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Highly purified CD1-3-4-8- human thymocytes were obtained by panning techniques combined with cell depletion with antibody-coated magnetic beads. Most of these cells expressed cytoplasmic CD3 antigen, as assessed by mAbs known to react with the CD3 epsilon chain. After culture with low doses of PMA (0.5 ng/ml) and subsequent addition (at 24 h) of recombinant interleukin 2 (rIL-2; 100 U/ml) cells underwent extensive proliferation (40-60-fold of the initial cell input after 2 wk). The majority of the proliferating cells were CD3-TCR-. The remaining cells (5-40%) were represented by CD3+ TCR gamma/delta+ (BB3- A13+) cells. Further removal of CD3+ TCR-gamma/delta+ cells resulted in highly purified CD3- populations that further proliferated in culture with no substantial phenotypic changes. When CD3+ thymocytes were cultured under the same experimental conditions, only CD3+ TCR-alpha/beta+ cells could be detected, thus indicating that PMA did not affect the surface expression of the CD3/TCR complex, but rather induced preferential growth of CD3- thymocytes. Surface marker analysis of cultured CD3- thymocytes showed that they were homogeneously CD7+, whereas low proportions of cells expressed CD2 and CD8 antigens. Among the natural killer (NK) cell markers, CD56 was highly expressed by all cells, whereas CD16, CD57, CD11b, NKH2, and GL183 were absent. Importantly, these cells were different from peripheral NK cells, as 80-95% of them expressed cytoplasmic CD3 antigen. Functional analysis revealed a strong cytolytic activity against both NK-sensitive (K562) and NK-resistant (M14, Daudi) human target cells. In a redirected killing assay against the Fc gamma R+ P815 cells, mAbs specific for triggering molecules including CD3, CD2, and CD16 failed to augment target cell lysis, while a strong cytolytic effect was induced by PHA. In addition, PHA alone or in combination with PMA induced tumor necrosis factor alpha (TNF-alpha) and interferon gamma (IFN-gamma) (but not IL-2) production by CD3- thymocytes. Cloning of fresh CD1-3-4-8-thymocytes in the presence of PMA and rIL-2 resulted in CD3-CD56+ clones that displayed a pattern of cytolytic activity and lymphokine production similar to that of the polyclonal populations. Northern blot analysis of transcripts coding for CD3/TCR molecules revealed the presence of CD3 zeta, epsilon, and gamma transcripts, while CD3 delta was undetectable. Mature transcripts for both gamma and delta TCR chains could be detected, whereas no TCR-alpha mRNA and only a truncated (1.0 kb) form of TCR-beta mRNA were revealed.(ABSTRACT TRUNCATED AT 400 WORDS)

The term of "natural killer" (NK) cells was originally assigned on a merely functional basis to lymphoid cells capable of lysing certain tumors in the absence of prior stimulation. However, both their origin and the molecular mechanism(s) involved in their function remained a mystery for many years 1. Regarding their origin, clear evidence has now been provided both in mouse and in man that NK and T cells may derive from a common precursor 2-5. Thus, mature NK cells can be obtained in vitro from CD34(+) cells isolated from umbilical cord blood, bone marrow (BM) and even human thymus 6 when cultured in the presence of appropriate feeder cells or IL-15. The molecular mechanism allowing NK cells to discriminate between normal and tumor cells, predicted by the "missing self hypothesis" 7, has been clarified only in recent years. Thus, NK cells recognize MHC class I molecules through surface receptors delivering signals that inhibit, rather than activate, NK cells. As a consequence, NK cells lyse target cells that have lost (or express insufficient amounts of) MHC class I molecules, as frequently occurs in tumors and in cells infected by certain viruses.

Page 1980 Due to a printing error the affiliation given for Michele Cilli was wrongly indicated; the correct affiliation is: Istituto Scientifico Tumori and Centro Biotecnologie Avanzate, Genova, Italy

Aim of this study was the characterization of SCLC lines genetically engineered to express the immunostimulatory cytokine IL-2. Preliminarily, we examined by immunofluorescence and RT-PCR the expression of IL-2 receptor on 10 different SCLC lines to evaluate possible effects of IL-2 on the tumour cells. Only one SCLC cell line (GLC1) out of 10 tested expressed IL-2R α , β and γ chain while the others were negative. Human IL-2 gene was cloned by RT-PCR and inserted into the RSV.5 expression vector containing the neo resistance gene. By electroporation or lyposome-mediated transfection we have selected stable transfectants of 4 different SCLC lines (GLCI, N592, NCI-H69 and NCI-H164) secreting 25–200 U/ml of biologically active IL-2 in the supernatant. All IL-2 transfected tumour cell lines, including the IL-2R+ GLC1, displayed an “in vitro” proliferative potential (by MTT proliferation assay) similar to that of untransfected cell lines. Further analyses of the tumourigenic potential in nude mice are in progress. IL-2 transfected cells were able to stimulate the proliferation and to induce anti-tumour cytotoxic activity in peripheral blood lymphocytes.

Monoamine oxidase type B (MAO-B) was similarly active in the hypothalamus of 60-day-old male and female Charles River rats. A single, 2 mg/kg IP injection of deprenyl, however, resulted in a significantly greater inhibition of hypothalamic MAO-B in normal males than in normal females. Repeated administration of estrogen (estradiol valerate) to intact males postnatally, a treatment which disrupts the masculinization process, although not provoking true "feminization," decreased the inhibition of MAO-B, thus abolishing the sex-specific difference. The intensity of deprenyl-provoked hypothermia and ptosis in males exceeded that of females; neonatal and postnatal estrogenization of males resulted in diminution of these effects. Androgen administration to neonate females did little affect the biochemical and in vivo parameters of MAO inhibition. It is concluded that sex-specific, biochemical differences in MAO-B inhibition may have pharmacological correlates, and both facets of MAO inhibition are sensitive to neonatal exposure to estrogen.

Transcription of the T cell receptor (TcR) delta gene was detected in clones and in enriched populations of CD3- major histocompatibility complex-unrestricted cytotoxic cells. The transcripts were derived, at least in part, from unrearranged TcR delta genes, as shown by hybridization to a synthetic oligonucleotide probe corresponding to germ-line sequences just upstream of J delta 1. Transcripts from unrearranged TcR delta genes are similar in size to those from productively rearranged genes. The fact that the bulk of delta transcripts was derived from unrearranged genes was supported by the observation that TcR delta gene rearrangements were not detected in DNA isolated from an enriched CD3- cytotoxic cell population. Transcription of the TcR delta gene in CD3- cytotoxic cells is consistent with the notion that these cells belong to a T cell lineage.

Hypothyroidism of mild intensity was obtained with prenatal and neonatal submission of Long-Evans rats to an iodide-rich diet. Chronic daily administration of methimazole to iodide-supplemented Long-Evans pups or to iodine-deprived Charles-River rats through the first 29-30 days of age provoked severe hypothyroidism. Monoamine oxidase type A (MAO-A) and not type B (MAO-B) activity was consistently, although slightly (by approximately 20%), increased in the hypothyroid brain. Triiodothyronine (T3)-induced hyperthyroidism did not affect MAO activity. Replacement therapy with T3 did not normalize MAO-A activity in hypothyroidism. Methimazole displayed a competitive and reversible in vitro inhibition of MAO-A but not MAO-B activity. Although this effect was obtained at concentrations far higher than those estimated to reach the brain after a single injection of the goiterogen, the occurrence of accumulation processes in the metabolism-deficient hypothyroid neonate rats cannot be excluded. Thus, MAO-A activity might be either directly depressed during the goiterogenic treatment, or increased as the result of some kind of rebound effect after interruption of methimazole administration.

The control of production of the membrane (m) vs secreted (s) forms of immunoglobulin heavy chains was investigated in a panel of cell lines expressing different heavy chain classes but identical light chains (lambda) and variable regions. These cell lines could be induced towards Ig secretion by mitogen treatment. During this process a shift from m to s heavy chain production takes place. Here we show that, similarly to IgA- and IgE-producing B cells, in IgG2a-producing I.29 cells the gamma m-gamma s shift was accompanied by a shift in the corresponding mRNAs, with a decrease of gamma m mRNA and an increase of the gamma s mRNA in LPS-stimulated cells. By contrast, the micron mRNA was increased in LPS-stimulated IgM-producing cells, albeit these cells synthesized reduced amounts of micron polypeptides. The utilization of the translational level in the early steps of B lymphocyte maturation thus distinguishes the mode of regulation of mu chains from those of the other isotypes. In addition, in B cells a post-translational event blocks the secretion of IgM but not of IgG or IgE.

Tyrosine hydroxylase (TH) was studied in the entire hypothalamus of 60-day-old male and female rats, and its activity was similar in both sexes. Administration of estrogen or androgen to male or female pups soon after birth and during development did not affect the kinetic parameters of TH. It is, thus, suggested that sexual dimorphism in the levels and turnover of catecholamines (CA) and their sensitivity to fluctuations in gonadal steroids during development do not depend on corresponding sex-related or steroid-sensitive differences in the synthesis of hypothalamic CA.

T lymphocytes were isolated from ascitic fluid of three patients with ovarian carcinoma at III-IV stage. Surface markers analysis of such purified T cells revealed that T8+ cells were well represented among ascitic T lymphocytes (from 35 to 56%). Low percentages of activated T cells, as indicated by HLA-DR and TAC (interleukin-2 receptor) positivity, were also present. However, fresh ascitic T lymphocytes failed to lyse autologous tumor target cells in a 4-h 51Cr release assay. Furthermore, by applying a limiting dilution microculture system that allows optimal conditions for cloning of human T lymphocytes, we derived clones from these populations. From 41 to 63% of clones so obtained had cytolytic activity in a lectin-dependent assay allowing detection of cytolytic T cells of any specificity. More importantly, in all three patients several clones were found to lyse autologous tumor target cells as well. Some of these clones have been studied in more detail: 9 out of 10 expressed the T8+/T4- phenotype, whereas only one was T8-/T4+; 6 out of 9 clones had a definite NK-like activity, while none of them lysed autologous PHA-lymphoblasts.

Cyclosporin A (CsA) is an immunosuppressive drug that acts, at least in part, by blocking IL-2 release. Since IL-2-producing human T cells are heterogeneous with respect to their functional capabilities and surface phenotype, we investigated whether differences in sensitivity to CsA existed among different IL-2-producing T-cell clones. Preliminary dose/response experiments showed that 100 ng/ml CsA completely inhibited the PHA- or OKT3-induced IL-2 production by four representative T4+/T8- clones. On the other hand, this drug concentration had virtually no inhibitory effect on the proliferation of CTL-L murine indicator cells to exogeneous IL-2. Clones were derived directly from peripheral blood by applying a microculture system that allows clonal expansion of essentially all T cells: under these experimental conditions growing clones are therefore highly representative of the starting T-cell populations. Among clones so derived, 28 were selected according to their capability to release IL-2 upon PHA stimulation. Six of such clones displayed cytolytic activity in a PHA-dependent assay against P815 murine target cells. CsA (100 ng/ml) abrogated IL-2 production of all clones, including those displaying cytolytic activity and expressing the T4-/T8+ phenotype.

Long-Evans, male and female rats born of mothers kept on an iodide-rich diet prior to delivery and during lactation, were fed this diet after weaning, thus becoming slightly hypothyroid. A more severe hypothyroidism was also provoked with the chronic administration of methimazole to Long-Evans iodide-supplemented, or Charles River iodine-deprived pups through the first month of age. Additional Long-Evans rats were made hyperthyroid with a daily injection of triiodothyronine (T3) through the first 29 days of age. Severe hypothyroidism in both strains of rats markedly increased the density of serotonin type 1 (5-HT1) and type 2 (5-HT2) receptors in the brain (less cerebellum, corpus striatum and olfactory bulbs) at 31–32 days of age. Receptor alterations were not correlated to either the rise in thyrotropin (TSH) levels in hypothyroidism or the direct influence of residual methimazole after the last treatment, or to neonatal malnutrition. This increase in 5-HT receptor density might represent an adaptive (supersensitivity) postsynaptic response to the state of central serotonergic hypofunction occurring in hypothyroidism. Though receptor alterations might be important, their precise functional role in the etiogenesis of hypothyroid-associated mental disturbances is difficult to ascertain.

Cells from the monoclonal B cell lymphoma I.29 expressing surface IgM (mu +) are capable of differentiating in vitro to IgM secretion and of switching to IgA or IgE production in response to lipopolysaccharide (LPS) stimulation. To determine whether a single mu + B cell is capable of undertaking both differentiative pathways (isotype switch and plasma cell differentiation) I.29 mu + cells were cloned by limiting dilution and a panel of clones were analyzed by immunofluorescence, endogenous labeling and Northern blotting. While 100% of the clones could differentiate toward IgM secretion, only a proportion of them (greater than 70%) also switched to IgA and/or IgE production. Certain clones switched preferentially to a specific isotype. Taken together with the observation that C gamma genes were never the target of switching in our experiments, these data suggest that individual mu + clones from the I.29 lymphoma are "precommitted" as for their switching potentials. The subclones that showed a high frequency of switching to IgA transcribed the germ line C alpha gene(s), suggesting a role for chromatin structure in determining the isotype switch specificity. Switch variant clones expressing either IgA or IgE on the cell surface were isolated and found capable of further differentiating toward Ig secretion in response to LPS. On the contrary, we could not induce switch to IgA in IgE-producing cells. Unlike mu + and alpha + cells, all the switch variant clones expressing IgE tested by endogenous labeling constitutively secreted large amounts of IgE in the supernatants even in the absence of LPS stimulation.

T lymphocytes isolated from ascitic fluid of patients with stage III-IV ovarian carcinoma were cloned by means of a microculture system that allows cloning of virtually all peripheral blood human T lymphocytes. Under these experimental conditions, 15% to 42% of ascitic T cells gave rise to clonal progenies that were analyzed for different functional capabilities. Of the clones obtained, 36%-70% had cytolytic activity in a PHA-dependent assay (using P815 as target cells) that allowed detection of cytolytic cells of any specificity. About one-half of the cytolytic clones lysed the NK-sensitive K562 target cells as well. In addition, 30%-50% of the total clones released IL-2 upon stimulation with PHA for 24 hr. In all patients analyzed a variable proportion (11%-56% of all cytolytic clones) had cytolytic activity against autologous tumor cells. Some of these clones have been analyzed in more detail: 18/21 expressed the T4-T8+ phenotype, whereas the remaining 3 were T4+ T8-. Only one out of 6 clones tested lysed allogeneic ovarian carcinoma cells as well, while 5/8 had a definite NK-like activity. Finally, all 8 clones tested were inhibited by anti-T11 and 7/8 by anti-T8 monoclonal antibody.

Blymphocytes undergo during their development several sequential rearrangements at the immunoglobulin (Ig) loci (1). The first event is a D-JH recombination that usually takes place at both the heavy chain (IgH) loci, and is then followed by rearrangement of one of the numerous VHgenes to the DJHcomplex (2). When a functional VDJ rearrangement has taken place, synthesis of μ Polypeptide ensues and rearragments of the light chain loci begin. A K-λ hyerarchy has been demonstrated in both human and mouse B cells (3,4). A successful heavy chain gene recombination stops any further rearrangement at the IgH locus (5,6), and possibly stimulates IgL recombination. Similarly the creation of a productive light chain gene blocks further recombinations at the IgL loci (5). Subsequently IgM molecules are synthetized and expressed on the cell surface by lymphocytes (7). The latter may coexpress IgD through alternative splicing of the VDJ - Cμ -C δ transcription unit (8). IgM+ B cell may undergo a second recombinatorial event at the IgH locus, termed isotype switching. This event generally implies the deletion of Cμ, Cδ and all the CHgenes which are located 5′ to the one CH gene that will be expressed (9,10). Although the sequences mediating switch recombination have been isolated and determined (9–11), rather little is known about the mechanisms regulating isotype switching. The question of how an IgM+B lymphocyte decides which CH gene to recombine is still open. It is well known that certain antigens or routes of immunization result in vivo in the predominance of a given isotype (12). On the other hand several in vitro experiments have shown that more than one isotype can be produced by the progeny of a single B cell (13–15). Isotype switching is not the only differentiative option of an IgM+ B cell. The latter may infact respond to antigen or mitogen by differentiating into IgM secreting plasmacells (7). It is generally accepted that plasmacell may undergo a very limited number of divisions.

T cells isolated from thyroid tissue and peripheral blood of 2 patients with Hashimoto’s thyroiditis were studied by a high cloning efficiency microculture technique. Clonal efficiences of 37 and 24% were obtained from thyroid-derived T cell cultures, while 40 and 90% efficiencies resulted from peripheral-blood-derived cultures. A prevalence of T4––/T8+ T cell clones were found in thyroid infiltrates. The functional analysis of the clones demonstrated significantly higher proportions of clones with cytolytic activity in a lectin-dependent assay in thyroid-derived microcultures, as compared to peripheral blood-derived ones. The proportion of clones displaying natural-killer-like activity was increased in 1 patient only. Cytolytic activity was displayed not only by all T4––/T8+, but also by several T4+/T8–– intrathyroid clones. Remarkable proportions of cytolytic clones were also able to release interleukin-2 upon phytohemagglutinin stimulation. Finally, the proportion of T cell clones able to release gamma-interferon following mitogen stimulation was significantly higher in thyroid- vs. peripheral-blood-derived microcultures. These results provide further data about the possible pathogenetical role of both regulatory and effector T lymphocytes in human autoimmune thyroiditis.

The sex-dependent differentiation of monoamine oxidase (MAO) in the hypothalamus of 60-day-old, Charles River rats was found to involve only type A (MAO-A), and not type B (MAO-B) enzyme. In vivo inhibition of type A by clorgyline, and type B by (−)deprenyl, however, tended to decrease the specific activity of both types of MAO to a smaller extent in the female than in the male hypothalamus. When masculinization was prevented by neonatal administration of estradiol (E) to males, hypothalamic MAO-A and MAO-B activities increased in both control and MAO-inhibited rats. Androgenization of females, however, had little effect on the MAO activity. Whereas the effects of neonatal estrogenization were attributable neither to a direct influence of E nor to a sexual difference in the peripheral clearance of the MAO-inhibitor used, single, high doses of steroids to adult, but not to newborn rats, did acutely affect the kinetics of MAO-A. The activity of MAO-A was also decreased by high concentrations of E or TS in vitro. The imprinting for patterns of hypothalamic MAO-A and MAO-B in the two sexes results, probably, from genetic predetermination. Neonatal changes in the homeostasis of gonadal hormones may result in type-MAO nonspecific effects in adulthood, whereas the short-term effects of high concentrations of steroids may be selective for the A form.

Expression of the human T-cell receptor (TCR) γ chain has been detected on subpopulations of peripheral T cells (1–5), a thymus-derived clone (6), and the T-cell line PEER (7). At least three forms of γ chains are known to exist in γδ TCRs (Table 1): [1] disulfide-linked Cγ1 chains have been detected on T-cell clones derived from the periphery (2–5,8); [2] a 55 kD Cγ2 chain, nondisulfide linked to a 40 kD δ chain is expressed on a cell line derived from an immunodeficiency patient (1) and on the PEER cell line (7); and [3] a second nondisulfide-linked ~ 41 kD Cγ2 chain has been found on a peripheral blood-derived cell line (8) and on a thymic clone (6). This latter receptor is poorly characterized and, furthermore, it is unknown if all three types of γ chains can be expressed by a single individual. Here we report that the 41 kD nondisulfide-linked γ chain can be separated into two components that are indistinguishable in charge, but differ slightly in size. This type of γ chain associates with a 36 kD putative δ chain. We show that all three forms of γδ TCRs can be expressed on CD3+WT31− lymphocytes from one individual. However, the 55 kD γ chain was not expressed by all individuals, which most likely reflects the absence of one of the Cγ2 alleles (9,10).

T cell clones (n = 456) were derived from 9 patients following allogeneic bone marrow transplantation (BMT) with or without acute graft versus host disease (aGVHD) and from 4 healthy donors. The cloning efficiency was 63.2% in controls, 13.2% and 12.1% in patients with or without aGVHD. Once established, T cell clones were typed for surface markers (CD3, CD4, CD8) and tested for production of IL-2 and expression of cytolytic activities in a lectin-dependent cellular cytotoxicity assay (LDCC) and against the K562 target cell line to detect natural killer activity. We found the expected imbalance of CD4/CD8 clones in BMT patients, as compared to controls. A higher proportion of IL-2-producing clones was observed in patients with aGVHD (83.5%; P less than 0.02) as compared to patients without aGVHD (64.8%) and controls (68.5%). No major differences were found in terms of LDCC, whereas an increased percentage of clones with NK-like activity was found in patients with aGVHD (34.7%, P less than 0.05) as compared to patients without aGVHD (29.5%) and controls (21.3%). The clones were also tested for inhibition of IL-2 production mediated by cyclosporine. Such inhibition could be obtained in virtually all clones both in patients with or without aGVHD, suggesting that the latter is probably not due to the emergence of CsA-resistant clones. In conclusion, this study demonstrates a low cloning efficiency in BMT patients associated with the well-known CD4/CD8 imbalance. A higher production of IL-2, an increased NK activity, but not the presence of CsA-resistant clones appear to differentiate patients with from patients without aGVHD.

Human CD3- lymphocyte populations were obtained by treating peripheral blood lymphocytes with mAbs directed to CD3, CD4, and CD8 surface antigens. The resulting populations were cultured with irradiated allogeneic cells; at day 4, 100 U/ml IL-2 were added and cultures continued for an additional 10 d. The resulting populations were CD3-CD2+CD7+ and displayed cytolytic activity against PHA-induced blast cells bearing the stimulating alloantigens but not against autologous or unrelated allogeneic blast cells. When CD3- populations were cultured with irradiated autologous cells, no cytolytic activity could be detected either against autologous or allogeneic blast cells. On the other hand, K562 target cells were lysed by both MLC-derived CD3- cell populations regardless of the origin (autologous or allogeneic) of the stimulating cells. CD3- clones were further derived from MLC-stimulated CD3- populations. These clones displayed a cytolytic pattern similar to the original MLC populations as only specific PHA blasts could be lysed. These clones did not express detectable surface TCR-alpha/beta or -gamma/delta molecules and lacked productive mRNA for TCR alpha and beta chains, while small amounts of TCR-gamma mRNA were detectable in one of four clones tested. Also mRNA for CD3 gamma and delta chains were undetectable in all clones, however, CD3 epsilon mRNA was consistently present.

Human Natural Killer Receptors and Their Ligands (Roberto Biassoni and Cristina Bottino, Istituto Nazionale per la Ricerca sul Cancro, Genova, Italy; Claudia Cantoni, Universita degli Studi di Genova, Istituto Giannina Gaslini, Genova, Italy; Alessandro Moretta, Universita degli Studi di Genova, Genova, Italy). Natural killer (NK) cells are a lymphocyte subpopulation that are important effectors of innate immune responses against infectious pathogens. They are thought to play an important role in host defense, not only against virally infected cells, but also in killing of tumor cells. Recent progress indicates that NK cells express an array of receptors, some of them clonally distributed, able to modulate the natural cytotoxicity. Three NK-specific activating receptors have been characterized; they belong to a novel receptor family called natural cytotoxicity receptors (NCR) and are represented by NKp46, NKp44, and NKp30. These receptors, upon engagement by their specific ligands, induce a strong activation of NK-mediated cytotoxic activity. This overview discusses the receptors (both activating and inhibitory) expressed by NK cells and their ligands. Finally, the dysfunction of one of these molecules occurring in a genetically inherited immunodeficiency is discussed.