Growth inhibition of human T cells by antibodies recognizing the T cell antigen receptor complex

Monoclonal antibodies that bind to the T cell MHC-antigen recognition complex (anti-T3 or anti-Ti) are known to either mimic ligand binding and activate T cells or block ligand binding, leading to an inhibition of T cell activation. In the present experiments, we demonstrate a direct inhibitory effect on the growth of human T cells by anti-T3 or anti-Ti antibodies. The proliferation of human peripheral blood T cells preactivated by exposure to PHA was inhibited in a specific manner by anti-T3. Colony formation in soft agar by REX cells, a leukemic cell line of early T cell phenotype, was completely inhibited by anti-T3 or anti-Ti antibodies, whereas isotype-matched antibodies to a variety of other T cell markers had no effect. Growth of REX cells in suspension culture was not affected by anti-T3 or anti-Ti. A cell line, T3.N1, was established from an agar colony of anti-T3-resistant REX cells. T3.N1 was phenotypically identical to REX except for failure to express any detectable T3 or Ti surface antigen. T3.N1 colony formation in soft agar was not inhibited by anti-T3 or anti-Ti. There was no rise in [Ca2+]i of T3.N1 cells after anti-T3 or anti-Ti exposure. These results indicate that in addition to the well-known positive regulatory effects of ligand binding to the T3/Ti complex, T3/Ti binding can also result in a down-regulatory signal for human T cell growth.     

The effect of substance P on the regional lymph node antibody response to antigenic stimulation in capsaicin-pretreated rats

The undecapeptide substance P (SP) contained in primary afferent nerves is thought to mediate that part of the neurogenic inflammatory response consisting of vasodilation and plasma extravasation. This response is diminished in rats pretreated as neonates with the neurotoxin capsaicin. It is not known whether primary afferent nerves influence cellular responses of the immune response to antigenic stimulation. Using 6- to 12-wk-old Sprague-Dawley rats pretreated as neonates with capsaicin, we examined the regional lymph node response to a s.c. antigenic stimulus of sheep red blood cells. The number of cells secreting antigen-specific antibody in these animals was reduced by more than 80% using direct and indirect plaque assay methods. The reduced antibody response in capsaicin-pretreated animals was reversed by a s.c. infusion of SP given over a 4-hr period at the injection site immediately after antigen stimulation. This response had a threshold at approximately 1.0 X 10(-5) M SP. SP1-7 (1.0 X 10(-5) M) was without effect but an infusion of SP5-11 (1.0 X 10(-5) M) reversed the effects of capsaicin treatment indicating a carboxyl-terminal effect of SP. The results suggest that the reduced response of capsaicin-treated animals to an antigenic stimulus is due to an effect of capsaicin on the SP-containing primary afferent nerves rather than a toxic effect of capsaicin on the immune system.     

Cell-associated tumor necrosis factor (TNF) as a killing mechanism of activated cytotoxic macrophages

Different macrophage populations were investigated for their abilities to secrete tumor necrosis factor (TNF) and to lyse TNF-susceptible tumor cells. In this way we could demonstrate that TNF-secretion, although a feature of all activated macrophage populations, is no absolute requirement for the killing of the TNF-sensitive Wehi 164 target. Macrophage cytotoxicity against this cell but not against the TNF-resistant P815 mastocytoma, was completely inhibitable by a specific anti-TNF serum also in the absence of measurable secreted TNF. Moreover the TNF-dependent lysis of tumor cells could also be performed by activated macrophages that had been fixed with paraformaldehyde before the addition of the target cells. In the indirect radioimmunoassay, TNF could be demonstrated on the surface of fixed effector cells. Our results must be interpreted in terms of membrane-associated TNF as the lytic principle for TNF-susceptible tumor cells.     

Cytotoxic T lymphocytes from mice with soluble class I Q10 molecules in their serum are not tolerant to membrane-bound Q10

Q10 is a class I Qa-2 region-encoded molecule that is secreted by the liver and present in serum at high concentrations (about 10 to 60 micrograms/ml) in most strains of mice. The amino terminal portion of this molecule can also be expressed as an integral membrane protein by splicing the 5' end of the Q10 gene to the 3' end of H-2Ld and transfecting the hybrid gene into murine L cells. Because CTL primarily recognize polymorphic determinants controlled by the alpha 1 and alpha 2 domains of class I molecules and because the Q10d/Ld product expressed by transfected L cells includes the alpha 1 and alpha 2 domains of Q10d, we could address whether mice bearing serum Q10 were tolerant to this molecule at the CTL level. The results of these experiments demonstrate that Q10+ mice are able to generate H-2-unrestricted CTL activity against Q10d expressed on transfected L cells, and this response was not inhibitable by the addition of Q10-containing normal mouse serum. It is unlikely that this CTL activity is due to possible polymorphic differences in Q10 alleles, since semisyngeneic BALB/c (H-2d) mice, from which the Q10d hybrid gene construct was derived, are able to generate anti-Q10d effector cells. The Q10d molecule was shown to cross-react with H-2Ld, lending support to the concept that Qa genes can serve as donors for polymorphic sequences found in H-2K, -D, and -L. That mice can generate anti-Q10 CTL activity suggests that this soluble class I protein does not act as a toleragen for these cells. The implications of these findings for an understanding of self-tolerance are discussed.     

Lymphokine-mediated induction of antigen-presenting ability in thymic stromal cells

We have established and characterized long term thymic stromal cultures from BALB/c (H-2d) and CBA/J (H-2k) mice. All cultures contained multiple adherent cell types, whereas some also contained thymic macrophages (TM). Culture supernatants from all cultures tested contained macrophage colony-stimulating factor activity, whereas only cultures with TM had soluble or membrane-associated interleukin (IL)-1. However, a thymic epithelial cell line (3D . 1), cloned from one of these cultures, produced IL-1 bioactivity. Further analysis confirmed the production of IL-1 alpha mRNA by the epithelial cell. No IL-2 or IL-4 (formerly called B cell stimulatory factor 1) activity was detected in any of the cultures. Antigen-presenting (AP) ability was determined using the chicken ovalbumin (OVA)-specific, I-Ad-restricted T cell hybridoma 3DO-18.3. Harvested TM exhibited antigen-specific, Ia-restricted AP ability which was enhanced by IL-4 as well as interferon-gamma (IFN-gamma). In contrast, AP ability was detected in non-macrophage stromal cell cultures (NMSC) only after preincubation with IFN-gamma. AP by preinduced NMSC was also Ia-restricted and could be blocked by anti-I-Ad antibodies. Since the T cell receptor of 3DO-18.3 is known to recognize a peptide produced by CNBr degradation of OVA, these observations suggest that both TM and NMSC can process OVA to produce this peptide. Glutaraldehyde-fixation experiments confirmed that NMSC must process native OVA into antigenic peptides for successful AP. Assays using several cloned stromal cell lines of different lineages suggested that only epithelial cells could be induced with IFN-gamma to exhibit competent AP. Given the possible role for IFN-gamma in the maintenance of Ia in the thymus, we investigated whether IFN-gamma production could be ascribed to a subpopulation of thymocytes. Culture supernatants from calcium ionophore and phorbol ester-stimulated peanut agglutinin-negative, but not peanut agglutinin-positive, thymocytes induced AP ability in NMSC. Thus, some thymocytes can produce an Ia-inducing lymphokine (most likely IFN-gamma) which may play an important role in T cell ontogeny through its effects on both thymic macrophages and thymic epithelial cells.     

The molecular basis of the hyaluronic acid-mediated stimulation of granulocyte function

Previous investigations have demonstrated that the function of polymorphonuclear leukocytes (PMN) is stimulated by hyaluronic acid (HA). The aim of the present investigation was to study the molecular basis for the effect of HA. HA fragments of m.w. in the range from 792 (tetrasaccharide) to 3,000,000 all stimulated the chemotactic and phagocytic function of PMN. The active concentration ranged from 4 to 64 pmol/liter, irrespective of the molecular size. Further investigations demonstrated that N-acetyl-D-glucosamine (NAGA) was the smallest active fragment of HA. NAGA is one of the components from which HA is built up; the other component glucuronic acid was without effect, and so were the other glycosaminoglycans, N-acetyl-D-mannosamine, N-acetyl-D-galactosamine, and D-glucosamine. Finally, Con A, the glucopyranosyl and glucomannosyl binding lectin, inhibited the stimulatory effect of NAGA. As is the case with HA, fibronectin also acts as a necessary cofactor to NAGA when incubations are made in the absence of whole blood or serum. The present results strongly indicated that the combined action of NAGA and fibronectin worked directly on the PMN by an interaction at the cellular membrane level. We conclude that the stimulatory action of HA on granulocyte functions is mediated through one of its two structural components, i.e., NAGA.     

Acquisition of non-MHC restricted cytotoxic function by IL 2 activated thymocytes with an "immature" antigenic phenotype

Culture of human thymocytes in interleukin 2 (IL 2) results in the generation of cytotoxic T lymphocytes (CTL) that kill tumor cell targets without major histocompatibility complex (MHC) restriction. Thymic non-MHC restricted CTL expressed Leu-19 antigen, but were generated from thymic precursor cells that lacked expression of Leu-19. In contrast, short term culture in Il 2 of peripheral blood lymphocytes depleted of Leu-19+ lymphocytes did not result in the generation of cytotoxic activity. IL 2 was necessary and sufficient for the generation of cytotoxic thymocytes and induction of Leu-19 antigen expression. Thymic non-MHC restricted CTL were generated from precursor cells expressing CD1, an antigen present on the majority of thymocytes. Furthermore, cytotoxic activity was detected in IL 2 cultured thymocyte populations with an "immature" antigenic phenotype, i.e. CD1+ and CD4+, CD8+. Upon subsequent culture, thymic non-MHC restricted CTL lost expression of CD1, and developed an antigenic phenotype similar to peripheral blood non-MHC-restricted CTL, suggesting that peripheral non-MHC-restricted CTL may originate from these thymic precursors.     

Evidence for negative regulation of T cell growth by low affinity interleukin 2 receptors

Two experimental situations have been studied, and the results provide evidence for a negative regulatory role for the low affinity interleukin 2 receptor (LA-IL 2R). The IL 2-dependent T helper cell line L-14, deprived of IL 2, becomes quiescent and expresses comparable numbers of high affinity IL 2R (HA-IL 2R) and LA-IL 2R. After activation by recombinant IL 2, this cell line preferentially expresses LA-IL 2R. The IL 2 responsiveness of the L-14 cell line was found to vary according to the ratio of LA-IL 2R to HA-IL 2R: the relative predominance of the LA-IL 2R coincides with a hyporeactivity of cells to IL 2. In contrast, a predominance of HA-IL 2R is accompanied by an increase in cellular IL 2 reactivity. Treatment of three IL 2-dependent T cell lines (L-14, HT-2, and C30.1) with limited amounts of recombinant IL 2 and moderate concentrations of anti-IL 2R monoclonal antibodies stimulates T cell growth. This treatment was shown to selectively diminish the expression of membrane LA-IL 2R. The stimulation was attributed to the decrease of expression of LA-IL 2R.     

A synthetic peptide homologous to the envelope proteins of retroviruses inhibits monocyte-mediated killing by inactivating interleukin 1

The synthetic peptide CKS-17 has homology to a highly conserved region of the immunosuppressive retroviral envelope protein P15E, to envelope proteins of HTLV I, II, III, and to that encoded by an endogeneous C-type human retroviral DNA. CKS-17 inhibits the immune response of lymphocytes and the respiratory burst of human monocytes. Because P15E-related antigens are present in human malignant cell lines and cancerous effusions, we sought to determine the effect of CKS-17 on monocyte-mediated tumor cell lysis. Lysis of A375 tumor cells by lymphokine or lipopolysaccharide-activated human monocytes was inhibited by 10 microM CKS-17 (control, 79%; CKS-17-treated, 19%). Another synthesized peptide, CS-2, which has partial homology to CKS-17, failed to block monocyte-mediated killing. Thus, the inhibition by CKS-17 appeared to be specific. Because interleukin 1 (IL-1) is a cytocidal factor produced by activated monocytes, we also investigated the effect of CKS-17 on IL-1 production by monocytes and on direct IL-1-mediated cytotoxicity. CKS-17 did not block production or secretion of IL-1 by lipopolysaccharide- or interferon-gamma-activated monocytes. However, the direct cytocidal activity of both recombinant IL-1 alpha and IL-1 beta against A375 tumor cells was blocked by CKS-17. The cytotoxic activity of IL-1 was inhibited by CKS-17 if (a) IL-1 was preincubated with CKS-17 for 1 hr at 37 degrees C or (b) the A375 cells were incubated with CKS-17 for 1 hr prior to the addition of IL-1. CKS-17 also blocked IL-1-induced proliferation of murine thymocytes, the D10 T cell line, and an IL-1-responsive astrocytoma cell line. These data suggest that CKS-17 may be a potent inhibitor of IL-1.     

Polypeptides of the Golgi Apparatus of Neurons from Rat Brain

An antiserum was raised against fractions of the Golgi apparatus of neurons from rat brain. Immunoblots of these fractions with the antiserum showed two principal bands of 185 and 150 kilodaltons (kd) in apparent molecular mass. The antiserum reacted with five or six bands of 200, 150, 130, 100-110, 64, and 40 kd in apparent molecular mass in immunoblots of several crude brain membrane fractions. Affinity-purified antibodies from the different gel bands transferred to nitrocellulose paper were used in immunoblot and immunocytochemical studies. Antibodies eluted from the 200-, 150-, 100-110-, and 64-kd bands reacted not only with the corresponding band but also with the other three bands. Antibodies eluted from the 40-kd band stained only the corresponding band. On light and/or electron microscopic immunocytochemistry, the antiserum stained the Golgi apparatus of rat neurons, glia, liver, and kidney tubule cells. Weaker, segmented, and less consistent staining was observed in nuclear envelopes, rough endoplasmic reticulum, and plasma membranes of neurons. Antibodies eluted from the bands at 200, 150, 100-110, and 64 kd stained intermediate cisterns of the Golgi apparatus of neurons. These findings suggest that a group of related polypeptides of brain membranes is preferentially expressed or enriched in the Golgi apparatus of neurons. Polypeptides with apparent molecular masses of 185 and 150 kd probably represent moieties endogenous to membranes of the neuronal Golgi apparatus.