Development of a murine lymphoma model system for the characterization of natural killer:tumor cell interactions.

We have developed a model system which can be utilized for characterizing both the molecular basis of natural killer (NK):tumor interactions and the consequences of these interactions on tumor growth in vivo. This model system consists of several tumor lines which were derived from the murine lymphoma ASL1w under conditions which permitted the isolation of clonal lines that differ in their susceptibility to NK-mediated lysis. The NK-resistant clones used in this study (AW5J and AW5E) were susceptible to cytotoxic T-lymphocyte-mediated lysis and thus appear to be resistant to lytic processes utilized uniquely by NK cells. In competitive (cold target) inhibition assays, the AW5J clone did not inhibit NK recognition as well as the NK-sensitive clones. Thus AW5J may not be efficiently recognized by NK cells. The AW5E clone, on the other hand, competitively inhibited NK recognition as efficiently as the NK-sensitive clones; therefore, AW5E appears to evade a post-recognition event which is required for NK-mediated lysis. The susceptibility of these clones to killing by NK cells directly correlated with their lethality, suggesting that NK cells regulated the growth of these tumors in vivo. The level of host NK activity was also an important determinant of the level and site of tumor localization. Two-step immunofluorescence assays and flow cytometric analysis were used to quantitate the number of tumor cells in the bone marrow, spleen, and lymph nodes of mice with augmented or depleted NK activity. Increasing host NK activity decreased the number of tumor cells in each organ which could support the growth of the particular tumor tested. Furthermore, the extent of NK regulation was dependent, in part, upon the susceptibility of the particular tumor to NK-mediated lysis and the site of tumor growth. Thus, the data presented here demonstrate the utility of the ASL1w-derived clones as a model system which can be used to delineate the requirements and consequences of NK:tumor interactions.

The role of NK cells in the regulation of experimental metastasis in a murine lymphoma system.

This report describes the role of natural killer (NK) cells in regulating the localization of two closely related murine lymphomas (ASL1 and ASL1w) to various organ sites. These tumors are phenotypically similar yet differ in their overall malignancy and experimental metastatic pattern. Both tumors show similar patterns of migration in kinetic studies, however, they vary in their NK susceptibility. Cold target inhibition analysis and assessment of susceptibility to cytotoxic T lymphocytes (CTL) suggested that the NK resistance of ASL1 was due to the lack of expression of a relevant target structure(s) required for NK recognition. The role of NK cells in regulating the growth of the tumors in vivo was studied by altering NK cell levels in recipient mice prior to tumor injection. Neither NK cell depletion nor augmentation affected the growth of the NK resistant ASL1 tumor but NK depletion significantly shortened the mean survival time (MST) of recipients of the NK sensitive ASL1w tumor as well as changed its metastatic pattern; conversely, NK augmentation significantly increased the MST of ASL1w recipients. These data suggest that NK cells play a critical role in determining the metastatic characteristics of the ASL1w lymphoma.

Pre-thymic transcription of TCR genes by adult murine bone marrow cells.

In the adult mouse, the earliest thymocytes are derived from bone marrow-resident T lymphocyte precursor (pre-T) cells that immigrate to the thymus. There they undergo maturation through a series of developmental steps that include rearrangement and expression of the TCR genes, positive and negative selection, and functional maturation. Although these intrathymic processes have been extensively characterized, little is known about the T cell-specific events that take place in the bone marrow microenvironment. Of particular interest are the events surrounding transcription and rearrangement of the various TCR chains that are required for functional TCR expression. We have previously reported the transcription of incompletely rearranged TCR beta genes in pre-T cell-containing fractions of adult bone marrow. Here we demonstrate that the TCR gamma chain genes are also transcriptionally active in these cells. Like the TCR beta transcripts, TCR gamma transcripts are sterile, originating from unrearranged gamma loci. Interestingly, both RAG-1 and RAG-2 transcripts were also detected in this cell fraction, suggesting that sterile TCR transcription might be dependent upon the presence of a functional recombinase system. However, both C beta and C gamma sterile transcripts could be detected from the same bone marrow cell population isolated from RAG-1 gene deficient mice. Therefore, the expression of TCR genes can initiate at the earliest stages of T cell development, prior to exposure to the thymic microenvironment, and a functional recombinase system is not required for the production of these sterile TCR transcripts.

Interleukin 7 induces TCR gene rearrangement in adult marrow-resident murine precursor T cells.

Rearrangement of the T cell antigen receptor genes is a complex, highly regulated process. To gain a better understanding of the extracellular factors involved in the regulation of TCR beta and gamma gene rearrangement in adult murine bone marrow-resident precursor T cells, several cytokines were tested for their ability to induce gene recombination. A selected population of C58/J bone marrow cells (Thy 1(low), CD3, CD8, B220) that is enriched for pre-T cell activity was propagated in vitro in medium supplemented with IL-3 and mast cell growth factor (MGF, also referred to as stem cell factor, Steele factor and c-kit ligand). These cytokines were required for the maintenance of pre-T cell activity in culture, but had no effect on TCR gene expression. Several additional cytokines were added to the culture medium. Of all those tested, only IL-7 induced complete rearrangement of the TCR gamma locus. Complete rearrangement of the TCR beta locus was not induced under any of the culture conditions analysed here. The bone marrow cells cultured in IL-3, MGF and IL-7 did not begin to express mature T cell proteins and maintained their in vivo progenitor potential. Furthermore, IL-7 cultured bone marrow cells were capable of differentiation in vivo into all phenotypic subpopulations of T cells, without an apparent bias toward the gammadelta lineage. The data presented here suggest that TCR gamma gene rearrangement in adult pre-T cells is regulated by IL-7, but that the TCR beta locus requires additional or alternative signals for the induction of complete rearrangement.

Blood circulating microparticle species in relapsing-remitting and secondary progressive multiple sclerosis. A case-control, cross sectional study with conventional MRI and advanced iron content imaging outcomes.

BACKGROUND: Although multiple sclerosis (MS) is thought to represent an excessive and inappropriate immune response to several central nervous system (CNS) autoantigens, increasing evidence also suggests that MS may also be a neurovascular inflammatory disease, characterized by endothelial activation and shedding of cell membrane microdomains known as 'microparticles' into the circulation. OBJECTIVE: To investigate the relationships between these endothelial biomarkers and MS. METHODS: We examined the relative abundance of CD31(+)/PECAM-1, CD51(+)CD61(+) (αV-ß3) and CD54(+) (ICAM-1) bearing microparticles in sera of healthy individuals, patients with relapsing-remitting MS, and secondary-progressive MS. We also investigated the correlation among circulating levels of different microparticle species in MS with conventional MRI (T2- and T1-lesion volumes and brain atrophy), as well as novel MR modalities [assessment of iron content on susceptibility-weighted imaging (SWI)-filtered phase]. RESULTS: Differences in circulating microparticle levels were found among MS groups, and several microparticle species (CD31(+)/CD51(+)/CD61(+)/CD54(+)) were found to correlate with conventional MRI and SWI features of MS. CONCLUSION: These results indicate that circulating microparticles' profiles in MS may support mechanistic roles for microvascular stress and injury which is an underlying contributor not only to MS initiation and progression, but also to pro-inflammatory responses.

Cytokine and adhesion molecule expression in SCID mice reconstituted with CD4+ T cells.

The objectives of this study were to quantify colonic cytokine and endothelial cell adhesion molecule (ECAM) expression in the colons of severe combined immunodeficient (SCID) mice reconstituted with different subsets of CD4+ T lymphocytes. We found that animals injected with CD45RBhigh but not CD45RBlow T cells or phosphate-buffered saline (PBS) developed clinical evidence of colitis at 6-8 weeks following reconstitution, as assessed by loss of body weight, development of loose stools and/or diarrhea, and histopathology. Concurrent with the onset of distal bowel inflammation was enhanced expression of a variety of Th1 and macrophage-derived cytokines including interferon gamma, tumor necrosis factor-alpha, interleukin (IL)-1beta, IL-6, IL-12, and IL-18 lymphotoxin-beta. In addition, message levels and vascular surface expression of ICAM-1, VCAM-1, and MAdCAM-1 were all significantly enhanced in the colitic SCID mice reconstituted with CD45RBhigh T cells compared with SCID mice reconstituted with PBS or CD45RBlow T cells that did not develop disease. Significant increases in some of these ECAMs were also noted in the cecum and stomach and to a lesser degree in the small bowel. Our data confirm that reconstitution of SCID mice with CD45RBhigh but not CD45RBlow T cells induces chronic colitis, and that the colonic inflammation is associated with enhanced expression of proinflammatory cytokines and different ECAMs in the colon. Furthermore, our studies demonstrate that reconstitution of SCID mice with CD45RBhigh T cells enhances ECAM expression in tissues distant from the site of active inflammation.

In vitro maintenance of bone marrow-resident T cell precursors in cultures supplemented with exogenous IL-3.

T cell precursors (pre-T cells) from adult murine bone marrow were propagated in vitro using a newly developed cell culture system. This system differs from "classical" bone marrow culture techniques in that it does not require the establishment of a monolayer of bone marrow stromal cells nor the presence of an irradiated feeder layer for the maintenance of the pre-T cell compartment, but rather depends on the continuous presence of exogenous interleukin-3 (IL-3) in the culture. Cells harvested from these cultures retain the capacity to repopulate the thymuses of sublethally irradiated recipient mice, following intrathymic transfer, for at least 2 weeks after the initiation of the cultures. Interestingly, pre-T cells maintained under these culture conditions rapidly lose their capacity for thymic repopulation following intravenous transfer, suggesting a potential defect in recirculation and/or homing properties. This culture system should prove useful in the investigation of bone marrow-resident pre-T cells.

Peanut lectin binding as a marker for activated T-lineage lymphocytes.

A series of experiments was undertaken to ascertain the reliability of peanut agglutinin (PNA) binding as a means of distinguishing mouse pre-T cells from other cell types. Normal bone marrow, thymus and spleen cells were stained with graded doses of fluoresceinated lectin (FITC-PNA) and subsequently examined by fluorescence microscopy to determine the number of lectin-binding cells. In addition, the PNA binding properties of mitogenically activated lymphocytes were examined in a similar manner. The results of this study show that PNA binding does not define a fixed population of cells, as the percentage of PNA+ cells detected in normal mouse tissues varied directly with the concentration of PNS used for staining. Nor is PNA able to distinguish between pre-T cells and mature T cells after mitogenic stimulation, both of which are shown to bind PNA. This lectin may , however, have value as a general marker of lymphoid precursors and activated lymphocytes of the T lineage.

Influence of MHC on thymus repopulation following intrathymic transfer of mouse T-cell precursors.

T-cell precursors (pre-T cells) traditionally have been detected by their ability to repopulate the thymus of heavily irradiated mice following intravenous injection. Recently, an assay system involving the direct injection of pre-T cells into the thymus of sublethally irradiated animals has been described. Here we report the results of experiments designed to evaluate the ability of bone marrow cells to produce thymic repopulation following intrathymic injection in a wide range of donor-host strain combinations. Irradiated (600 R) mice were injected intrathymically with 2 X 10(6) bone marrow cells which differed from the recipient with respect to their Thy-1 allotype and the percentage of thymus cells expressing either donor- or recipient-type Thy-1 was determined 9 to 23 days after injection. The results of these experiments showed that thymocytes expressing the Thy-1 allotype derived from the donor marrow were only detected when the donor and host were matched at MHC. By contrast, thymic repopulation by MHC-mismatched donor marrow cells could readily be observed when these cells were given intravenously.

Target cell expression of MHC antigens is not (always) a turn-off signal to natural killer cells.

Recent evidence has demonstrated that the lytic function of natural killer cells might be regulated by potential target cells through the target cells' expression of cell surface components that are able to inhibit the lytic process. Specifically, it has been shown in many target cell systems that the expression of class I MHC proteins by target cells is inversely proportional to their susceptibility to lysis by NK cells. It has been suggested, therefore, that MHC proteins may act as important negative regulatory elements in the ongoing control of NK cell function. Herein, we examined two closely related murine lymphoma cells (ASL1 and ASL1w), both in terms of their susceptibility to lysis by NK cells as well as their expression of both H-2K and H-2D class I MHC proteins. The results of these studies showed that whereas ASL1 and ASL1w cells differed greatly in their susceptibility to NK cell lysis (ASL1 was much more NK resistant than ASL1w), both expressed high levels of H-2K and D proteins. In contrast to what might have been predicted base on reports from other target cell systems, the more NK susceptible ASL1w cells expressed somewhat higher levels of H-2K Ag than did ASL1 cells. These results indicate that expression of H-2 class I proteins by target cells, in and of itself, is not sufficient to inhibit the lytic activity of murine NK cells.

Effects of maternal alcohol consumption on milk and blood lymphocytes and IgG antibody levels from Trichinella spiralis-infected rats.

Immunity to the intestinal parasite Trichinella spiralis can be transferred from the mother to the neonate during lactation. Previous studies in our laboratory showed that the passage of immunity to pups from ethanol-treated dams was depressed. This study examined the effect of ethanol consumption during pregnancy and lactation on the T. spiralis-associated immune components in milk and blood. Groups of female rats were fed either ethanol-containing or isocaloric liquid diets for 30 days before T. spiralis infection, mated and maintained on corresponding diets through pregnancy and lactation. Two-color flow cytometric analysis was performed for lymphocyte populations, enzyme-linked immunoabsorbent assay for specific IgG, and radial immunodiffusion assay for total IgG. The percentage of total T cells and their subsets, T helper cells and T cytotoxic/suppressor cells in milk and those in blood were similar between pair-fed and ethanol-treated animals. However, the percentage of natural killer cells in milk from ethanol animals was significantly reduced compared with the pair-fed group (33% vs. 54%). The percentage of activated or memory type T helper cell subset (OX2-W3/25+) was significantly increased in the blood of the ethanol-treated group. Pair-fed animals showed higher T. spiralis-specific IgG antibody levels in both in milk and blood compared with ethanol-treated animals. In ethanol-treated animals, specific IgG levels and total IgG concentration in milk were significantly lower than those in blood, whereas in pair-fed animals, only total IgG concentration in milk was lower than that in blood. This study indicates that ethanol consumption during pregnancy and lactation alters the maternal immune system.(ABSTRACT TRUNCATED AT 250 WORDS)

Reliable method for the simultaneous detection of cytoplasmic and surface CD3 epsilon expression by murine lymphoid cells.

During their development, T-cell precursors (pre-T cells) undergo a variety of changes with respect to their expression of specific surface proteins. Among the most critical of the surface markers acquired by developing T cells is the T-cell receptor (TCR)/CD3 complex. Prior to the assembly and transport of complete TCR/CD3 multimeric complexes to the plasma membrane, the individual constituent subunits are expressed in the cytoplasm (ER-Golgi). In order to study the expression of the T-cell receptor TCR/CD3 complex during pre-thymic T-cell differentiation, we have developed a flow cytometric technique for the simultaneous detection of surface (sCD3 epsilon) and cytoplasmic CD3 epsilon (cCD3 epsilon). This technique, which employs fixation in 1% paraformaldehyde and permeabilization with 1% saponin and 0.025% digitonin, features reliable internalization and low nonspecific binding of anti-CD3 epsilon in murine lymphoid cells, as well as tissue culture cell lines. The combination of saponin and digitonin treatment was also compatible with the staining of sCD3 and other lymphocyte surface antigens such as Thy1, CD4, CD8, B220, and IgM. In contrast, permeabilization of cells with the detergents Tween 20 and Triton X-100 was shown to remove surface-bound anti-CD3 epsilon. The present technique permitted the detection of discernible sCD3 epsilon and cCD3 epsilon double and single positive lymphocytes and may prove useful in defining bone marrow-resident pre-T cells.

Colony-forming units in the spleen and bone marrow of young (C57BL/6xDBA/2)F1 hybrid mice.

Spleen or bone marrow cells from (C57BL/6 X DBA/2)F1 hybrid mice ranging in age from 1 to 32 days were transplanted into syngeneic irradiated adult recipients. By enumeration of macroscopic hemopoietic spleen colonies 8 days after transplantation, the colony-forming unit(CFU) content of the spleen and bone marrow of the young donor mice was determined. The CFU concentration in the bone marrow, initially at a level of 15.1--17.0 CFU/105 nucleated cells, increased during the third week of life and reached a value comparable to that of the adult during week 4. In contrast, the CFU concentration of the spleen decreased during the first 2 weeks and reached a plateau at a level of 4.45--6.66 CFU/105 nucleated cells during the third and fourth weeks of age. This was still significantly higher than that of the adult hybrid. The rise in prominence of the bone marrow as a hemopoietic organ during this period appeared to be due to an increase in total cellularity coupled with an increase in CFU concentration in this hybrid. While the cellularity of the spleen also increased during this time, the CFU concentration decreased, resulting in little change in the total CFU content of this organ.

Clonal abortion of bone marrow T cell precursors: T cells acquire specific antigen reactivity prethymically.

Lethally irradiated (900 R) mice were reconstituted with bone marrow cells from syngeneic donors that had been tolerized 2 to 3 wk earlier to either DNP or TNP compounds. Five weeks after reconstitution, these animals were tested for their ability to mount a delayed hypersensitivity (DH) response to the tolerizing haptens. Recipient mice were specifically tolerant to the hapten that was used to induce tolerance in the marrow donor. Mixing experiments in which mice were reconstituted with marrow from DNP-tolerant and TNP-tolerant donors showed no indication of active suppression or effective antigen carry-over in this system. This observation held true even in experiments in which mice were reconstituted with a mixture of marrow from tolerant and normal donors at a ratio of 5:1. Thus the reduced responsiveness in recipient mice seemed to be due to the functional elimination of hapten-responsive T cell precursor (pre-T) clones. Recipient unresponsiveness was also shown to be MHC restricted. Maintenance of unresponsiveness appeared to be due to the restricted access of regenerating pre-T cell clones to the maturational influence of the recipient's thymus.

Differences in target cell DNA fragmentation induced by mouse cytotoxic T lymphocytes and natural killer cells.

Fragmentation of YAC-1 target cell DNA during cytolysis mediated by mouse natural killer (NK) cells and cytotoxic T lymphocytes (CTL) was compared. Cleavage of nuclear chromatin was always an extensive and early event in CTL-mediated cytolysis, whereas with NK cell-mediated killing the degree of DNA fragmentation showed an unexpected relationship to the effector:target (E:T) ratio. At low NK:YAC-1 ratios, DNA fragmentation and 51Cr release were equivalent and increased proportionately until a ratio of about 50:1 was reached; at higher ratios, 51Cr release increased as expected but DNA fragmentation decreased dramatically. Comparison of time course data at E:T ratios producing similar rates of 51Cr release showed that the target cell DNA fragmentation observed in NK killing was not nearly as rapid nor as extensive as that observed with CTL effectors. These results suggest that NK cells induce target cell injury via two different mechanisms. One mechanism would involve lysis mediated by cell-to-cell contact, while the other may induce DNA fragmentation via a soluble mediator. In support of this notion, cell-free culture supernatants containing NK cytotoxic factor (NKCF) induced DNA fragmentation in YAC-1 cells. The DNA fragments induced by NK cells and NKCF-containing supernatants consisted of oligonucleosomes indistinguishable from those induced by CTL. The results presented here show distinct differences in target cell DNA fragmentation induced by CTL and NK cells, and suggest that these two effectors use different mechanisms to achieve the same end. CTL seem to induce DNA fragmentation in their targets by direct signaling, whereas NK cells may do so by means of a soluble factor.