Activity of host-derived T cells which differentiate in nude mice grafted with co-isogenic or allogeneic thymuses.

If nude mice are grafted with a neonatal thymus, host type precursor cells develop within the graft thymus and after about 6 wk the T-cell population of the thymus, spleen, and lymph nodes is of host type. However, immunological responsiveness produced in nude mice in this manner is incomplete: (a) the ability to react to T-cell mitogens in vitro is greater than in untreated nudes but lower than in normal mice; (b) the response to T-cell dependent antigens is less than normal; and (c) the rejection of skin grafts is slower than in normal animals. Whether host precursor cells which differentiate in an allogeneic thymus are able to reject skin grafts from thymus donor strain appears to depend on the strain combination used.

Differentiation of T-cell precursors in nude mice demonstrated by immunofluorescence of T-cell membrane markers.

When the thymus from an AKR mouse (TL(-), theta-AKR) is grafted to a BALB/c-nu/nu mouse (TL2, theta-C3H), the grafted thymus is rapidly repopulated by host lymphocytes, i.e., lymphocytes having the TL2 and theta-C3H T-lymphocyte membrane antigen markers. theta-C3H lymphocytes also appear rapidly in the spleen and lymph nodes. After a few weeks, BALB/c nude mice grafted with AKR thymus and normal BALB/c mice could not be distinguished on the basis of the number of TL-positive thymocytes or theta-C3H-positive lymphocytes in thymus, spleen, or lymph nodes. These experiments give a definitive proof of the existence of precursor cells for the T compartment of the lymphoid system in the nude mouse. They strongly suggest the involvement of host-derived T cells in the recovery of some T-cell functions by nude mice grafted with allogeneic thymuses.

Switch in immunoglobulin class production observed in single clones of committed lymphocytes.

Mouse spleen cells, after stimulation with lipopolysaccharide, were cloned in culture. After 4 to 5 days, the daughter cells were stained and examined for immunoglobulin class with double immunofluorescent reagents. A switch of the stained color of these cells was observed, implying a switch from imunoglobulin M to immunoglobulin G production in the progeny of a single B cell.

Restoration of taxol sensitivity of multidrug-resistant cells by the cyclosporine SDZ PSC 833 and the cyclopeptolide SDZ 280-446.

BACKGROUND: Taxol, a promising agent for the treatment of cancer, has entered phase II clinical trials. Nevertheless, it belongs to the class of compounds that show impaired retention in multidrug-resistant cells expressing P-glycoprotein (Pgp), a drug efflux pump. Chemosensitizers like verapamil modulate multidrug resistance by interfering with the efflux action of Pgp and thus can decrease drug resistance or can restore drug sensitivity by restoring normal drug accumulation and distribution within the multidrug-resistant tumor cell. The two strongest, nearly equipotent chemosensitizers identified to date are the cyclosporine derivative SDZ PSC 833 and the semisynthetic cyclopeptolide SDZ 280-446. PURPOSE: This study was designed to investigate the capacities of verapamil, SDZ PSC 833, and SDZ 280-446 to decrease resistance of two multidrug-resistant cell lines to taxol. METHODS: We studied in vitro the growth of two multidrug-resistant tumor cell lines displaying high resistance to taxol: multidrug-resistant Chinese hamster ovary cells and murine monocytic leukemia P388 cells. We determined the taxol concentration that produced 50% inhibition of cell growth (IC50) in the two multidrug-resistant cell lines and in the parent cell lines, in the presence of a range of chemosensitizer concentrations (0-30 microM). IC50 values were determined in the presence and in the absence of verapamil, SDZ PSC 833, or SDZ 280-446. RESULTS: At nontoxic concentrations (0.3-1 microM), SDZ PSC 833 and SDZ 280-446 produced an almost complete reversal of the high taxol resistance of the multidrug-resistant tumor cells, whereas only partial restoration of sensitivity to taxol was achieved with verapamil. CONCLUSION: SDZ PSC 833 and SDZ 280-446 can restore the normal taxol sensitivity of highly resistant multidrug-resistant tumor cells. IMPLICATIONS: The combination of taxol with SDZ PSC 833 or SDZ 280-446 may be recommended for treatment of multidrug-resistant cancers.

In vivo circumvention of P-glycoprotein-mediated multidrug resistance of tumor cells with SDZ PSC 833.

The new nonimmunosuppressive cyclosporin analogue, SDZ PSC 833, is a very potent multidrug-resistance modifier. In vitro, it was shown to be at least 10-fold more active than cyclosporin A (Sandimmune), itself more active than verapamil, on most P-glycoprotein-expressing multidrug-resistant (MDR) tumor cell lines. In vivo, SDZ PSC 833 was tested in a few protocols of combined therapy with either Vinca alkaloids or doxorubicin as anticancer drugs, using the homologous tumor-host system (P388 cells of DBA/2 origin grafted into DBA/2 or B6D2F1 mice). Although these MDR-P388 tumor cells belong to a highly resistant variant that in vitro required about 150-fold more anticancer drug for 50% cell growth inhibition than the parental P388 cells, significant prolongation of survival times of the MDR-P388 tumor-bearing mice was obtained when treated with a combination of SDZ PSC 833 p.o. were otherwise ineffective doses of anticancer drugs given i.p. This chemosensitizing effect of SDZ PSC 833 was dose-dependent and was most effective in a protocol combining administration of SDZ PSC 833 p.o. 4 h before a doxorubicin i.p. injection: in comparison with the survival of MDR-P388 tumor-bearing mice treated with the anticancer drug alone, the pretreatment with SDZ PSC 833 at 25 and 50 mg/kg gave 2- to 3-fold increases of survival times. Since the MDR-P388 tumor cells used in our studies belong to a highly resistant variant, with a much higher degree of drug resistance than the one known to occur in cancer patients, SDZ PSC 833 appears to be a very promising chemosensitizer.

Establishment and characterization of long-term cultured cell lines of murine resident macrophages.

Murine resident macrophages can proliferate in vitro when they are grown in coculture on a layer of mesothelial or endothelial type feeder cells. Resident macrophages were obtained from lung explants of C57Bl/6 lpr/lpr mice and from spleen explants or peritoneal washing of Balb/c mice; the cells were seeded without further washing. After 3-4 weeks of culture, the macrophages began to proliferate on a confluent layer of feeder cells. The macrophages then could be collected in the fluid phase and reseeded for permanent culture after generation of a new feeder layer. These cells were characterized as macrophages by the following criteria: 1) their morphology, ultrastructure, and adherence properties; 2) more than 90% of the macrophages phagocytized yeasts compared with less than 1% of the feeder cells; 3) the presence of functional Fc and mannose receptors, nonspecific cytoplasmic esterases, and membrane ectoenzymes such as nicotinamide adenine dinucleotide (NAD) glycohydrolase and nucleotide pyrophosphatase; 4) by cytofluorographic phenotype analysis with monoclonal antibodies, characterizing a normal macrophage population (MAC1+, Fcrec+, H-2K+, THY1-, LYT2-, L3T4-). 5) by functional studies proving that the expanded macrophages could function as accessory cells in the induction of lymphocyte proliferation in response to concanavalin A (Con A), that they generated reactive oxygen radicals and that they were cytotoxic for tumor cells. During coculture, growth or activating factors such as macrophage colony-stimulating factor or gamma-interferon were released in the medium. Long-term cultured macrophages had chromosomal abnormalities. Our study suggests that tissue macrophages can proliferate in vitro and hence that it is possible to establish long-term cultured cell lines of macrophages of defined and reproducible characteristics.

An enzyme-linked lectin-binding assay on cells (CELLBA) for the comparison of lectin receptor expression on cell surfaces.

Lectins can be used to specifically detect some cell surface glycans. Their expression on different cells or on cells of a given lineage throughout differentiation or following treatment with drugs can be compared using lectins labelled with radioactive, fluorescent or enzymatic probes. We describe a new method which, by analogy with CELISA (ELISA on cells), is called CELLBA (or ELLBA on cells) for cellular, enzyme-linked lectin-binding assay. It permits the comparison of the expression of specific glycans in a large number of different cell samples. As an example, it was able to detect alterations of cell surface glycan expression caused by inhibitors of N-linked oligosaccharide trimming.

A quick procedure for identifying doubly homozygous immunodeficient scid beige mice.

This article describes a rapid and reliable procedure for identifying mice which are doubly homozygous at the scid and beige (bg) loci starting from CB17 scid (no T and B cells) and B6 bg mice (no NK activity). The [scid, bg] mice are directly identified in the F2 progeny by monitoring (1) the hypogammaglobulinemia for the scid gene and (2) the prolonged bleeding associated with the bg gene. Like CB17 scid mice, the [scid, bg] mice show a high susceptibility to infections and die early in life unless they are protected against potential infections. This is achieved by a graft of splenocytes plus bone marrow cells from (B6 bg x CB17 scid) F1 mice. These [scid, bg] mice combine the bg and scid immunodeficiencies and should be better recipients for xenografts than classical scid mice.

A comparison of five different methods for the detection of TNP specific mouse IgE: ELISA, ELISA on cells, rosetting, granule enzyme release assay and passive cutaneous anaphylaxis.

Using the same anti-TNP hybridoma supernatant pool as IgE antibody source (2 micrograms/ml), several methods were compared for their sensitivity in IgE detection and convenience for screening purposes. Using rat basophilic leukemia (RBL) cells as specific receptor cells, one out of two rosetting methods with TNP-sheep red blood cells allowed the detection of 0.5 ng IgE/ml (50 pg/assay) while the other was much less sensitive (60 ng/ml). More convenient for screening was an ELISA method performed on cells, in which the IgE bound to the RBL cell surface could be detected either by enzyme-anti-Ig or by enzyme-antigen conjugates with a similar, albeit low, sensitivity of 10 ng IgE/ml (500 pg/assay). Using the same antibody and cell sources, a very convenient and more sensitive method for screening purposes was found to be the measurement of antigen-induced basophil granule beta-N-acetylglucosaminidase release by IgE sensitized RBL cells: 0.5 ng IgE/ml and 50 pg/assay. For the same anti-TNP IgE source, this compares with a detection limit by ELISA of 0.2 ng IgE/ml (10 pg/assay) and by passive cutaneous anaphylaxis in rats of 2 ng IgE/ml (100 pg/assay).

The MultiScreen filtration system to measure chemoattractant-induced release of leukocyte granule enzymes by differentiated HL-60 cells or normal human monocytes.

A variety of chemoattractants initiate chemotaxis by selective binding to chemoattractant receptors (CARs), a subfamily of seven transmembranous G-protein coupled receptors (7TM-GPCRs) expressed in the leukocyte plasma membrane. Whatever the chemoattractant, signaling leading to chemotaxis involves several common biological steps which occur within seconds to minutes of CAR ligand binding. Though each step can be used to study the progress of the chemotaxis activation process. only certain biological events are suitable for monitoring chemotaxis signaling on large sample numbers as required for drug screening. An example of such is the release of granule enzymes by leukocytes in response to a CAR ligand. In this study, promyelocytic HL-60 cells were employed to set up a 96-well microplate methodology using filtration instead of centrifugation to collect the extracellular fluid together with the cell-released enzymes. Undifferentiated HL-60 cells were found not to respond to any of the CAR ligands. With various types of HL-60 cells which had differentiated along the neutrophilic or monocytic pathways, a large enzyme release was dose-dependently triggered by fMLF or C5a, but none of the tested CC or CXC chemokines. The highest responsiveness was found for neutrophilic HL-60 cells differentiated with dibutyryl cyclic AMP. With normal human monocytes (prepared from the blood of healthy donors by leukapheresis and elutriation), the granule enzyme release response was large to fMLF or C5a, substantial to MCP-1, low to RANTES or MIP-1alpha, but insignificant to Eotaxin, IL-8 and GROalpha. The method readily measures N-acetyl-beta-D-glucosaminidase, beta-D-glucuronidase and elastase activities, and requires approximately five times fewer cells than classical methods, a very important feature when normal human cells are to be used in screening assays. The method was also adapted to large scale screening of antagonists such as cyclosporins A and H for fMLF-mediated signaling using HL-60 cells and monocytes, and truncated (9-76) MCP-1 for MCP-1-mediated signaling using monocytes.

An indirect asymmetrical sandwich ELISA using anti-allotype antibodies for the specific and quantitative measurement of mouse IgG2a of Igh-1b allotype.

We recently described an indirect double sandwich ELISA (Klein-Schneegans et al., J. Immunol. Methods (1989) 119, 117) which permits the specific and quantitative measurement of mouse IgM, IgA and IgG subclasses with one major exception: IgG2a of the b allotype (Igh-1b in mouse strains such as C57BL/6) could not be reliably quantitated even by a very specific and sensitive asymmetrical sandwich ELISA (using two different anti-IgG2a isotype antibodies for capture and for detection). We now describe a similar method based on the use of two different anti-IgG2a allotype antibodies for the capture and detection of IgG2a in the serum of Igh-1b mouse strains.

Indirect double sandwich ELISA for the specific and quantitative measurement of mouse IgM, IgA and IgG subclasses.

We have developed sensitive enzyme-linked immunosorbent assays (ELISA) which measure mouse serum heavy chain immunoglobulin isotypes in nanograms per milliliter. In each case the specific isotypic Ig is sandwiched between an isotype-specific antibody used for coating and another isotype-specific antibody coupled to biotin for detection (with alkaline phosphatase coupled to avidin). These methods are simple to perform, specific for each isotype, reproducible with an average coefficient of variation of 5% for IgG1, 3% for IgG2a, 7% for IgG2b, 10% for IgG3, 3% for IgA and 7% for IgM, and at least 100 times more sensitive than radial immunodiffusion. The assays have been used to determine the absolute concentrations of mouse serum heavy chain Ig isotypes.

Derivatives of a novel cyclopeptolide. 2. Synthesis, activity against multidrug resistance in CHO and KB cells in vitro, and structure-activity relationships.

A series of derivatives of the novel cyclopeptolide 1 was prepared, and their ability to chemosensitize multi drug resistant CHO and KB cells in vitro was evaluated. In contrast to the parent compound, several of the derivatives were found to be highly active. In particular, conversion of the R-lactic acid residue of 1 into its S-isomer via lactone ring cleavage and recyclization with inversion resulted in a marked enhancement of activity. Some of these derivatives (e.g., 15a, SDZ 280.446) belong to the most potent resistance modulating compounds known so far.

Aureobasidins: structure-activity relationships for the inhibition of the human MDR1 P-glycoprotein ABC-transporter.

Cyclic depsipeptide cyclo-[D-Hmp(1)-L-MeVal(2)-L-Phe(3)-L-MePhe(4)-L-Pro(5)-L-aIle+ ++(6)-L-MeVal(7)-L-Leu(8)-L-betaHOMeVal(9)], the antifungal antibiotic aureobasidin A (AbA), was reported to interfere with ATP-binding cassette (ABC) transporters in yeast and mammalian cells, particularly the MDR1 P-glycoprotein (Pgp), a transmembrane phospholipid flippase or "hydrophobic vacuum cleaner" that mediates multidrug resistance (MDR) of cancer cells. In a standardized assay that measures Pgp function by the Pgp-mediated efflux of the calcein-AM Pgp substrate and uses human lymphoblastoid MDR-CEM (VBL(100)) cells as highly resistant Pgp-expressing cells and the cyclic undecapeptide cyclosporin A (CsA) as a reference MDR-reversing agent (IC(50) of 3.4 microM), AbA was found to be a more active Pgp inhibitor (IC(50) of 2.3 microM). Out of seven natural analogues and 18 chemical derivatives of AbA, several were shown to display even more potent Pgp-inhibitory activity. The Pgp-inhibitory activity was increased about 2-fold by some minor modifications such as those found in the naturally occurring aureobasidins AbB ([D-Hiv(1)]-AbA), AbC ([Val(6)]-AbA), and AbD [gammaHOMeVal(9)]-AbA). The replacement of the [Phe(3)-MePhe(4)-Pro(5)] tripeptide by an 8-aminocaprylic acid or the N(7)()-desmethylation of MeVal(7) led to only a 3.3-fold decreased capacity to inhibit Pgp function, suggesting that the Pgp inhibitory potential of aureobasidins, though favored by the establishment of an antiparallel beta-sheet between the [D-Hmp(1)-L-MeVal(2)-L-Phe(3)] and [L-aIle(6)-L-MeVal(7)-L-Leu(8)-] tripeptides, does not critically depend on the occurrence of the [L-Phe(3)-L-MePhe(4)-L-Pro(5)-L-aIle(6)] type II' beta-turn secondary structure. In contrast, the most potent Pgp inhibitors were found among AbA analogues with [betaHO-MeVal(9)] residue alterations, with some data suggesting a negative impact of the [L-Leu(8)-L-betaHOMeVal(9)-D-Hmp(1)] gamma-turn secondary structure on Pgp inhibitory potential. The [2,3-dehydro-MeVal(9)]-AbA was the most potent Pgp inhibitory aureobasidin, being 13-fold more potent than AbA and 19-fold more potent (on a molar basis) than CsA. Finally, there was no correlation between the SAR for the human MDR1 Pgp inhibition and the SAR for Saccharomyces cerevisiae antifungal activity, which is mediated by an inositol phosphoceramide synthase activity.

Impaired antibody response of C57BL/6 beige mutant mice to a thymus-independent type 2 antigen.

The murine equivalent of the human Chediak-Higashi Syndrome is the beige (bg) mutant in the C57BL/6 (B6) background. Besides the well-known lack of natural killer (NK) activity in bg-homozygous mice, functional abnormalities of T cells, macrophages and various granulocytes have been reported. With the exception of one study indicating a decreased in vitro response to lipopolysaccharide, there is no report concerning the B cell compartment of the beige mutant. The in vivo anti-trinitrophenyl antibody response to a TI-2 antigen (TNP-Ficoll) was found here to be significantly lower in B6 beige than in B6 wild mice, although both strains responded similarly to an analogous TD antigen (TNP-ovalbumin). Since the marginal zone macrophages of the spleen were previously shown to be essential for the initiation of antibody responses to TI-2 antigens, they might be another target of the beige mutation.

Common and individual features of the humoral immunity dysregulation of mice homozygous at the viable motheaten (mev) mutation.

The "viable motheaten" (mev) mutation in the C57BL/6 (B6) mouse causes in mev-homozygous B6 mice a rapidly progressing and fatal autoimmune syndrome, combined with a generalized immunodeficiency. Serological analyses of a large number of mev/mev individual mice revealed profound abnormalities of the levels of various spontaneously occurring, natural antibodies and of the levels and distribution of the different classes of immunoglobulins, some common to all mev/mev mice and some specific to individual mice. Although very high serum IgM levels (70X the normal B6 mouse serum level) and high anti-ssDNA titers are common features, the increases in the other immunoglobulin isotypes are not so marked (4.7X for IgG3, 2.7X for IgA, 1.6X for IgG1 and IgG2b, and even 0.6X for IgG2a), and the occurrence of other natural antibody specificities shows more individual variability.

Adoptive transfer of the gld syndrome in double congenic nude lpr mice.

Homozygosity for either the lymphoproliferation (lpr) or the generalized lymphoproliferative disease (gld) mutation of mice causes the development of strikingly similar autoimmune and lymphoproliferative syndromes. The relationship between the lpr and gld mutations was studied by grafting B6 gld spleen cells (SC) to athymic B6 nude lpr mice (B6 nulpr) or to B6 nude (B6 nu) mice as controls. The injection of B6 gld SC, but not of B6 wild SC, to B6 nulpr mice caused a prolongation of survival of the short living B6 nulpr recipients. This was associated with elevated anti-single stranded DNA antibody titers and a serum hyperglobulinemia, as well as by a splenomegaly which was nearly as high as in genetically B6 gld mice, and by a marked lymphadenopathy (though milder than that of B6 gld mice). In contrast the [gld----nu] chimaeras showed a more attenuated form of gld-induced syndrome. These results suggest that the lpr environment supplied in athymic lpr recipients is compatible with--and may even favour--the development of the gld-induced syndrome.

Serological abnormalities induced by engraftment of viable motheaten hematopoietic cells in nude beige recipients.

C57BL/6J (B6) mice homozygous for the viable motheaten (mev) mutation are short-lived and display severe immunodeficiency, autoimmunity and inflammatory disease. B6 mice doubly homozygous for the nude (nu) and beige (bg) mutations (nubg mice) are also short-lived and immunodeficient. Nevertheless, grafts of mev lympho-hematopoietic cells increased life expectancy of nubg recipients. Such [mev --> nubg] chimeras did not develop any mev-like inflammatory pathology but showed autoimmunity features, particularly hyperglobulinemia which, unlike the mev one, was due to IgG rather than IgM. Serological studies of [mev IgHb --> nubg Igha] chimeras surprisingly revealed the exclusive host B-cell origin of the IgG2a overproduced by these chimeras. Yet, about half of such chimera serum IgM being IgMb, mev B cells had actually engrafted the nubg hosts. Together with the lack of transfer of the inflammatory pathology, this suggests that a non-mev environment might succeed acting as a regulator of some mev-induced dysfunctions.

Different nature of the proliferation defects of GLD, LPR and MEV C57BL/6 mouse lymphoid cells.

The three non-allelic gld, lpr and mev mutations in the mouse all lead to profound immunodeficiency besides a splenomegaly and a generalized autoimmunity. Spleen cells from young B6 gld, B6 lpr and B6 mev mice all display a decreased proliferative response to the T-cell mitogen concanavalin A (ConA), but the nature of the deficiency seems very different. No restoration of proliferation could be obtained by adding exogenous recombinant rIL2 to ConA-treated mev spleen cells, this lack of IL2-responsiveness suggesting a lack of (functional) IL2-receptors. In young mice of both gld and lpr strains, a B6 wild-type level of proliferation could be reached by rIl2 addition to ConA-treated spleen cells, this normal responsiveness to exogenous IL2 suggesting a normal expression of IL2-receptors. The endogenous IL2 production by ConA-treated spleen cells decreased very much with ageing in both B6 gld and B6 lpr mice. Yet, IL2 production in young mice revealed an earlier deficiency of the B6 lpr mice: the young B6 gld IL2 levels reached about 60% of age-matched B6 wild cell levels, but the B6 lpr levels reached 14% only. Finally the addition of exogenous rIL2 to ConA-pretreated cells from old B6 gld and B6 lpr mice, while enhancing the proliferative responses, could not restore the B6 wild-type levels. This suggests that, with ageing, the expression of functional IL2-receptors may become as abnormal in these gld and lpr mutants as it is from birth in the mev mutant mice.

Interactions of B6 wild and B6 gld cells engrafted within athymic nude beige recipients.

The murine gld mutation is targetted to the gene coding for the ligand of the Fas receptor for apoptosis. Gld mice display a lymphoproliferative and autoimmune syndrome that can be transferred in both irradiated euthymic wild and athymic beige (nubg) recipients. In order to test whether a supply of normal wild cells could correct the development of the gld syndrome, nubg mice were grafted with mixtures of gld and wild spleen cells from congenic donors which differed for the allotypes of the T-cell Thy1 membrane glycoprotein and/or of the B-cell Ig heavy chain. In the nubg chimeras, the wild spleen cells could down-regulate the hyperactivation of the B cells and the proliferation of the gld T cells, but this was not due to total eradication of the gld T-cell subset. Since this occurred in an athymic recipient, the correction of the gld syndrome did not require wild stem cell differentiation within a thymic environment, but should only depend on a sufficient Fas ligand supply by normal wild cells. Since the gld cells could proliferate in the nubg environment, the nubg environment could not provide sufficient Fas ligand to regulate the gld cell proliferation. Thus, the nubg B cells might lack Fas ligand expression, or express it but to a lower extent that T cells.