Samuel D. Waksal

T lymphocytes with promiscuous cytotoxicity.

CYTOTOXIC T lymphocytes generated during a unidirectional mixed lymphocyte culture (MLC) lyse target cells which have the antigenic phenotype of the allogeneic stimulating cells. The cytotoxic effect is restricted to cells that bear the same major histocompatibility antigens (H–2 antigens in mice) as the stimulating cells1. H–2 restriction of cytotoxicity is also seen when immune T lymphocytes react in vitro to cells bearing viral2, chemical3, or minor histocompatibility antigens4 or following autosensitisation against unmodified fibroblasts5. Thus, H–2 restriction of cytotoxic lymphocytes has been observed in many circumstances. We now report that cultures of normal spleen cells generate T lymphocytes that damage target cells regardless of their H–2 phenotype.

INDUCTION OF T-CELL DIFFERENTIATION IN VITRO BY THYMUS EPITHELIAL CELLS*

Thymus-reticular epithelial cells (TE-cells) were grown in a cell culture devoid of any lymphocytic elements. These cells were able to induce T-cell differentiation in spleen cells from T-dificient mice as expressed by con-A responsiveness and GvH reactivity. It was also shown that xenogeneic rat TE cells were as effective in the induction of T-cell differentiation in vitro as syngeneic TE cells. This system is therefore ideal for the study of T-cell development.

Analysis of intrathymic differentiation patterns during the course of AKR leukemogenesis

Abstract Thymocytes from AKR mice in different stages of leukemia development were analyzed with the fluorescence-activated cell sorter (FACS) using monoclonal antisera to Lyt-1, Lyt-2, Thy-1.1, H-2K k , and Ia k . In addition, the number of cells bearing receptors for peanut agglutinin (PNA) was assessed. The results were correlated with the expression of murine leukemia virus (MuLV) antigen. Thymocytes from late preleukemic and leukemic stages were found to have a phenotype characteristic of a more mature cell population in that there was an increase in the expression of determinants encoded within the K end of H-2 k and Ia k . This was associated with a decrease in the number of thymocytes bearing receptors for PNA during the leukemic stage. Simultaneously, a shift from a Lyt-1 + 2 + thymocyte population to cells with varying expressions of Ly antigens was observed. Analysis of Lyt determinants on thymomas indicated that they could arise from cells bearing any of the different possible combinations of Ly phenotypes. The cell surface antigen changes occurred in temporal correlation with an increased expression of MuLV antigens.

Ultrastructural Localization of Concanavalin A Binding Sites on the Surface of Differentiating Hemopoietic Cells

SummaryThe surface characteristics of hemopoietic cells in normal human bone marrow have been explored by the use of the ultrastructural concanavalin A-peroxidase-diaminobenzidine (CAPD) procedure for the detection of specific carbohydrate residues (α-D-mannopyranoside, α-D-glucopyranoside, and α-N-acetyl-D-glycosaminide) associated with the cell surface. All cells present in the bone marrow were capable of binding Con A to their surfaces. The extent of binding proved cell specific and could be related to the stage of morphological development of each cell line. Maximum surface reactivity in the bone marrow cell population occurred with the most immature cells (myeloblast and erythroblast), lymphocytes, eosinophils, monocytes, macrophages and platelets, while mature neutrophils, basophils, and erythrocytes showed only minimum surface reactivity. These findings serve not only to expand our knowledge of the chemical nature of the surface of differentiating hemopoietic cells but provide striking evidence that modifications in the surface of hemopoietic cells occur during the process of normal cell differentiation in the bone marrow.

Phenotypic alteration in retroviral gene expression by leukemia-resistant thymocytes differentiating in leukemia-susceptible recipients

(AKR x NZB)F1 mice possess the dominant genes, Akv-1, Akv-2, Nzv-1a and Nzv-2a, which determine the expression of ecotropic and xenotropic viruses. Nevertheless, their thymic lymphocytes fail to produce these agents, and these mice are resistant to leukemia. We investigated the mechanism of this cell-specific restriction in radiation chimeras. (AKR x NZB)F1 thymocytes that had differentiated in lethally irradiated AKR recipients produced high levels of ecotropic and xenotropic viruses and showed marked amplification of MuLV antigen expression. Polytropic viruses could also be isolated from such thymocytes. These virological changes in chimeric thymocytes were donor- and host-specific and occurred only when (AKR x NZB)F1 bone marrow cells were inoculated into AKR recipients. This inductive capacity of the host environment could be detected in irradiated AKR recipients as early as age 2 months. The phenotypic changes brought about in leukemia-resistant (AKR x NZB)F1 thymocytes by the leukemia-susceptible AKR thymic microenvironment may be the result of a three-component inductive system.

Thymus-derived lymphocyte differentiation and lymphocytic leukemias: I. Evidence for the existence of functionally different subpopulations of thymus-derived cells in leukemic AKR mice

Abstract The effects of transplanting thymic (LTC), splenic (LSC), and lymph node (LLNC) lymphocytes derived from overtly leukemic AKR mice into preleukemic syngeneic animals were studied. Each of these thymus-derived (T cell) populations produced a different and distinct pathology in recipient mice. Animals receiving LTC exhibited thymoma and enlargement of peripheral lymphoid tissues. Gross organomegaly was also noted in mice given LSC, but thymic atrophy was uniformly observed. The thymus appeared normal in mice receiving LLNC, but marked enlargement of peripheral lymphoid tissues again were observed. The differences noted in disease pathologies correlated with the “homing” patterns of the subpopulations investigated. These findings suggest that subpopulations of T cells exist in mice with a thymus-derived neoplastic disorder.

Evidence for Conformational Changes in Concanavalin A Upon Binding of Saccharides as Determined from Solvent Water Proton Magnetic Relaxation Rate Dispersion Measurements

In previous studies of the interaction of solvent water molecules with the Mn++ ion in Mn-Con A by observation of the dispersion of the spin-lattice relaxation rate (T) of the solvent water protons over a wide range of magnetic fields (Koenig et al., 1973), we have shown that this rate is dominated by the residence time of a single exchanging water ligand on the Mn++ ion. In limited measurements at low fields, we also observed that the binding of α- or β- methyl-D-glucopyranoside to Mn-Con A decreased the relaxation rate by approximately 15 percent. In the present study, we have measured the effects of binding of a series of mono- and oligosaccharides on the solvent water proton relaxation rate over a range of magnetic fields from 5 Oe to 12 KOe and show that the observed decrease in the relaxation rate is due to an increase in the residence time of the single exchanging water ligand. This effect is consistent with a conformational change in the protein upon binding of saccharides. We find that the binding of α- and β- methyl-D-glucopyranoside, α- methyl-D-mannopyranoside and β-(o-iodophenyl)-D-glucopyranoside produce the same increase in residence time and therefore the same conformational change in the protein, whereas galactose and β- (o-iodophenyl)-D-galactopyranoside show no effects. The same reduction in relaxation rate as that caused by the above monosaccharides was observed with the following oligosaccharides: D-maltose, D-maltotriose, D-maltotetraose, o-α-D-mannopyranosyl-(1→2)-D-mannose, o-α-D-mannopyranosyl-(1→2)-o-α-D-mannopyranosyl-(1→2)-D-mannose, o-α-D-mannopyran-osyl-(1→2)-o-α-D-mannopyranosyl-(1→2)-0-α-D-mannopyranosyl-(1→2)-D-mannose and melezitose. As observed by Goldstein and co-workers, the first three oligosaccharides have nearly the same affinity constant, whereas the α-(1,2) linked mannans show increasing affinity constants with increasing chain length. Melezitose also shows enhanced binding by a factor of four relative to α-methyl-D-glucopyranoside. The water relaxation data suggest that the above mono- and oligosaccharides bind to Con A by a similar mechanism involving only a single saccharide residue combined with the protein at one time. Determination of the enthalpy (∆H) and entropy (∆S) of binding of maltotriose and melezitose indicates that the factor of 8 difference in their affinity constants is due to different ∆S values. This suggests that the greater affinity of melezitose is due to the presence of two glucose residues in the molecule, either of which is capable of binding to the same site on the protein. The increased probability of binding for melezitose results in a larger forward rate constant relative to maltotriose which has only one glucose residue which can bind to the protein. Thus, the greater affinity of the α-(1→2)-mannose oligosaccharides appears to be due to a statistical increase in probability of binding because of the presence of more than one binding residue in the chain and not to an extended binding site on the protein.

Brain-associated-theta antiserum. Differential effects on lymphocyte subpopulations.

Abstract Subpopulations of lymphocytes were delineated, both in vitro and in vivo , using brain associated-θ antisera. Lymphocytes were depleted from the cortical areas of the thymus and the T-dependent regions of the spleen. No depletion occurred in the medullary area of the thymus or in the deep cortical areas of the lymph node. These histological findings correlated well with the effects of brain associated-θ antisera using an in vitro cytotoxicity assay. Greater concentrations of antisera were required to cause cell death of lymphocytes from lymph node than lymphocytes from either spleen or thymus. These data would suggest that the θ antigen may be either lost or modulated during the process of lymphocyte differentiation.

Natural cytotoxicity in AKR/J mice during adjuvant induced amyloidogenesis☆

Abstract The induction of amyloidosis in AKR mice has previously been shown to be associated with a decrease in the incidence of spontaneous thymic leukemia (P. Ebbesen, Brit. J. Cancer 29 , 76, 1974). Amyloid induction with azocasein depresses the activity of the natural killer (NK) cell, a cell believed to be important in the protection against the development of malignancy. In the present studies, therefore, we examined the response of the NK cell to the induction of amyloidosis in AKR mice. Rapid and long-term depression of NK cell activity against YAC-1 tumor cells was noted following intraperitoneal administration of complete Freunds adjuvant enriched with Mycobacterium butyricum . Mixing studies suggested that active suppression did not account for the observed decrease in NK cell activity. Although some NK cell activity was noted in ascitic fluid, redirection was not felt to account for the rapid and dramatic reduction in splenic NK cell activity. Furthermore, serum from adjuvant-treated but not control mice was found to significantly inhibit NK cell activity in vitro . These studies therefore suggested a role for a serum factor in the depression of this activity. The apparent paradox of decreased NK cell activity in a setting of diminished leukemogenesis is considered in relation to previous studies describing impaired T-cell functioning after the induction of amloidosis.

Thymocyte Maturation in AKR Leukemia

The central role played by the thymus during leukemogenesis in the AKR mouse is well established (1, 2). The AKR mouse develops lymphocytic lymphoma and leukemia with an incidence of 90% (2). Although this strain is infected with gross virus prior to birth, manifestations of the disease are not apparent until six months of age (3). Therefore, there appears to be a latent period before the onset of thymoma in these animals. The intrathymic events during this latent period are of prime importance since neonatal thymectomy reduces the incidence of leukemia (4).