A monoclonal antibody specific for a common determinant of the human T cell receptor gamma/delta directly activates CD3+WT31- lymphocytes to express their functional program(s).

In an attempt to select mAbs specific for human TCR-gamma/delta, a polyclonal CD3+ 4-8-WT31- (TCR-gamma/delta+) cell line (MV1) was used for mice immunization. An mAb, termed BB3, reacted with MV1 cells but not with a large panel of CD3+ WT31+ (TCR-alpha/beta+) cell populations or clones. In addition, BB3 mAb reacted with the majority of CD3+ WT31- clones derived from six different donors. Double-color fluorescence experiments and FACS analysis showed that BB3+ cells were restricted to the CD3+ fraction of peripheral blood lymphocytes; in addition, in several donors the percentages (0.5-8% of total PBL) of BB3+ cells paralleled those of CD3+ WT31- cells. Surface molecules recognized by BB3 were susceptible to antibody-induced modulation; in addition, cell treatment with either BB3 or anti-CD3 mAb caused the simultaneous downregulation of the two molecules. That BB3 molecules are physically linked to CD3 antigen was further supported by immunoprecipitation experiments. Thus, under conditions that preserve the TCR-CD3 association, both BB3 and anti-CD3 mAb precipitated from 125I-labeled MV1 cells the same set of molecules. These consisted in the 18-28-kD CD3 molecules and in three bands of approximately 44, 42, and 38 kD under reducing conditions. When cell lysis was performed in 1% NP-40, the molecules immunoprecipitated by BB3 mAb were represented by an 80-kD band under nonreducing conditions, which resolved, under reducing conditions, in the three 44-, 42-, and 38-kD bands. Similar disulphide-linked forms of the TCR molecules were revealed in all of the other eight CD3+ WT31- BB3+ clones analyzed. Analysis of TCR molecules by electrophoresis (NEPHGE) showed that BB3 or anti-CD3 precipitated a 44-kD molecule displaying a basic PI (approximately 7.5) and two more acidic proteins (PI approximately 6) with a mol mass of 42 and 38 kD. Studies aimed to define whether stimuli directly acting on TCR-gamma/delta could induce CD3+ WT31- cell activation revealed that (a) In the presence of PMA, soluble BB3 mAb induced IL-2 production by MV1 cell line and by three other CD3+ WT31- BB3+ clones analyzed. (b) BB3 mAb-producing hybridoma used as triggering target, was efficiently lysed by CD3+ WT31- BB3+ effector cells (but not by CD3+ WT31+ BB3- conventional CTL). (c) Soluble BB3 mAb induced CD3+ WT31- BB3+ effector cells to lyse the Fc receptor-positive P815 target cells. (d) BB3-TCR-gamma/delta interaction on CD3+ WT31- BB3+ cells induced a rapid increase of [Ca2+]i levels, similar to that observed in response to anti-CD3 mAbs.

Interferon-gamma and IL-10 may protect from allergic polysensitization in children: preliminary evidence.

BACKGROUND: A functional defect of T regulatory cells (Treg) has been proposed as pathogenic mechanism of allergic reaction. Polysensitization is a common feature of allergic patients. AIM OF THE STUDY: It was to investigate the possible role of Treg-Th1 cytokines, in the development of new sensitizations in childhood. METHODS: Forty monosensitized (MS) children with allergic rhinitis were evaluated and followed-up for 2 years. New sensitizations were investigated. IL-10 and IFN-gamma were evaluated in in vitro experiments. RESULTS: Children remaining MS showed significant higher production of both IL-10 and IFN-gamma. CONCLUSION: This preliminary study provided evidence that IL-10 and IFN-gamma production could be defective in allergic children prone to develop polysensitization.

Normal levels of soluble CD4 in sera from patients with juvenile chronic arthritis.

Sera from a group of patients with juvenile chronic arthritis (JCA) were tested for soluble CD4 (sCD4). In most cases normal levels of the molecule were detected independent of disease activity. Similar results were obtained when sera from a population of adult rheumatoid arthritis (RA) patients were analyzed. Immunophenotypic studies of circulating mononuclear cells from seven JCA patients with active disease showed that T cells did not express activation markers. Finally, preliminary experiments showed that sCD4 levels were high in the synovial fluids from 3 RA patients as compared with paired serum determinations.

Levels of soluble CD27 in sera and synovial fluid and its expression on memory T cells in patients with juvenile idiopathic arthritides.

OBJECTIVE: CD27 is a member of tumour necrosis factor receptor family. Its expression is predominantly confined to mature lymphocytes and is strongly enhanced after cell activation. Shedding of the CD27 from the surface of activated cells is related to their effector phase. The aim of the present study was to evaluate the levels of soluble CD27 in sera and synovial fluids, together with its expression on circulating and synovial fluid (SF) memory T cells, in children with JIA. METHODS: Sera from 40 patients with active JIA were studied for soluble CD27. Paired SF samples were available in 20 patients. Sera from 12 age-matched patients affected with various acute infectious diseases and 12 age-matched healthy subjects were used as controls. In 8 JIA patients freshly isolated circulating and SF lymphocytes were stained for CD27 in CD4+CD45 RO+ T cell subpopulation and analyzed by cytometry. RESULTS: Soluble CD27 serum levels were significantly higher in patients with polyarticular JIA and acute systemic infectious diseases than in patients with active oligoarticular or healthy controls. Both polyarticular and oligoarticular JIA patients showed increased levels of soluble CD27 in SF when compared with paired serum samples (p = 0.01). In all the patients tested a significant enrichment of CD27- T cells was seen in the SF (median 39.5%, range 18-56%) when compared to paired CD4+CD45RO+ peripheral lymphocytes (median 19.5%, range 5-43%; p = 0.01). CONCLUSIONS: A clear enrichment of CD4+ memory SF T cells with a CD27-phenotype is observed when compared to correspondent circulating T lymphocytes. This issue is conceivably related to re-activation and recruitment of memory T cells to the site of inflammation, and to the subsequent expansion of a subpopulation of "effector" memory T cells.

Targeting of "T" lymphocytes against human hepatoma cells by a bispecific monoclonal antibody: role of different lymphocyte subsets.

In an attempt to construct bispecific monoclonal antibodies (bimAbs) able to target cytotoxic T lymphocytes against human hepatoma cells, an HGPRT-deficient mutant of the Hepama-6 hybridoma, which produces an antihuman-hepatoma mAb, was directly fused with splenocytes from Balb/C mice immunized by a polyclonal cytotoxic T-cell line. Hybrid hybridomas were selected in HAT medium, and their supernatants were directly screened for the ability to induce IL-2-cultured cytotoxic T lymphocytes to kill hepatoma cells in a 51Cr-release assay. The selected hybrid hybridoma, termed DQ-33, secretes a bimAb, which reacts with a CD3-associated determinant. When resting peripheral-blood lymphocytes were used as effector cells, virtually no cytolytic activity could be induced by DQ-33, whereas phytohemagglutinin-activated lymphocytes that had been expanded in vitro in IL-2-containing medium could be efficiently targeted against hepatoma cells. Targeting by DQ-33 bimAb was analyzed on different subsets of IL-2-cultured lymphocytes. It was evident that CD+4-8+ TCR alpha/beta+ and CD3+4-8-TCR gamma/delta+ lymphocytes were efficiently induced by bimAb to lyse human hepatoma cells, whereas no induction of cytolysis could be observed when CD3 + 4 + 8-TCR alpha/beta+ cells were used as effectors. DQ-33 bimAb was also able to induce lymphokine secretion (IL-2, GM-CSF and TNF-alpha) by all the different subsets of lymphocytes analyzed in the presence of target cells expressing the relevant antigen, independent of the expression of cytolytic activity.

Outgrowth of interleukin-2-dependent T cell lines with cytotoxic activity against mesothelial cells from cultures of peritoneal effluent cells of children on CPD.

Previous studies on the peritoneal immune system described the presence of activated T lymphocytes in peritoneal effluents (PE) from patients on chronic peritoneal dialysis (CPD), and showed that mesothelial cells (MC) can present antigens to T cells. In order to better define phenotypic and functional characteristics of T cells and their interactions with MC, we isolated PE cells from 15 children. At the immunophenotypic analysis, high percentages of activated T cells were identified (mean value: 15% double staining for CD3/DR; 12% CD25+). T cells with gamma/delta T cell receptor (mean 5%) and natural killer cells (mean 17%) were also present in elevated numbers. MC lines (n = 7) and interleukin-2-dependent T cell lines (9 CD4+; 1 CD8+) were also obtained by incubating PE cells under different conditions. Two cell lines showed a major histocompatibility complex (MHC) restricted cytotoxic activity against autologous MC; two lines killed allogeneic MC; one line killed both autologous and allogeneic MC. Although the hypothesis that activated T cells could kill MC after recognition of surface structures modified by dialysis fluid, or during antigen presentation, needs to be further investigated, our data suggest that the subsets of lymphocytes we identified could play an important role in the mechanisms of peritoneal membrane defense.

Peritoneal mesothelial cells produce complement factors and express CD59 that inhibits C5b-9-mediated cell lysis.

The CD59 membrane protein confers protection from C5b-9-mediated cell lysis. Because evidence exists for complement (C) activation and generation of C5b-9 in the peritoneal cavity during chronic peritoneal dialysis (CPD), we investigated, on mesothelial cell (MC) lines, the expression of CD59 and the production of C components. Four MC lines were obtained from children on CPD, and two from non uremic children. CD59 expression on MCs was investigated with anti-CD59 monoclonal antibody (mAb) and polyclonal goat immunoglobulin G (IgG). MC lines were positive for staining with anti-CD59 mAb. Western blotting analysis of MC membrane demonstrated a band with the same molecular weight as CD59. Incubation of MC with anti-CD59 mAb abrogated the protective effect of CD59 (100% cytotoxicity). C3, C4, and C6 were detected in the supernatants of MC; in non uremic MC supernatants, C5, C7, C8, and C9 were also detectable, and C4 concentration was tenfold higher. CD59 expression confers to MCs protection from C5b-9-mediated lysis. MCs produce C factors. These findings suggest that production of complement components and expression of CD59 on MCs could play a role both in peritoneal cavity infection (decreased complement production) and in peritoneal membrane damage (decreased CD59 expression and reduced remesothelialization owing to MC lysis).

Induction of functional activities of human lymphocytes by monoclonal antibodies.

Monoclonal antibodies (MoAbs) specific for surface molecules involved in lymphocyte activation, represent an useful tool for the analysis of the activation pathways of different effector lymphocytes. We have analyzed the ability of MoAbs directed against "triggering" surface molecules, such as CD3/TCR, CD2 and CD16 to induce activation of the lytic machinery in T and NK lymphocytes, using a redirected killing assay. In addition, we have constructed bispecific monoclonal antibodies (BiMoAbs) directed against both the CD3/TCR complex (or the CD16 molecule) and a tumor associated antigen expressed on the surface of ovarian cancer cells. BiMoAbs were constructed by two different approaches: a) conjugation of two distinct MoAbs by a chemical heterobifunctional agent; b) selection of hybrid-hybridomas secreting BiMoAbs. BiMoAbs were able to focus appropriate lymphoid effector cells on tumor cells expressing the relevant antigen, and to induce tumor cell lysis. Therefore, these reagents were able to confer a new specificity for a given antigen to effector lymphocytes, independently from the original one. For these properties, BiMoAbs may represent a suitable tool for new strategies of adoptive immunotherapy, based on the use of effector cells that have been specifically armed by BiMoAbs against the tumor.

[Rational bases for new approaches to the therapy of pediatric solid tumors: immunotherapy and gene therapy]. / Le basi razionali per nuovi approcci alla terapia dei tumori solidi pediatrici: immunoterapia e terapia genica.

Neuroblastoma is one of the commonest solid tumors in children. Conventional therapeutic approaches, such as surgery, chemotherapy and radiotherapy, fail to control tumor progression in stage III and IV patients. The search for novel therapeutic strategies should necessarily take into account immunotherapy and gene therapy. Here the theoretical bases for the development of such approaches are discussed. Studies carried out with neuroblastoma (NB) cell lines have shown that neoplastic cells express a wide array of potential tumor associated antigens (TAA) but are devoid of HLA molecules which are necessary for TAA presentation to the host immune system. Transfection of NB cells with the interferon gamma gene appears a promising approach, since this cytokine up-regulates the expression of class I HLA molecules in NB cells. Other cytokines of potential interest for gene transfer studies are interleukin 2 (IL2) and interleukin 12 (IL12).

Cytolytic activity of T lymphocytes isolated from ovarian carcinoma ascitic fluid. Analysis at the population and clonal level.

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.

Clonal analysis of peripheral blood lymphocytes from three patients with advanced neuroblastoma receiving recombinant interleukin-2 and interferon alpha.

In this study we have investigated, at the population and the clonal levels, the immunophenotypes and the non-specific cytotoxic functions of peripheral blood lymphocytes from three stage IV neuroblastoma patients receiving treatment with recombinant interleukin-2 (IL-2) and interferon alpha (IFN alpha). Both IL-2 alone and the combination of IL-2 and IFN alpha caused an in vivo expansion of CD56+, CD3- NK cells most of which expressed the p75 molecule, i.e. the beta chain of the IL-2 receptor. Peripheral blood mononuclear cells (PBMC), drawn after treatment, displayed an increased NK activity, but no lymphokine-activated killer (LAK) activity. However, the subsequent in vitro culture of PBMC with high-dose IL-2 induced the generation of a potent LAK activity, which was mediated by an expanded population of CD3+, CD8+ T cells. Finally lymphocytes that had been isolated after cytokine therapy were cloned, in the presence of low-dose phytohemagglutin, immediately or following culture with IL-2. Clones derived from LAK cells expanded in vitro had predominantly a CD3+, CD8+ immunophenotype, whereas those raised from freshly separated lymphocytes were either CD3+, CD4+ or CD3+, CD8+ in equal proportions. Most of the above clones were poorly or not at all cytolytic against NK-sensitive or NK-resistant targets. In contrast, the few NK clones obtained (CD3-, CD56+) lysed all targets with high efficiency.

Toxoplasma gondii-specific CD4+ T cell clones from healthy, latently infected humans display a Th0 profile of cytokine secretion.

Human Toxoplasma gondii (Tg)-specific T cell clones were raised by infecting peripheral blood mononuclear cells (MNC) from two healthy, latently infected individuals with Tg trophozoites. All of the clones had a CD4+ immunophenotype and produced simultaneously interleukin (IL)-2, interferon (IFN)-gamma, IL-4 and IL-5 upon mitogen or antigen stimulation. Tg-specific T cell clones were classified as T helper of type 0 (Th0) since most of them released roughly comparable amounts of IFN-gamma and IL-4. In some clones, a trend to an increased production of IFN-gamma following antigen-specific as compared to non-specific stimulation was observed. The Th0 phenotype was also expressed by T cell clones that had been raised from bulk cultures performed in the presence of IL-4 or IFN-gamma. All of the Tg-specific T cell clones were cytolytic in a non-specific assay which involves the triggering of the CD3-T cell receptor (TcR) complex. Some clones specifically lysed an autologous lymphoblastoid cell line (LCL) that had been infected with Tg trophozoites. Finally, most of the Tg-specific T cell clones produced IL-10, irrespective of whether they had been raised from bulk cultures incubated in the presence or absence of IL-4 or IFN-gamma. Taken together, these findings suggest that Tg-specific Th0 helper cell clones from healthy, latently infected individuals, beside activating toxoplasmacidal mechanisms through IFN-gamma release, might limit the magnitude of the immune response of the parasite by killing Tg-infected antigen-presenting cells and by releasing IL-10.

Graves' disease: phenotypic and functional analysis at the clonal level of the T-cell repertoire in peripheral blood and in thyroid.

We have investigated at the clonal level the repertoire of intrathyroid and peripheral T lymphocytes in three patients with Graves' disease using a high efficiency cloning technique. Clonal efficiencies ranged from 10 to 31% for intrathyroid, and from 19 to 100% for peripheral T cells. In Graves' disease the phenotypic analysis showed similar percentages of CD3+ CD4+ CD8- and CD3+ CD4- CD8+ clones in thyroid infiltrates and peripheral blood. The functional evaluation showed similar or lower proportions of cytolytic clones in thyroid infiltrates with respect to peripheral blood. Furthermore, the proportions of intrathyroid and peripheral T-cell clones capable of releasing interleukin-2 and/or gamma-interferon in response to mitogen stimulation were similar. Finally, 44% of intrathyroid clones were neither cytolytic nor able to release IL-2 and gamma-interferon. These results are strikingly different from those obtained in Hashimoto's thyroiditis, where the large majority of intrathyroid T-cell clones are cytolytic and the proportions of clones able to release gamma-IFN are remarkably increased in thyroid infiltrates when compared to those obtained from peripheral blood. Taken together, these data suggest a different role for T lymphocytes in the pathogenesis of the two major human autoimmune thyroid diseases.

CD3-negative lymphokine-activated cytotoxic cells express the CD3 epsilon gene.

The expression of genes encoding different polypeptide chains of the TCR-CD3 complex was analyzed in a panel of cloned MHC-unrestricted cytotoxic cells. The clones were derived from CD3+ and CD3- human PBL. After expansion in rIL-2, all clones were able to lyse the NK-sensitive target cell line K562. In contrast, lysis of fresh tumor cells was achieved almost exclusively by CD3- clones. To test whether a known TCR-CD3 complex may be involved in MHC-unrestricted cytotoxicity, total RNA from nine CD3+ and 11 CD3- clones was isolated and hybridized with DNA probes for the TCR alpha-, beta-, and gamma-chains and for the CD3 gamma-, delta-, and epsilon-chains. TCR gamma transcripts were present at high levels in CD3+CD4- CD8- clones but were undetectable in all CD3- clones. Lysis of fresh tumor cells is an activity which can be independent of the TCR alpha beta and TCR gamma complexes because the CD3- clones did not express these TCR genes. Interestingly, all CD3- clones expressed CD3 epsilon transcripts, but not CD3 gamma- or delta-transcripts. CD3- lymphokine-activated cytotoxic cells may therefore be derived from immature T cells which do not yet express a complete CD3 complex. The CD3 epsilon chain, if expressed in CD3- cells in association with other molecules, could be involved in the activation and lytic function of these MHC-unrestricted cytotoxic cells.

Functional and molecular characterization of tumour-infiltrating lymphocytes and clones thereof from a major-histocompatibility-complex-negative human tumour: neuroblastoma.

Neuroblastoma (NB) is a major-histocompatibility-complex(MHC)-negative neuroectodermal tumour that is often infiltrated with lymphocytes. A detailed characterization of NB-associated tumour-infiltrating lymphocytes (TIL) has never been carried out. Here we have investigated the immunophenotype and the cytotoxic activities of TIL from nine and seven NB patients respectively. Furthermore, the T cell receptor (TcR) variability and the patterns of cytokine gene expression of fresh versus recombinant (r) interleukin (IL)-2-cultured TIL were studied in four NB cases. The results obtained showed the following: (1) freshly isolated TIL were comprised of a mixture of CD4+ and CD8+ T cells partially expressing HLA-DR and/or CD25. The CD4/CD8 ratio ranged from 0.5 to 5 in the different cases. Upon culture of TIL with rIL-2, an increased proportion of CD56+ and CD8+ lymphocytes was consistently observed; (2) IL-2-expanded TIL lysed natural-killer(NK)sensitive and lymphokine-activated-killer(LAK)-sensitive target cell lines; (3) reverse-transcriptase/polymerase-chain-reaction (RT-PCR) experiments showed that most TcR V beta genes were expressed both in fresh and in cultured TIL, suggesting that such cell populations were polyclonal; (4) interferon gamma, IL-4, IL-5, tumour necrosis factor (TNF) alpha, IL-8, IL-10 mRNA and, to a lesser extent, IL-2 mRNA were expressed by cultured TIL, as assessed by RT-PCR; the corresponding tumour samples consistently contained TNF alpha, IL-8 and IL-10 mRNA, whereas IL-2 and IFN gamma mRNA were faintly expressed in some NB tumours and IL-4 and IL-5 mRNA were never detected. A total of 90 clones were subsequently raised from IL-2-expanded TIL from six NB patients; 87/90 clones were of T cell lineage with a CD4+ or CD8+ immunophenotype, whereas the 3 remaining clones were of NK cell origin. Upon triggering of the CD3-TcR complex, 64% CD4+ and 77% CD8+ T cell clones killed the murine P815 mastocytoma cell line. Virtually no T cell clone lysed a LAK-sensitive NB cell line whereas 15% CD4+ and 17% CD8+ clones mediated NK-like activity against the K562 cell line. Finally, the patterns of cytokine production by CD4+ clones were roughly consistent with those of a T helper (TH) 1 profile and similar to those observed in CD8+ clones.

A novel surface molecule expressed by long-term cultured T and natural killer cells is involved in cell activation.

Two monoclonal antibodies (mAb), termed ED6 and LD6, were obtained by immunizing mice with cytotoxic T cell lines expressing the T cell receptor (TcR) gamma/delta. These mAb were selected according to their ability to trigger the cytolytic program of the immunizing cell lines in a redirected killing assay. Both mAb recognized molecule(s) expressed on the surface of most long-term cultured TcR gamma/delta +, TcR alpha/beta + and CD3-CD16+ lymphocytes, while it was absent on resting peripheral blood lymphocytes. In addition both mAb reacted with neoplastic B cell lines, Epstein-Barr virus-transformed B cell lines, small cell lung cancer and glioma cell lines, while no surface reactivity was detected on ovarian, breast, colon and non-small cell lung cancer lines. The functional activity of these mAb was studied by two cytolytic assays. Both mAb were able to trigger the cytolytic program of CD3+TcR gamma/delta + polyclonal cell lines and of a CD3-CD16+ NK cell clone against the murine mastocytoma target cell line P815 (Fc receptor+) in a 4-h 51Cr-release assay. In addition, ED6 and LD6 hybridomas were lysed by TcR gamma/delta + effector cells while other hybridomas (obtained from the same fusion) were not lysed. ED6 and LD6 mAb (in the presence of submitogenic doses of the phorbol 12-myristate 13-acetate) also induced the secretion of interleukin 2 by ED6/LD6+ T cell clones expressing TcR gamma/delta or alpha/beta. mAb-induced surface antigen modulation experiments showed that the antigenic determinant recognized by ED6 and LD6 co-modulated, thus indicating that the two mAb probably recognize the same or closely associated molecules. The molecular characteristics of the antigen recognized by the mAb were investigated by Western blot analysis. The LD6 mAb recognized a major band of approximately 65 kDa, both under nonreducing and reducing conditions. These data indicate that ED6 and LD6 mAb recognize a novel non-lineage-specific activation antigen which is involved in the induction of the functional program of long-term cultured T or natural killer cells.

Re-targeting of human lymphocytes expressing the T-cell receptor gamma/delta to ovarian carcinoma cells by the use of bispecific monoclonal antibodies.

TcR gamma/delta+ lymphocytes represent a small subset homogeneously composed of cytolytic T cells displaying unique motility and homing properties. Since the lytic machinery of these cells can be efficiently triggered by monoclonal antibodies (MAbs) directed to the TcR gamma/delta, such MAbs were used for the construction of bispecific MAbs in conjunction with an MAb specific for ovarian carcinoma cells. Hybrid hybridomas were obtained by fusing the Mov19 MAb (IgG2a)-producing hybridoma with either GI (IgG2a) or A13 (IgG1) hybridomas, secreting MAbs specific for 2 peripheral blood subsets of TcR gamma/delta+ lymphocytes. Hybrid hybridomas producing bispecific MAbs were screened according to their ability to induce ovarian carcinoma (IGROVI) target cell lysis by GI+ or A13+ T cell clones, respectively. The GI-derived GM33.9 bispecific MAb induced selective lysis of Mov19+ ovarian carcinoma target cells only by GI+ clones, whereas the A13-derived AM18.4 MAb was effective only in combination with A13+ clones. Neither the anti-TcR gamma/delta nor the Mov19 parental MAbs (used alone or in combination) induced target-cell lysis. The hybrid nature (IgG1/IgG2a) of the AM18.4 bispecific MAb was indicated by 2-color immunofluorescence experiments. Thus, both ovarian carcinoma and A13+ effector cells were double stained by AM18.4 bispecific MAb followed by PE-conjugated anti-IgG1 and FITC-conjugated anti-IgG2a second reagents. Polyclonal TcR gamma/delta+ cells were obtained by direct stimulation of peripheral blood mononuclear cells with Sepharose bead-conjugated anti-TcR gamma/delta MAbs and IL-2. The lines so obtained contained more than 90% of TcR gamma/delta+ cells after 4 weeks of culture, with an increase in TcR gamma/delta+ cell numbers ranging from 200 to 1,000-fold. These TcR gamma/delta+ cell lines efficiently lysed ovarian carcinoma target cells in the presence of bispecific MAb and may therefore represent a suitable source of effector cells for induction of ovarian carcinoma cell lysis.

Clonal analysis of T lymphocytes infiltrating the thyroid gland in Hashimoto's thyroiditis.

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 efficiencies 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.

Phenotypic and functional analysis of human CD3+ and CD3- clones with "lymphokine-activated killer" (LAK) activity. Frequent occurrence of CD3+ LAK clones which produce interleukin-2.

Clones capable of lysing fresh, uncultured tumor cells ("lymphokine-activated killer": "LAK" activity) were selected from microcultures derived from either E-rosette-positive or E-rosette-negative cell populations. All the selected clones displayed a strong cytolytic activity against the NK-sensitive K562 cell line. Two major phenotypic groups of clones could be identified: a first group expressed the CD3 differentiation antigen, present exclusively on mature T lymphocytes; however, in contrast to typical cytolytic T lymphocytes, the majority of these clones expressed the unusual CD4- CD8- phenotype, whereas the remainder were CD4- CD8+. A second group was represented by CD3- clones which, in most instances, expressed the T-cell-lineage-specific CD2 antigen. Following stimulation with phytohemagglutinin (PHA), most of the CD3+ LAK clones produced Interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) whereas those expressing the CD3- phenotype did not. Since previous studies indicated that PHA may be inefficient in inducing lymphokine production by T-cell variants lacking the CD3/T cell receptor complex (TCR), CD3- clones were further stimulated with the calcium ionophore A23187 plus phorbol 12-myristate 13-acetate (PMA). Only 2/11 CD3- LAK clones produced small amounts of IL-2, whereas the majority released IFN-gamma. Given the peculiar phenotypic and functional properties of many CD3 + LAK clones, we suggest that they may belong to a T-cell subset distinct from typical CTLs.