluated for their ability to stimulate immature (CD4-8-) murine thymocytes. CD4-8- thymocytes could be induced to proliferate by several anti-Thy-1 mAb provided that exogenous interleukin 1 or interleukin 2 was provided. Apparently non-stimulatory anti-Thy-1 mAb could be rendered stimulatory if cross-linked by a rabbit anti-rat immunoglobulin reagent. Anti-Thy-1-stimulated CD4-8- thymocytes were found to uniformly express CD3 but did not stain positively with F23.1, a mAb reactive with alpha/beta T cell receptors utilizing the V beta 8 gene family. Immunoprecipitation analysis of such stimulated CD4-8- thymocytes indicated that they expressed a CD3-associated heterodimer of 45 kDa/35 kDa most likely corresponding to the product of the gamma and (putative) delta T cell receptor genes. Taken together, these data demonstrate that anti-Thy-1 mAb selectively activate a subset of CD4-8- thymocytes committed to the gamma/delta T cell receptor lineage.

Animals↗
PubMed1987

Perforin is present only in normal activated Lyt2+ T lymphocytes and not in L3T4+ cells, but the serine protease granzyme A is made by both subsets.

We show that among the subsets of peripheral T lymphocytes (Lyt2+ L3T4- and L3T4+ Lyt2- cells) activated in short-term cultures by stimulation with H-2 incompatible leukocytes 97% of the cytolytic activity and all detectable perforin activity resides in the Lyt2+ cells. But both populations contain approximately equal amounts of a serine protease, granzyme A, the expression of which was previously thought to be restricted to cytolytic T lymphocytes.

Animals↗
PubMed1987

Biochemical characterization of a T-lymphoma-specific 90,000 molecular weight disulfide linked dimeric glycoprotein.

The biochemical features of a membrane antigen detected by a mouse monoclonal antibody (A1) raised against the murine thymoma cell line EL4 are described. This reagent detected a novel disulfide-linked 90,000 mol. wt dimeric membrane glycoprotein composed of two chains of approx 45,000 mol. wt. Endo-beta-N-acetylglucosaminidase F digestion generated a single 28,000 polypeptide, thus suggesting that the A1 molecule is a homodimer. No structural homology between the A1 molecule and the human T 90/44 protein (9.3 antigen) could be revealed by peptide mapping analysis. In view of the fact that three polypeptides of mol. wts 28,000-30,000, 21,000 and 15,000 respectively co-precipitated with the A1 antigen, the possible relationship of the A1 molecular complex to other known T-cell surface antigens including the antigen receptor is discussed.

Animals↗
PubMed1987

Abelson virus transformation of an interleukin 2-dependent antigen-specific T-cell line.

Abelson murine leukemia virus (A-MuLV) carries the gene v-abl, one of a group of oncogenes with structural and functional (tyrosine kinase) homology to three growth factor receptors. Work in this and other laboratories has shown that A-MuLV infection can render myeloid and lymphoid cells independent of the growth factors interleukin 3 and granulocyte-macrophage colony-stimulating factor. We have now shown that v-abl can also relieve interleukin 2 (IL-2) dependence in T cells. We infected a cloned IL-2-dependent antigen-specific cell line. Transformed cells were generated which were factor independent and tumorigenic. The transformants each bore unique v-abl DNA inserts and expressed v-abl mRNA. No elevation of expression of either IL-2 or its receptor could be detected in these cells. Thus, A-MuLV can short-circuit the dependence of hematopoietic cells on IL-2, IL-3, and possibly granulocyte-macrophage colony-stimulating factor, none of whose receptors are known to be of the tyrosine kinase type.

Abelson murine leukemia virus↗
PubMed1986

Point mutations of human interleukin-1 with decreased receptor binding affinity.

Interleukin-1 (IL-1) is a monocyte-derived polypeptide hormone that interacts with a plasma membrane receptor. We have used oligonucleotide-directed mutagenesis to construct mutant human IL-1 proteins. Three different point mutants in a unique histidine residue (position 30) exhibited varying degrees of reduced IL-1 receptor binding affinity, whereas point mutants at five other residues behaved normally. Structural analysis of these mutant proteins by nuclear magnetic resonance spectroscopy detected no (or only minor) conformational changes relative to wild-type IL-1. These data suggest that the unique histidine residue influences the architecture of the receptor binding site on human IL-1.

Animals↗
PubMed1986

Cultured Lyt-2- L3T4- T lymphocytes from normal thymus or lpr mice express a broad spectrum of cytolytic activity.

T lymphocytes expressing the surface phenotype Lyt-2- L3T4- represent a minor population of immature thymocytes that appear to be the precursors of mature T cells. Cells with the same apparent surface phenotype also accumulate in vast numbers in the lymphoid tissues of the autoimmune lpr mouse. Lyt-2- L3T4- T lymphocytes from lpr lymph node (LN) or normal thymus express low to undetectable levels, respectively, of surface antigen receptor. In addition, they produce reduced amounts of lymphokines compared with normal T cells and lack precursors of alloantigen-specific cytolytic T lymphocytes. We previously showed that after culture with phorbol esters and interleukin 2, lpr Lyt-2- L3T4- T lymphocytes proliferate and differentiate, acquiring increased levels of surface antigen receptor by most cells, as well as Lyt-2 by a portion. We now show that cultured Lyt-2- L3T4- T cells from lpr LN or normal thymus are very efficiently cytolytic toward not only allogeneic tumor targets, but also natural killer (NK)-susceptible targets and syngeneic targets. Such killing was not inhibited by antibodies to H-2 or Lyt-2. In contrast, cultured mature Lyt-2+ L3T4- T cells from normal LN, thymus, or lpr LN were cytolytic only toward allogeneic targets and were dependent on Lyt-2 expression and H-2 recognition. The similarities of cultured Lyt-2- L3T4- T cells to NK and lymphokine-activated killer cells are discussed.

Animals↗
PubMed1986

Different structural constraints for recognition of mouse H-2Kd and -Kk antigens by alloimmune cytolytic T lymphocytes.

We have constructed a new series of hybrid genes among the H-2Kd,-Kk, and -Kb. The site of recombination occurs in the third exon, encoding the alpha 2 domain, and divides this domain into two parts, alpha 2A and alpha 2B. The novel genes differ only in the COOH-terminal half of the alpha 2 domain, i.e., the alpha 2B region. This region, comprising residues 142-182, contains a limited number of amino acid differences between the three alleles. The hybrid genes have been introduced into 1T 22-6 cells (H-2q), and cell surface expression of hybrid antigens was verified. Cells expressing different types of hybrid antigens have been examined for their susceptibility to lysis by cytotoxic T lymphocytes directed either against the H-2Kd antigen or the H-2Kk antigen. Our results show that the alpha 1 and alpha 2A domains of the H-2Kk antigen can constitute target molecules for alloimmune anti-Kk T cells, whereas the alpha 2B region, when exchanged for Kd or Kb sequences, plays only a limited role. In contrast, the alpha 1 and alpha 2A domains of Kd are not sufficient to be recognized by alloimmune anti-Kd T cells. In this instance, the alpha 2B domain seems to play an essential role. This region has undergone several amino acid substitutions involving charged residues.

Amino Acid Sequence↗
PubMed1986

Role of interleukin 1 in early T cell development: Lyt-2-L3T4- thymocytes bind and respond in vitro to recombinant IL 1.

Thymocytes that bear neither Lyt-2 nor L3T4 differentiation antigens (2-4- thymocytes) contain the precursors for mature L3T4+Lyt-2- and Lyt-2-L3T4+ T cells. In the present study we determined the capacity of 2-4- cells to respond to recombinant interleukin 1 (rIL 1) in vitro. The presence of rIL 1 enhanced IL 2-dependent proliferation to the lectins Con A and PHA by threefold to eightfold. In a second assay, rIL 1 enhanced proliferation and IL 2 production by 2-4- cells in response to phorbol myristate acetate (PMA) and the calcium ionophore, ionomycin. Using a direct IL 1 binding assay, we were able to detect both high-affinity (Kd approximately 5 pM) and low-affinity (Kd approximately 200 pM) classes of IL 1 receptors on freshly isolated 2-4- cells. Bound IL 1 was rapidly internalized, suggesting that such receptors were functional. These results are compatible with a role for interleukin 1 during thymocyte maturation.

Animals↗
PubMed1986

Functional status of interleukin 2 receptors expressed by immature (Lyt-2-/L3T4-) thymocytes.

A subpopulation of phenotypically immature (Lyt-2-/L3T4-) thymocytes express receptors for the polypeptide hormone interleukin 2 (IL 2); however, these cells do not proliferate in vitro in response to IL 2. In investigating this phenomenon in greater detail, we observed that the IL 2 receptors (IL 2-R) on freshly isolated immature thymocytes bound IL 2 with about fivefold lower affinity (Kd approximately 100 pM) than IL 2-R on activated mature T cells and T cell lines (Kd approximately 20 pM). Furthermore, in contrast to activated T cells, Lyt-2-/L3T4- thymocytes did not endocytose bound IL 2. When stimulated in short-term culture with a combination of phorbol ester (PMA) and calcium ionophore, Lyt-2-/L3T4- thymocytes proliferated in a largely IL 2-dependent fashion. IL 2-R expression on these activated cells initially disappeared (at 24 hr) and subsequently reappeared (at 48 to 72 hr). Reexpressed IL 2-R on activated thymocytes resembled those on mature T lymphocytes in that they bound IL 2 with high affinity (Kd = 15 to 25 pM) and were capable of endocytosing IL 2. Taken together, these data place certain constraints on the putative physiologic role of IL 2 in intrathymic growth regulation.

Animals↗
PubMed1986

Binding and internalization of interleukin 1 by T cells. Direct evidence for high- and low-affinity classes of interleukin 1 receptor.

In this report we describe, on the basis of direct IL-1 binding assays and IL-1 internalization studies, the existence of two classes of IL-1-R on a variety of T cell types. Cells of the EL4-6.1 thymoma express large numbers (approximately 20,000 per cell) of IL-1-R that have a Kd of approximately 300 pM for IL-1. Even though these receptors make up 98-99% of the total IL-1-R per cell, they appear to be nonfunctional, based on their inability to endocytose IL-1. A minor class of IL-1-R (200-400/cell) has an approximately 100-fold higher affinity for IL-1 (Kd, approximately 5 pM) and can rapidly internalize the ligand upon binding. All of the biological activity of IL-1 can be shown to occur via binding to high-affinity IL-1-R since the IL-1 concentration giving half-maximum biological activity in EL4-6.1 cells corresponds precisely to the Kd of this class of receptor. Other cell types, including normal T cells, also express both high- and low-affinity IL-1-R, but the absolute number of receptors per cell is considerably less.

Cell Line↗
PubMed1986

Interleukin 1-dependent induction of both interleukin 2 secretion and interleukin 2 receptor expression by thymoma cells.

The macrophage-derived product, interleukin 1 (IL 1) is thought to play an important regulatory role in the proliferation of T lymphocytes; however, its mechanism of action is unknown. We describe in this report a variant subline of EL4 thymoma cells (EL4-6.1) that displays a high degree of responsiveness to IL 1. We show that recombinant IL 1 can induce both the secretion of interleukin 2 (IL 2) and the expression of IL 2 receptors (IL 2-R) by these cells. EL4-6.1 cells do not constitutively secrete IL 2, nor do they express IL 2-R; but when cultured in the presence of recombinant IL 1, they secrete detectable amounts of IL 2 (5 to 15 U/ml). In the presence of either suboptimal levels of phorbol ester (PMA) or Ionomycin, the addition of IL 1 resulted in up to an 80-fold enhancement in the amount of IL 2 secreted. Stimulation with IL 1 alone or in combination with Ionomycin was unable to induce detectable IL 2-R expression by EL4-6.1 cells. However, in the presence of suboptimal concentrations of PMA, IL 1 induced expression of about 3000 high affinity (dissociation constant, Kd of 31 pM) and 50,000 low affinity (Kd of 2800 pM) IL 2-R. These IL 2-R were functional, based on their ability to rapidly internalize IL 2. This model system will allow a detailed analysis of the mechanisms involved in the regulation of the immune response by IL 1 and IL 2.

Animals↗
PubMed1986

Separation of tumor-infiltrating lymphocytes from tumor cells in human solid tumors. A comparison between velocity sedimentation and discontinuous density gradients.

The separation of viable tumor-infiltrating lymphocytes (TIL) from surgical biopsies of human solid tumors was achieved by velocity sedimentation at unit gravity or by discontinuous density gradients. The two methods were adapted to small volumes and cell numbers not exceeding 1 X 10(8). The recovery, purity and composition of the TIL-enriched fractions were comparable in the two methods. Density gradients were more rapid, simpler and more practical for preparation under sterile conditions of TIL from clinical material than velocity sedimentation. Lymphocytes in the TIL-enriched fractions obtained by either of the methods were poorly responsive to mitogens. This poor responsiveness is a characteristic of the human TIL and seems to be related to effects exerted by tumor cells.

Breast Neoplasms↗
PubMed1986

Phorbol ester enhances both interleukin 2 receptor expression and immunoglobulin secretion in human Epstein-Barr virus-immortalized B cells.

The enhanced expression of interleukin 2 (IL2) receptors by 12-O-tetradecanoylphorbol 13-acetate (TPA) on sublines of an Epstein-Barr virus-immortalized human B17 B cell line (M. Steinitz et al., Immunobiology 1979. 156: 41.) was studied by immunofluorescence using the anti-Tac monoclonal antibody and by binding studies with purified radiolabeled IL2. These studies show that TPA at a final concentration of 5 ng/ml greatly increased Tac antigen expression on a number of sublines of B17. IL2-binding studies revealed that TPA induced an increase in not only the number of IL2 receptors per cell but also the affinity of the receptors for IL2. The number and affinity of IL2 receptors on the C76 subline treated with TPA appear to be similar to those of activated normal human peripheral T cells. Furthermore, TPA-induced differentiation of these B cell lines was measured by induction of immunoglobulin secretion using an enzyme-linked immunosorbent assay. The capacity of TPA to induce differentiation in human B cells and the biological significance of IL2 receptor expression by activated B cells are discussed.

Antigens, Surface↗