The generation of natural killer (NK) cells from NK precursor cells in rat long-term bone marrow cultures.

In this report, we describe a novel long-term bone marrow culture (LTBMC) system to study the origin and generation of natural killer (NK) cells from NK precursors. Rat bone marrow was cultured for 4 wk in RPMI 1640 with 5% fetal calf serum and 2-mercaptoethanol to allow the formation of an adherent stromal cell layer containing NK precursor cells. After addition of interleukin 2 (IL-2), the LTBMC generated high numbers (up to 100-fold expansion in 7 d) of pure 3.2.3+ large granular lymphocytes with lytic activity against NK-sensitive and -resistant tumor targets, as well as antibody-dependent cellular cytotoxicity. NK activity in LTBMC could be detected 3 d after addition of as little as 1 U/ml rIL-2, whereas lymphokine-activated killer activity was found 5 d after addition of at least 10 U/ml rIL-2. In vivo depletion and in vitro complement lysis studies showed that the NK precursor cells in LTBMC did not express the NK-associated surface markers asialo GM1 or 3.2.3. We also found that LTBMC cells did not exhibit colony growth in granulocyte/macrophage or spleen colony-forming unit assays. The generation of NK cells from NK precursors required, in addition to IL-2, a second growth/maturation factor(s), which was present in the conditioned medium of the LTBMC. This LTBMC system provides a unique in vitro model to study the development of NK cells from precursor cells, the role of the bone marrow stromal microenvironment in this development, and the lineage relationship of NK cells to other hematopoietic cells.

Cross-species bone marrow transplantation: evidence for tolerance induction, stem cell engraftment, and maturation of T lymphocytes in a xenogeneic stromal environment (rat----mouse).

Transplantation of untreated F344 rat bone marrow into irradiated B10 mouse recipients (non-TCD F344----B10) to produce fully xenogeneic chimeras resulted in stable xenogeneic lymphoid chimerism, ranging from 82% to 97% rat. Survival of animals was excellent, without evidence for GVH disease. The specificity of tolerance which resulted was highly donor-specific; MHC disparate third party mouse and rat skin grafts were promptly rejected while donor-specific F344 grafts were significantly prolonged (MST greater than 130 days). Multi-lineage rat stem cell-derived progeny including lymphoid cells (T- and B-lymphocytes), myeloid cells, erythrocytes, platelets, and natural killer (NK) cells were present in the fully xenogenic chimeras up to 7 months after bone marrow transplantation. Immature rat T-lymphocytes matured and acquired the alpha/beta T-cell receptor in the thymus of chimeras in a pattern similar to normal rat controls, suggesting that immature T-lymphocytes of rat origin could interact with the murine xenogeneic thymic stroma to undergo normal maturation and differentiation. This model may be useful to study the mechanisms responsible for the induction and maintenance of donor-specific transplantation tolerance across a species barrier.

Bone marrow as a potential source of hepatic oval cells.

Bone marrow stem cells develop into hematopoietic and mesenchymal lineages but have not been known to participate in production of hepatocytes, biliary cells, or oval cells during liver regeneration. Cross-sex or cross-strain bone marrow and whole liver transplantation were used to trace the origin of the repopulating liver cells. Transplanted rats were treated with 2-acetylaminofluorene, to block hepatocyte proliferation, and then hepatic injury, to induce oval cell proliferation. Markers for Y chromosome, dipeptidyl peptidase IV enzyme, and L21-6 antigen were used to identify liver cells of bone marrow origin. From these cells, a proportion of the regenerated hepatic cells were shown to be donor-derived. Thus, a stem cell associated with the bone marrow has epithelial cell lineage capability.

Hematopoietic stem cells with high proliferative potential. Assay of their concentration in marrow by the frequency and duration of cure of W/Wv mice.

THIS STUDY WAS DESIGNED TO APPROACH TWO PRIMARY QUESTIONS CONCERNING HEMATOPOIETIC STEM CELLS (HSC) IN MICE: what is the concentration of HSC with extensive proliferative potential in marrow, and how long can an HSC continue to function in an intact animal? The assay system was the W/W(v) mouse, a mouse with an inherited HSC defect, reflected in a reduction in all myeloid tissue and most particularly in a macrocytic anemia.A single chromosomally marked HSC will reconstitute the defective hematopoietic system of the W/W(v). The concentration of HSC in normal littermate (+/+) marrow was assayed by limiting dilution calculation using cure of W/W(v) as an end point (correction of anemia and erythrocytes' macrocytosis) and found to be approximately 10/10(5). This is significantly less than spleen colony forming cell (CFU-S) concentration: approximately 220/10(5) in +/+ and ranging from 50 to 270/10(5) in various other studies. Blood values were studied at selected intervals for as long as 26 mo. Of 24 initially cured mice, which were observed for at least 2 yr, 75% remained cured. However, of all cured mice, 17 lost the cure, returning to a macrocytic anemic state. Cured mice had normal numbers of nucleated and granulocytic cells per humerus and a normal concentration of CFU-S. However, cure of secondary W/W(v) recipients by this marrow was inefficient compared with the original +/+ marrow. These studies suggest the CFU-S assay over-estimates extensively proliferating HSC or perhaps does not assay such a cell. A single such HSC can not only cure a W/W(v), but can sustain the cure for 2 yr or more, despite a relative deficit of cells capable of curing other W/W(v). However, the duration of sustained cure may be finite.

5-Iodo-2-deoxyuridine administered into the lateral cerebral ventricle as a radiosensitizer in the treatment of disseminated glioma.

A rat brain tumor model (Fischer 344 rats) with the clinical and pathological features of dissemination via the cerebrospinal fluid (CSF) pathways was used to demonstrate the efficacy of 5-iodo-2-deoxyuridine (IUDR) as a radiosensitizer when it is administered directly into the CSF. Stereotaxic implantation of 9L gliosarcoma cells (5 X 10(5) into the CSF of the lateral cerebral ventricle resulted in widespread dissemination and median survival of 18.5 and 20 days (range, 10-22) in two experiments. A continuous 7-day infusion of IUDR into the CSF starting on the day of tumor implantation did not provide any beneficial effect. Irradiation of the cranial spinal axis with 800 rad on days 4, 6, and 7 after implantation achieved an increase in survival time that was modest but statistically significant. However, the combination of IUDR infusion and radiotherapy resulted in marked improvement in survival time and a 10% cure rate (two of 20 rats). This is the first demonstration in vivo that IUDR administered into the CSF can be a potent radiosensitizer.

In vivo sister chromatid exchange and cellular replication kinetics of normal and lymphoma AKR bone marrow cells.

The recent development of an AKR(Rb6.15)1Ald lymphoma model in our laboratory presents a unique means of comparing sister chromatid exchange (SCE) frequencies and cellular replication kinetics of normal and lymphoma cells in the same host environment. Lymphoma cells, distinguished from normal cells by the presence of two metacentric chromosomes, had an average cell cycle time of 8 hr compared to 11 hr for normal cells. Normal bone marrow cells from nonleukemic AKR/J, nonleukemic AKR(Rb6.15) Ald, and tumor passage recipients had similar baseline and 1,3-bis(2-chloroethyl)-1-nitrosourea-induced SCE frequencies following exposure to 4.4 mg 1,3-bis(2-chloroethyl)-1-nitrosourea per kg and 19 hr 5-bromo-2-deoxyuridine. While baseline SCE frequencies were only slightly higher, 1,3-bis(2-chloroethyl)-1-introsourea-induced lymphoma cell SCE frequencies were 3 times higher than normal induced SCE frequencies [34.06 +/- 6.6 (S.E.) versus 11.0 +/- 0.6 SCEs/cell].

Cellular replication kinetics and persistence of sister chromatid exchange-inducing lesions in normal and lymphoma AKR cells following exposure to 1,3-bis(2-chloroethyl)-1-nitrosourea.

The present studies were designed to evaluate the role of cell cycle time and time of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) administration on the persistence of sister chromatid exchange (SCE)-inducing lesions in normal and lymphoma second- and third-division AKR bone marrow cells. Normal second-division cells harvested from mice given injections of BCNU at the start of an 18-, 24-, or 28-hr 5-bromo-2-deoxyuridine (BrdUrd) infusion exhibited similar linear dose-dependent increases in SCE frequencies (p greater than 0.05). The faster-cycling lymphoma cells, harvested after 18-hr BrdUrd infusion, had significantly higher baseline (p less than 0.05) and BCNU-induced increases (p less than 0.001) in SCE frequencies than did normal cells. Dose-dependent increases in SCE frequencies were demonstrated in third-division normal and lymphoma cells from mice infused with BrdUrd for 24 or 28 hr. Whereas lymphoma cells from mice treated with 3.3 mg BCNU per kg exhibited 31.2 +/- 3.9 (S.E.) SCEs in second-division cells and 4.7 +/- 0.4 reciprocal and 22.9 +/- 2.0 nonreciprocal SCEs in third-division cells, a 5 times higher dose of BCNU was required to induce similar levels of 30.0 +/- 0.8 SCEs in second-division cells and 4.4 +/- 0.6 reciprocal and 22.6 +/- 1.2 nonreciprocal SCEs in third-division normal cells. BCNU dose-dependent increases in SCE frequencies were also observed following injection of BCNU 8 hr after the start of BrdUrd infusion. The unexpectedly higher levels of SCEs for both normal and lymphoma cells by this treatment protocol may be due to SCEs occurring at the same site in successive divisions in BrdUrd. Regardless of the protocol used, lower nonreciprocal SCE frequencies were observed in third-division cells relative to SCE frequencies in second-division cells; a possible consequence of the cytotoxicity of BCNU. Injection of BCNU produced significant changes in the proportions of normal and lymphoma cells completing one, two, and three or more divisions in BrdUrd. Lymphoma cells were consistently more sensitive to the specific type(s) of BCNU-induced damage leading to SCEs and cell death than were normal cells. These studies indicated that differences in SCE response were not due to cell cycle time, time of drug administration, or potential for repair. It is therefore suggested that increased sensitivity of lymphoma versus normal cells may be due to increased cellular uptake of BCNU.

NKR-P1+ cells localize selectively in Rat 9L gliosarcomas but have reduced cytolytic function.

To better understand immune responses to brain tumors and to develop possible approaches for immunotherapy, we have investigated the leukocyte populations infiltrating the rat 9L gliosarcoma. By immunocytochem-ical analyses of the cells infiltrating the tumor, we observed a substantial number of cells expressing natural killer cell receptor protein 1 (NKR-P1), a marker expressed only on rat lymphocytes capable of non-MHC-restricted cytotoxicity. Previous investigations have determined the existence of three populations of NKR-P1+ lymphocytes in normal rats, including NKR-P1bright/T-cell receptor (TCR)-/CD3-/CD5- (approximately 5-15%), NKR-P1dim-/TCRalphabeta+/CD3+/CD5+ (approximately 1-5%), and NKR-P1dim/TCRgammadelta+/CD3+/CD5+ (approximately 0.5-2%). By one-parameter flow cytometry, it was determined that NKR-P1+ cells constituted 30-60% of the lymphocytes in 9L tumors. Among splenic lymphocytes or peripheral blood leukocytes, NKR-P1bright cells are 1.5-4.5 times more numerous than NKR-P1dim cells. In striking contrast, NKR-p1dim cells were 4-5 times more numerous than NKR-P1bright cells among lymphocytes isolated from 9L tumors. Using quantitative analyses of laser confocal microscopic scans, we determined that NKR-P1dim cells were approximately 4 times as numerous as NKR-P1bright cells in situ, confirming flow cytometric findings. By two-color now cytometric analyses, it was observed that approximately 5-10% of the cells were NKR-p1bright/CD5-/TCR-, a phenotype representative of NK cells. Also, approximately 11-25% of the cells were NKR-P1dim/CD5+/TCR+ cells, corresponding to the T-cell subset with non-MHC-restricted lytic function. In addition, we observed a cell population among 9L-derived lymphocytes with a NKR- p1dim/CD5-/TCR- phenotype (approximately 15-25%). Cells of this phenotype have not been reported previously, and most likely represent NK cells down-modulated for expression of NKR-P1. Alternatively, they might represent cells of unknown origin or cells down-modulated for expression of T-cell markers in the microenvironment of 9L tumors. We also compared the lytic capacity of NKR-P1+ populations derived from normal animals and from 9L gliosarcomas. In these experiments, it was determined that, although cells isolated from 9L tumors had some capacity to lyse tumor target cells, they were clearly less efficient than cells isolated from normal splenocytes. Cumulatively, these data suggest that there is selective localization of cells capable of mediating antitumor responses in 9L, but that tumor-associated factors may down-regulate their function and expression of NKR-P1.

Effects of irradiation of CBA/CA mice on hematopoietic stem cells and stromal cells in long-term bone marrow cultures.

Following 200 cGy total body irradiation, 20-25% of CBA/Ca mice and their CBA/B and CBA/H sublines develop myeloid leukemia. To determine whether hematologic changes in vitro were detectable, long-term marrow cultures (LTBMCs) were established from the right and left hind limbs of 11 individual control and 11 CBA/B mice 100-114 days after 200 cGy total body irradiation. Individual cultures were studied weekly for cumulative production of nonadherent cells and colony-forming, hematopoietic progenitor cells. Control cultures produced significantly more nonadherent cells over 25 weeks in long-term marrow culture compared to those from irradiated (treated) mice. Permanent stromal cell lines were established from control and irradiated CBA/B mouse LTBMCs and clonal sublines were established. The stromal cell lines from LTBMCs of in vivo irradiated CBA/B mice had uniformly lower plating efficiencies, and only one formed a permanent clonal subline at 100-fold lower frequency compared to stromal cell lines from control mouse LTBMCs. The irradiation sensitivity of both uncloned and clonal sublines was similar by single-hit, multi-hit or by linear quadratic formula. Cocultivation of an IL-3 dependent hematopoietic progenitor cell line established from a control CBA/B, LTBMC with control of irradiated stromal cell lines derived from either a control (CC3) or the one successfully cloned in vivo irradiated (CT4) LTBMC, produced few cobblestone islands in the presence of IL-3. In contrast, formation of cobblestone islands in the presence of L cell-condition medium as a source of M-CSF was significantly greater, and these persisted for 21 days on both CC3 and CT4 stromal lines. The data provide evidence for irradiation induced changes in the bone marrow stromal cell compartment of CBA/B mice which persist and are detectable in vitro 6 months after explant of the cells to culture. These marrow stromal cell lines may provide valuable resources for analyzing the molecular biologic changes in the hematopoietic microenvironment during irradiation leukemogenesis.

Modulation of hematopoiesis and survival after high-dose chemotherapy or radiotherapy: review and discussion of possible mechanisms.

This paper reviews ways by which growth of transplanted or surviving hematopoietic stem cells might be enhanced to improve survival in the case of autologous marrow transplantation. Most of the treatments known to have such effects have been used in isologous mouse models and improve animal survival by enhancing hematopoietic recovery after high doses of whole body irradiation or chemotherapy. Although some of these were studied 20 years ago, the use of such treatments in man has awaited the realization that some disseminated cancer can only be eliminated by use of various treatments where hematopoietic damage is the limiting factor. Although increasing the number of transplanted pluripotent stem cells is the most certain way to hasten return of needed blood cells in transplant patients, several of the treatments listed in the paper have growth enhancing effects on both surviving host stem cells and transplanted cells. There are recent studies which indicate that such priming treatments may also be effective in other normal tissues such as gut and bladder epithelium. At least one study in man has intentionally applied this approach with the expected benefit and others may have done so inadvertently. Much work remains to find new combinations to select the best priming treatments and intervals in man and to determine whether or not such treatments are effective against tumors.

Efficient transformation and frequent single-site, single-copy insertion of DNA can be obtained in mouse erythroleukemia cells transformed by electroporation.

Electroporation has recently been shown to have advantages over the commonly used method of calcium phosphate precipitation for obtaining DNA-mediated transformation of certain types of cells. Although mouse erythroleukemia cells and other cells of hematopoietic origin are not transformed at useful frequencies by calcium phosphate-DNA precipitation methods, we obtained high frequencies of transformation (approximately 10(-5)) of these cells with electroporation. Even higher transformation frequencies (approximately 10(-3)) were obtained with human fibroblasts. Another advantage of electroporation was found when analysis of Southern blots of DNA from 243 transformed erythroleukemia cell colonies indicated that, under appropriate conditions, about 79% of the transformed cells had the exogenous DNA integrated in single copies at single sites. Under conditions of higher DNA and lower cell concentrations using fibroblasts, cotransformation was obtained with two plasmids that confer HAT or G418 resistance when integrated into cellular DNA. About 23% of the transformed cells developed both types of resistance. We describe a simple, inexpensive apparatus for carrying out electroporation.

Transplantation of chromosomally marked syngeneic marrow cells into mice not subjected to hematopoietic stem cell depletion.

The percentage of donor-host chimerism was determined 4-6 weeks or six months after injection of normal bone marrow cells into normal syngeneic or coisogeneic recipient mice. Donor-recipient pairs had chromosome markers that provided easy identification of metaphase cells. The percentage of donor cells in marrow or spleen ranged from 0 to 16% and this percentage was independent of the age of recipient or attempts to stimulate hematopoiesis in donor and/or host mice. In adult C57BL/6 mice there was a roughly linear dose-response relationship between cell dose and percentage of chimerism. There was no apparent dose-response relationship for AKR mice. The percentage of donor cells in the spleen was correlated to that seen in the marrow of recipients. Neonatal mice given the same intraperitoneal marrow cell dose as weanlings, but a larger number of cells relative to their own marrow mass, did not show a larger percentage of chimerism than weanlings. Similarly, weanlings given the same intravenous dose as adults showed no greater degree of chimerism than adults. Temporary anemia, induced by bleeding donors prior to cell collection, or more chronic hemopoietic stimulus (produced by injecting recipients with phenylhydrazine prior to cell injection with subsequent bleeding at intervals) did not result in an increased percentage of chimerism. These results indicate that there are "empty" sites in bone marrow of normal mice in which injected hematopoietic stem cells can lodge and grow.

Expression of murine CD34 by fetal liver NK cell progenitors.

Although 14.5-day murine fetal liver (FL) has few, if any, mature natural killer (NK) cells, culture of FL with recombinant human IL-2 (rhIL-2) and stroma from irradiated NK longterm bone marrow cultures (NK-LTBMC) allows proliferation and differentiation of NK cell progenitors. Using this system, NK cell progenitors were found in both CD34+ and CD34- sorted subpopulations of FL. The CD34 antigen was expressed by 14+/-1.3% of whole FL cells, while mature NK cells cultured from NK cell precursors in FL did not express the CD34 antigen. Anti-TER-119 mAb reacted with 84%+/-10.3% of the FL cells, and NK cell progenitors were enriched in the TER-119- subpopulation. After coculture with rhIL-2 and stroma, neither TER-119- nor TER-119+ cells expressed antigens associated with T cells (CD3, CD4, and CD8) or myeloid cells (Gr-1 and Mac-1). Only the TER-119 subpopulation generated NK1.1+ (77%) and B220+ (87%) cells. Within the TER-119 subpopulation, both CD34+ and CD34- cells generated cytolytic and NK1.1+ cells after culture. By a limiting dilution assay (LDA) of the Lin (i.e., negative for NK1.1, CD3, CD4, CD8, B220, Gr-1, and TER-119) CD34 positive or negative subpopulations, the calculated mean frequency of NK cell progenitors was about 1/100 for the CD34+Lin- subpopulation and about 1/(200-300) for the CD34-Lin- subpopulation. In kinetic studies, we found that NK1.1 antigen expression continued to increase with time in culture for both the CD34+Lin- and CD34-Lin- fractions. In contrast, the percentage of CD34+ cells decreased rapidly and produced CD34- cells, and the CD34- population remained CD34-. These data suggest that both CD34+ and CD34- subpopulations of FL can differentiate into NK cells when cocultured for 13 days with irradiated NK-LTBMC stroma and rhIL-2, and that CD34+ progenitors differentiate to CD34- precursors, which in turn differentiate to CD34- mature NK cells.

Addition of a bone marrow "facilitating cell" population increases stem cell-derived cobblestone area formation in impaired long-term bone marrow culture stroma.

Treatment of mouse bone marrow (BM) with rabbit anti-mouse brain serum (RAMBS) plus complement (C') depletes several cell types, including T cells and facilitating cells (FCs), that is, cells that facilitate engraftment of sorted allogeneic stem cells (SCs) in vivo. In the present study, treatment of BM with RAMBS+C' resulted in the depletion of approximately half of the late cobblestone area (CA)-forming stem cells as assayed on irradiated long-term bone marrow culture (LTBMC) stroma. In addition, LTBMC of RAMBS+C'-treated BM produced functionally impaired stroma with reduced ability to support CA formation by nontreated exogenous SCs. This stromal impairment was not due to depletion of TCRalphabeta T cells in the BM, because BM cultures from TCR alpha-chain knockout mice supported normal numbers of exogenous CAs. Because CD8+/TCR- cells are enriched for FCs, we tested the effect of adding these cells back to the treated BM prior to culture. The sorted FCs alone did not produce CAs, but did improve the ability of the impaired stroma to support late CA formation by sorted SCs. These studies provide a new model for dissecting the roles of different cellular components of BM in producing functional stroma that supports CA formation by SCs, and show that the number of CAs formed depends on the "quality" of the stroma as well as the number of SCs seeded. These findings further suggest that CD8+/TCR- BM cells may be important for the establishment of functional stroma.

The effect of syngeneic marrow injection upon recovery in sub- and near-lethally irradiated mice.

Mice were given sub-lethal (200-600 cGy) or near-lethal (800 cGy) whole body irradiation and the effect of injecting syngeneic marrow on subsequent hematopoietic recovery was studied. Marrow cell injection enhanced erythropoietic recovery after sub-lethal irradiation as reflected in hematocrit values and rate of appearance of 59Fe-labeled red cells in blood. However, this enhanced erythropoiesis was only seen in the spleen, and 59Fe uptake in marrow was reduced. When the irradiation dose was kept constant and the marrow dose increased from 10(5) to 10(6) to 10(7) cells, there was a somewhat erratic increase in spleen 59Fe and a decrease in marrow 59Fe uptake. When marrow cell number was kept constant and the dose of irradiation was increased from 200 to 400 to 600 to 800 cGy, there was an exponential increase in spleen 59Fe uptake but the marrow 59Fe uptake changed from depressed after lower doses to increased after 800 cGy. Cell injection after sub-lethal irradiation did not increase or decrease granulocytopoiesis. Injection of irradiated marrow cells also reduced marrow erythropoiesis and this was evident after both sub- and near-lethal irradiation. However, injection of irradiated cells did not increase splenic erythropoiesis. Following splenectomy, the depressed marrow erythropoiesis attending injection of viable cells was virtually eliminated but no increase was seen. These data suggest that the injection of autologous or syngeneic marrow may not be effective as a means of accelerating hematopoietic recovery after irradiation unless near-lethal or lethal dose have been received.

Latent deficiency of the hematopoietic microenvironment of aged mice as revealed in W/Wv mice given +/+ cells.

The macrocytic anemia of W/Wv mice can be cured by injection of +/+ bone marrow cells (BMC) from WBB6F1 mice. However, it has been observed that some W/Wv recipients appear to "lose" their cure with time, an effect that does not appear to be related to the age of the BMC donor. The present study was undertaken to determine the effect of recipient age on W/Wv responses to BMC injection. The effect of aging on erythroid parameters was similar in untreated W/Wv mice and +/+ controls. In both genotypes, hematocrit (HCT) and red blood cell count (RBC) decreased, and the modal red blood cell size (peak) increased between 13 and 150 weeks of age. As anticipated, mean HCT and RBC values were lower and peak values higher in W/Wv mice compared to +/+ controls at every age. However, the rate of decrease in HCT and RBC with age was the same for both genotypes, suggesting that the age effect and W gene effect were independent. Peak values increased slightly more with age for W/Wv than for +/+ controls. When female W/Wv mice in three age groups (23.5, 70, and 91.5 weeks old) were injected with 5 x 10(5) BMC from 20-week-old +/+ female donors and HCT, RBC, and peak were determined monthly, improvement was seen in most W/Wv recipients. However, in the older mice this improvement was slower and often was not sustained; 100% of the youngest recipients, 80% of the middle-aged, and only 30% of the older groups were cured after 3 months. Taken together, these data suggest a latent deficiency of the aging hematopoietic microenvironment that is revealed in W/Wv mice by the stress of continuing erythroid demand on the limited number of normal donor BMC.

Suppression of natural killer cell differentiation by activated T lymphocytes in long-term cultures of mouse bone marrow.

The goal of the present work was to study the regulatory role of T lymphocytes on natural killer (NK) cell generation in NK long-term bone marrow cultures (LTBMCs), an established mouse long-term bone marrow (BM) culture system used for the study of NK cell differentiation from precursors. Activation of the few T cells present in NK-LTBMCs by addition of anti-CD3 monoclonal antibody (mAb) together with interleukin (IL)-2 inhibited the generation of NK cells. Coculture with NK-LTBMCs of a pure population of preactivated BM T cells completely inhibited NK cell development even when the T cells were separated from the NK-LTBMCs by transwells. Depletion of IL-2 by activated T cells was not the mechanism of the negative regulation because anti-CD3 mAb added to the cultures inhibited the generation of NK cells even in the presence of 10-fold higher concentrations of exogenous IL-2 than that used in controls. Medium from cultures in which suppression had occurred was also suppressive, suggesting that one or more soluble factors released in the medium was responsible. That this effect was exerted on NK cell development from precursors was indicated by the finding that T cell-conditioned medium stimulated proliferation of mature NK cells. In our experimental conditions, monoclonal antibodies to IL-10, IL-13, transforming growth factor-beta, and tumor necrosis factor receptor failed to reverse the inhibitory effect.

Characterization of the stage in natural killer cell development in 14.5-day mouse fetal liver using adult bone marrow stroma.

Nonstimulated fetal liver (FL) from 14.5-day gestation mice had no natural killer (NK) cell activity and <3% expressed NK1.1. Even after short-term (3-4 day) culture of FL with the late-acting cytokines, interleukin (IL)-15 or IL-2, little or no NK activity was detected. However, longer-term (13 day) culture with IL-2 plus stroma derived from bone marrow (BM) of adult mice, resulted in extensive proliferation and differentiation to mature NK cells. Cell numbers began to increase after 4 days, and by day 13, they had increased 40-fold and 69% of the cells were NK1.1+ with high NK activity and 5%-10% were NK1.1- B220+. With stroma, but no IL-2, equivalent proliferation occurred, but differentiated cells were predominantly NK1.1- B220+, not NK cells. Culture for 13 days without stroma, but with either IL-2, IL-15, FLTK3-ligand (L) or stroma-conditioned medium, resulted in less than fivefold expansion, and minimal NK activity. Culture with combinations of FLTK3-L or ckit-L plus IL-15 or IL-2 increased both cell number and NK activity, but the increase in cell number was less than that seen with stroma plus IL-2. By limiting dilution assay on stroma plus IL-2, the precursor frequency was 1/(2660+/-292) whole FL cells and the absolute number, but not the frequency, increased during culture on stroma without IL-2. The NK cell progenitors were found in sorted NK1.1- and Sca-1+ c-kit+ lineage- subpopulations at a frequency of 1/(156+/-52.5). Together, these data suggest that the NK lineage cells in FL are primarily in early stages of development. They are highly proliferative, respond to early acting cytokines and express stem cell markers.

Granulocytic colonies on macrophage-coated cellulose acetate membranes (CAM) in S1/S1dmice.

The kinetics of formation of granulocytic colonies on macrophage-coated cellulose acetate membranes (CAM) were investigated in Sl/Sld mice, which have a genetic defect in their hematopoietic microenvironment. CAM were left in the peritoneal cavity of mice for 7 days to become coated with peritoneal cells. The mice were then sublethally irradiated to suppress endogenously derived granulocytic colonies, and bone marrow cells were injected i.p. within 1--2 hr irradiation. During the next 7 days, peroxidase-positive granulocytic colonies appeared on CAM. The number of colonies on CAM in Sl/Sld mice at 7 days was consistently less than that from littermate +/+ mice. Since it appeared that the CAM in the Sl/Sld provided an inferior microenvironment to that of the +/+, the effect of raising a CAM in one genotype and transferring it to another was investigated. CAM raised in +/+ or in Sl/Sld were transferred to an irradiated +/+ or Sl/Sld following which cells were injected into the secondary host and colonies determined subsequently. The number of granulocytic colonies on CAM was influenced primarily by the type of secondary host. Colony number was consistently less in the secondary Sl/Sld host than in the +/+ host whether the primary host was a Sl/Sld or a +/+. These observations confirm the concept of a microenvironmental defect in Sl/Sld mice. In addition, the studies indicate that the microenvironment on a CAM can be modified by a secondary host and suggest that "remodeling" of CAM may be a continuous, kinetic process.

Effect of rabbit antimouse brain serum on marrow cells capable of curing W/Wv mice.

The W/Wv mouse has a recessively inherited defect in hematopoietic stem cells (HSC) but can be cured of its hematopoietic abnormalities by infusion of marrow from a co-isogeneic, +/+ mouse. The "curative" cell for the W/Wv is thought to be a subcompartment of the HSC that is capable of forming hematopoietic spleen colonies (CFU-S) in irradiated mice. The curative HSC must have a very high proliferative potential and it is known that HSC with variable degrees of proliferative potential are found within the CFU-S compartment. Rabbit antimouse brain serum (RAMBS) was used to treat +/+ marrow and its effect upon CFU-S and upon curative cells was compared with the effect of normal rabbit serum (NRS) or of sham treatment. CFU-S were reduced to 70%-79% of control by NRS and to 8%-9% by RAMBS. Curative cells for the W/Wv were not detectably reduced by NRS; they were reduced by RAMBS, but to only approximately 20%-30% of control. Thus, it appeared to a certain degree that RAMBS spared HSC with a high proliferative potential when compared with its effect on the entire CFU-S compartment.