A phase I study of CR002, a fully-human monoclonal antibody against platelet-derived growth factor-D.

OBJECTIVE: To investigate the safety, pharmacokinetics (PK), binding activity and immunogenicity of CR002, a human monoclonal antibody (mAb) directed against platelet-derived growth factor-D (PDGF-D), administered as a single intravenous (i.v.) infusion over a range of doses. SUBJECTS: 40 healthy male subjects received increasing doses of CR002 at 0.3, 1, 3, 10, 30 mg/kg or placebo. METHOD: This was a randomized, double-blind, placebo-controlled, dose-escalation Phase I study. The trial had a duration of 90 days, with dosing on Day 1 and follow-up visits on Days 2, 4, 7, 14, 21, 30, 45 and 90. Serum was collected for PK, binding activity and immunogenicity analysis at screening and up to Day 90. Safety was recorded throughout the study by performing laboratory tests, recording vital signs and electrocardiograms (ECGs), by monitoring the occurrence of adverse events (AEs). The use of concomitant medications was also recorded. RESULTS: All 40 subjects received CR002 or placebo, and completed the trial. No dose-limiting toxicities (DLTs) occurred, the maximum tolerated dose (MTD) was not reached and was estimated as > 30 mg/kg. There were no deaths during this study and no SAEs or other significant AEs reported. The most frequent drug-related treatment-emergent AE (TEAE) was headache in 4 of 30 subjects (13.3%) in the CR002 group vs. 0 of 10 subjects in the placebo group. CR002 exhibited linear PK parameters, had a long half-life (t1/2 in the range 15.5 â 48.1 days) and a volume of distribution at steady state in the range 4.7 â 6.5. Free PDGF-D in the serum bound to CR002 in a reversible manner, as shown in the lowest dose cohort. However, levels of total circulating PDGF-D remained constant throughout the study. There were no anti-CR002 antibodies detected in subjects dosed with CR002. CONCLUSIONS: CR002 was safe and well-tolerated at all doses tested as a single i.v. administration. The MTD was estimated to be above 30 mg/kg, the highest dose tested. CR002 had a long half-life, low clearance and a limited tissue distribution. Although total levels of PDGF-D at all dose levels remained relatively constant, there was no detectable circulating free PDGF-D after CR002 administration. At the lowest CR002 dose tested (0.3 mg/kg), PDGF-D was detectable again by Day 21 and the levels increased near to pre-infusion levels by Day 90. In this study, CR002 was not immunogenic during the 90-day study period.

Human fibroblast growth factor 20 (FGF-20; CG53135-05): a novel cytoprotectant with radioprotective potential.

The aim was to evaluate the radioprotective properties of recombinant human fibroblast growth factor 20 (FGF-20; CG53135-05) in vitro and in vivo and to examine its effects on known cellular pathways of radioprotection. Relative transcript levels of the cyclooxygenase 2 (COX2), Mn-super oxide dismutase (SOD), CuZn-SOD, extracellular (EC)-SOD, nuclear respiratory factor 2 (Nrf2), glutathione peroxidase 1 (GPX1) and intestinal trefoil factor 3 (ITF3) genes, which are involved in radiation response pathways, were assessed by reverse transcriptase-polymerase chain reaction (RT-PCR) in NIH/3T3, IEC18, CCD-18Co, CCD-1070sk and human umbilical vein endothelial cells (HUVEC) cells exposed to FGF-20. Activation of the radioprotective signal transduction pathways initiating with the serine/threonine Akt kinase and the extracellular regulated kinase (ERK) were analysed. Levels of intracellular hydrogen peroxide and cytosolic redox potential were also measured in irradiated and unirradiated cells in the presence or absence of FGF-20. The effects of FGF-20 on cell survival in vitro following ionizing radiation were evaluated using clonogenic assays. To test the potential activity of FGF-20 as a radioprotectant in vivo, mice were administered a single dose of FGF-20 (4 mg kg(-1), intraperitoneally (i.p.) 1 day before lethal total-body irradiation and evaluated for survival. In vitro exposure to FGF-20 increased expression of the Nrf2 transcription factor and oxygen radical scavenging enzymes such as MnSOD, activated signal transduction pathways (ERK and Akt) and resulted in increased survival of irradiated cells in vitro. FGF-20 treatment also resulted in a concomitant reduction in intracellular levels of injurious reactive oxygen species (ROS) following acute ionizing irradiation. Finally, prophylactic administration of FGF-20 to mice before potentially lethal, whole-body X-irradiation led to significant increases in overall survival. FGF-20 reduced the lethal effects of acute ionizing radiation exposure in cells by up-regulating important signalling and free radical scavenging pathways. Survival-sparing effects of FGF-20 prophylaxis in acutely irradiated mice presumably are elicited by comparable mechanisms. These results indicate that FGF-20, has significant radioprotective attributes with potential applications in clinical and non-clinical exposure settings.

Huntingtin interacting protein 1 Is a clathrin coat binding protein required for differentiation of late spermatogenic progenitors.

Huntingtin-interacting protein 1 (HIP1) interacts with huntingtin, the protein whose gene is mutated in Huntington's disease. In addition, a fusion between HIP1 and platelet-derived growth factor beta receptor causes chronic myelomonocytic leukemia. The HIP1 proteins, including HIP1 and HIP1-related (HIP1r), have an N-terminal polyphosphoinositide-interacting epsin N-terminal homology, domain, which is found in proteins involved in clathrin-mediated endocytosis. HIP1 and HIP1r also share a central leucine zipper and an actin binding TALIN homology domain. Here we show that HIP1, like HIP1r, colocalizes with clathrin coat components. We also show that HIP1 physically associates with clathrin and AP-2, the major components of the clathrin coat. To further understand the putative biological role(s) of HIP1, we have generated a targeted deletion of murine HIP1. HIP1(-/-) mice developed into adulthood, did not develop overt neurologic symptoms in the first year of life, and had normal peripheral blood counts. However, HIP1-deficient mice exhibited testicular degeneration with increased apoptosis of postmeiotic spermatids. Postmeiotic spermatids are the only cells of the seminiferous tubules that express HIP1. These findings indicate that HIP1 is required for differentiation, proliferation, and/or survival of spermatogenic progenitors. The association of HIP1 with clathrin coats and the requirement of HIP1 for progenitor survival suggest a role for HIP1 in the regulation of endocytosis.

Fuc-TVII is required for T helper 1 and T cytotoxic 1 lymphocyte selectin ligand expression and recruitment in inflammation, and together with Fuc-TIV regulates naive T cell trafficking to lymph nodes.

To determine how the alpha(1,3)fucosyltransferases Fuc-TIV and Fuc-TVII, and the selectin ligands they control may contribute to the adaptive immune response, contact hypersensitivity (CHS) was characterized in mice deficient in either or both enzymes. We find a substantial CHS deficiency in Fuc-TVII(-/-) mice, and a complete deficiency in Fuc-TIV(-/-)/Fuc-TVII(-/-) mice. These defects are not accounted for by alterations in the number or function of epidermal Langerhans cells required for cutaneous antigen processing and presentation. By contrast, defective CHS in Fuc-TVII(-/-) mice or Fuc-TIV(-/-)/Fuc-TVII(-/-) mice is attributed in part to prominent, or nearly complete deficiencies, respectively, in the complement of naive T lymphocytes available in lymph nodes for antigen-dependent activation, expansion, differentiation, and dissemination. Fuc-TVII deficiency also deletes expression of E- and P-selectin ligands by Th1 and T cytotoxic 1 (Tc1) lymphocytes, annuls T cell trafficking to inflamed cutaneous sites in vivo, and thereby controls an essential component of the efferent phase of the cutaneous immune response. These observations indicate that collaborative contributions of Fuc-TIV and Fuc-TVII to L-selectin ligand synthesis, and to lymphocyte recruitment, are requisite components of the primary cellular immune response, and assign an essential role to Fuc-TVII in control of E- and P-selectin ligand expression by Th1 and Tc1 lymphocytes.

The alpha(1,3)fucosyltransferases FucT-IV and FucT-VII exert collaborative control over selectin-dependent leukocyte recruitment and lymphocyte homing.

E-, P-, and L-selectin counterreceptor activities, leukocyte trafficking, and lymphocyte homing are controlled prominently but incompletely by alpha(1,3)fucosyltransferase FucT-VII-dependent fucosylation. Molecular determinants for FucT-VII-independent leukocyte trafficking are not defined, and evidence for contributions by or requirements for other FucTs in leukocyte recruitment is contradictory and incomplete. We show here that inflammation-dependent leukocyte recruitment retained in FucT-VII deficiency is extinguished in FucT-IV(-/-)/FucT-VII(-/-) mice. Double deficiency yields an extreme leukocytosis characterized by decreased neutrophil turnover and increased neutrophil production. FucT-IV also contributes to HEV-born L-selectin ligands, since lymphocyte homing retained in FucT-VII(-/-) mice is revoked in FucT-IV(-/-)/FucT-VII(-/-) mice. These observations reveal essential FucT-IV-dependent contributions to E-, P-, and L-selectin ligand synthesis and to the control of leukocyte recruitment and lymphocyte homing.

Cell interaction molecules utilized in bone marrow.

Many aspects of blood cell formation can now be modeled in culture and rapid progress is being made in understanding how blood cell precursors interact with unique components of their environment. This brief review considers some cell interaction molecules that may be important for controlling the position of cells within, as well as their egress from, bone marrow.

The role of estrogen receptors and androgen receptors in sex steroid regulation of B lymphopoiesis.

Several observations suggest that sex steroids might participate in steady state regulation of B lymphopoiesis. B cell precursors decline dramatically in bone marrow of pregnant or estrogen-treated mice. Reciprocally, the same cell populations are increased in hypogonadal mice or male castrates. Estrogen treatment of hypogonadal mice reduced precursors to normal. However, questions remain about which hormones and receptors are the most important. Furthermore, these observations need to be reconciled with advances regarding new sex steroid receptors. We have now characterized B lymphopoiesis in androgen receptor-deficient testicular feminization (Tfm) mice. Testicular feminization mice had substantially elevated numbers of B cell precursors in the bone marrow and B cells in the spleen as compared with wild-type mice. The importance of one estrogen receptor (ER alpha) was evaluated in gene-targeted mice, and B cell precursors were found to be within the normal range. Our previous studies indicated that hormone receptors in stromal cells may be important for estrogen-mediated suppression of B lymphopoiesis. We now show that estrogen-mediated inhibition of B cell precursor expansion in culture was blocked by a specific estrogen receptor antagonist (ICI 182,780). Stromal cells derived from ER alpha-targeted bone marrow were fully estrogen responsive. RT-PCR analyses of these stromal cells revealed splice-variant transcripts of ER alpha, as well as message for a recently discovered estrogen-binding receptor, ER beta. Thus, androgens may normally inhibit B lymphopoiesis through the androgen receptor, whereas estrogens might utilize one or more receptors to achieve the same physiologic response.

Two models of murine B lymphopoiesis: a correlation.

During B cell genesis in mouse bone marrow (BM), precursor B cells pass through a series of developmental stages that have been defined by changes in expression of various marker molecules. The use of dissimilar phenotypic criteria in different laboratories, however, has led to the formulation of disparate models of B lymphopoiesis not fully reconciled with one another. We have directly compared two such models, one based on expression of intracellular mu heavy chain of IgM (c mu) and terminal deoxynucleotidyl transferase (TdT), the other monitoring cell surface leukosialin (CD43), heat-stable antigen (HSA; CD24) and the ectopeptidase BP-1. Each model uses cell surface B220 glycoprotein (CD45RA) to denote the B cell lineage. We have examined the cellular composition of four sorted BM fractions by immunofluorescent labeling of CD43, HSA and BP-1, using immunofluorescence microscopy of cytocentrifuged fractions to quantitate precursor B cell populations expressing either c mu or TdT. The results reveal a range of B cell differentiation stages within individual sorted BM fractions, providing a cross-reference between these two analytical methods and contributing to a unified model of B cell development in mouse BM.

Expression of bright at two distinct stages of B lymphocyte development.

The B cell regulator of Ig heavy chain transcription (Bright) is a DNA-binding protein that was originally discovered in a mature Ag-specific B cell line after stimulation with IL-5 and Ag. It binds to the intronic heavy chain enhancer and 5' of the V1 S107 family V(H) promoter. Several studies suggested that Bright may increase transcription of the heavy chain locus, and expression in cell lines was limited to those representing mature B cells. We have now analyzed normal hemopoietic tissues for the expression of Bright during B lymphocyte differentiation. We expected to find Bright expression in a subset of mature spleen cells, but also observed Bright in a subset of normal B lymphocytic progenitors in both adult bone marrow (BM) and in fetal liver as early as day 12 of gestation. Bright was also expressed in the small percentage of CD4(low) cells in the thymus that are newly arrived from the BM and are not yet committed to the T lymphocyte lineage, but was not observed at later stages of T cell differentiation in either the spleen or thymus. Bright mRNA was not detected in the immature B lymphocytes that initially populate the spleen after migration from the BM. In addition, new splice variants of Bright were observed in fetal tissues. Thus, Bright expression is highly regulated in normal murine lymphocytes and occurs both early and late during B cell differentiation. These findings may have important implications for the function of Bright in regulating Ig transcription.

Stromal cell modulation of negative regulatory signals that influence apoptosis and proliferation of B lineage lymphocytes.

The bone marrow microenvironment influences whether a given B cell proliferates, differentiates, or undergoes apoptosis. In this report, we demonstrate that apoptosis of primary murine B lymphocyte precursors can be regulated either positively or negatively by stroma. Several stromal lines that support lymphocyte outgrowth suppressed the spontaneous apoptosis of pre-B cells by as much as 90%. Direct contact with stromal cells more effectively protected lymphocytes than did stromal cell-CM or a collection of recombinant cytokines. In contrast, one unique stromal cell clone actually induced lymphocyte apoptosis, and a second line appeared inert. A survey of adherent cell lines suggested that expression of life-sparing molecules is widespread but not ubiquitous. Experiments with neutralizing Abs to CD44, vascular cell adhesion molecule-1 (VCAM-1), CD9, intercellular adhesion molecule-1 (ICAM-1), or ICAM-2 suggested that these interaction molecules do not deliver short-term survival signals to B cell precursors. Of particular interest, direct interaction with lymphocyte-supportive stromal cells minimized the negative regulatory effects of IL-1alpha, and a glucocorticoid, but not IFN-beta or PGE2. These results demonstrate that the effect of negative regulators depends upon the context in which these signals are presented. As molecules that influence B lymphopoiesis are better defined, it will be important to consider the role of each in combination with other stimuli.

Reassessment of germline heavy chain transcripts from two murine VH families.

While expression of functional heavy chain immunoglobulin mRNA requires rearrangement of variable (VH), diversity (D) and (JH) gene segments, these individual gene segments can be transcribed prior to their rearrangement. It has been proposed that the resulting germline, or sterile, transcripts play an important role in the rearrangement process because strong correlations between rearrangement frequency and sterile transcript levels have been observed in some studies. Murine VH genes have been grouped into families on the basis of coding sequence homology. VH families rearrange in a developmentally regulated manner, so that rearrangements of genes from several VH families are detected earlier than rearrangements of J558 family genes. Paradoxically, the only VH family for which sterile transcripts have been documented is the J558 family. We used RT-PCR analyses to ask whether sterile transcripts from other VH families could be detected in fetal liver samples prior to their rearrangement. While J558 family germline transcripts were easily detected, no sterile transcripts were observed from the S107 family. Our studies also revealed the ability of small quantities of degraded genomic DNA to nonspecifically prime cDNA synthesis, emphasizing the need for caution in interpreting RT-PCR data in which family-specific oligos are used for cDNA production. These results cast doubt on the idea that sterile transcripts are required for V(H)DJ(H) rearrangement.

Estrogen suppresses stromal cell-dependent lymphopoiesis in culture.

Numbers of pre-B cells change dramatically and reciprocally in response to estrogen levels in mice, suggesting that normal lymphopoiesis may be under hormonal control. However, little is known of the mechanisms involved in this process. We found that estrogen receptor mRNA was detectable by RT-PCR in lymphocyte supporting stromal cells as well as B lymphocyte precursors. Unlike glucocorticoids, estrogen did not induce apoptosis in isolated B lineage lymphocytes or interfere with their responsiveness to IL-7 in semisolid agar. Estrogen did inhibit clonal expansion of B cell precursors in a limiting dilution-type assay when the lymphocytes were cultured on a stromal cell clone. In other experiments, B cell precursors at particular stages of differentiation were isolated by cell sorting and cocultured with stromal cells for 4 days. This revealed that some subsets were more sensitive to an estrogen-containing environment than others. Although numbers of recovered cells were greatly reduced, the remaining lymphocytes had undergone relatively normal differentiation. The surviving population was enriched in cells that had acquired cytoplasmic mu chains, BP-1 Ag, and clonability with IL-7. Hormone-mediated inhibition occurred in serum and phenol-red free medium, and in cultures replete with IL-7. Direct contact between stromal cells and lymphocytes was not required. Furthermore, suppression resulted when stromal cells alone were treated with the hormone. These findings indicate that estrogen may regulate B lymphopoiesis via its influence on the microenvironment and that estrogen-induced stromal cell genes merit further study.

Pregnancy: a clue to normal regulation of B lymphopoiesis.

The large-scale production of lymphocytes in the bone marrow reflects a delicate balance between positive and negative regulatory signals. For instance, interleukin 7 (IL-7) provides a positive signal, and appears to be both essential and limiting in the mouse. However, much less is understood concerning the negative molecular signals that may limit the output of lymphocytes. Here, Paul Kincade and colleagues discuss how a chance observation with pregnant mice revealed that sex steroids can act as negative regulators of B lymphopoiesis, and may do so under normal steady-state conditions.

Increased B lymphopoiesis in genetically sex steroid-deficient hypogonadal (hpg) mice.

Interleukin 7 (IL-7) responsive B lineage precursors were greatly expanded in genetically hypogonadal female (HPG/Bm-hpg/hpg) mice that have a secondary deficiency in gonadal steroidogenesis. Estrogen replacement in these mice resulted in a dose-dependent reduction in B cell precursors. More modest increases were documented in genetically normal mice that were surgically castrated. These findings complement other recent observations that B lymphopoiesis selectively declines in pregnant or estrogen-treated animals. Sex steroids have long been known to influence such disparate processes as bone physiology and tumor growth, in addition to their importance for reproductive function. We now show that these hormones are important negative regulators of B lymphopoiesis.

Sex hormones as negative regulators of lymphopoiesis.

B lymphocytes, together with cells of seven other lineages, are made in large numbers from precursors in the bone marrow. Using cell culture models and recombinant proteins, progress has been rapid in identifying cytokines which could potentially regulate the proliferation, differentiation and migration of B-cell precursors. However, we still know little about molecular mechanisms which are important for maintaining steady-state conditions in vivo. B lymphopoiesis is severely diminished during pregnancy in normal mice and this provided a clue that sex hormones might be important negative regulators. Administration of estrogens alone, or in combination with progesterone, preferentially suppressed IL-7 responding cells and their progeny in bone marrow. There is precedent for these observations in the thymus, which transiently involutes during pregnancy, and also atrophies following estrogen treatment. The actual mechanism(s) through which sex steroids influence lymphopoiesis remain unclear, but cell culture experiments should be informative about potential interactions between hormones, the bone marrow microenvironment, and lymphocyte precursors. These findings raise a number of other important issues. For example, we need to learn if sex steroids are produced and/or concentrated locally within the marrow, if human lymphopoiesis is sensitive to these hormones, and if production of lymphocytes can be augmented in aging and in immunodeficiency by hormone manipulation.

Suppression of B lymphopoiesis during normal pregnancy.

We describe a dramatic reduction in numbers and activity of committed B lymphocyte precursors in the bone marrow of normal pregnant mice. Changes in cells responsive to IL-7 were evident as early as 6.5 d of pregnancy and values were < 10% of normal at parturition. B lineage precursors, identified by display of CD45R and absence of surface IgM, were also substantially depressed, and subpopulations representing different stages in the B lineage were assessed by three-color flow cytometry. Early pro-B cells are medium to large in size and have been previously characterized by low expression of the heat-stable antigen (HSA). This category of cells was not reduced, and in fact may have been slightly elevated, during pregnancy. In contrast, all subsequent populations of B lineage precursors, defined by patterns of expression of heat-stable and CD43 antigens, were substantially depressed. The immediate precursors of B cells (small pre-B cells) were identified by small size, expression of CD45R, absence of CD43, and lack of surface IgM. These were the most reduced of any phenotypically defined population in bone marrow. Numbers of newly formed B cells, characterized by the presence of sIgM, but not sIgD, were also diminished. However, B cells with a mature phenotype (sIgM+, sIgD+) were present in normal to somewhat elevated numbers. Mitogen-responsive B cells clonable in a semisolid agar assay were not significantly affected. A bromodeoxyuridine (BrdU) labeling technique was used to evaluate mitotic activity, which revealed an increased proportion of long-lived lymphocytes in the bone marrow of pregnant mice. These observations indicate that B lymphopoiesis is markedly downregulated during pregnancy and that all precursor populations beyond the early pro-B cell stage are affected. The pregnancy-related changes in bone marrow were selective for B lineage precursors, as cells expressing myeloid and erythroid markers were not reduced. In spleen, evidence was obtained for partial depletion of one subset of B cells. These cells, which have been reported to be recent immigrants from marrow, are characterized as having high levels of sIgM and HSA. Changes in other major B lymphocyte subsets in the spleen were less remarkable. When considered with results from the BrdU labeling procedure, the findings indicate that both production and export of lymphocytes from marrow may be substantially decreased. Numbers of B cell precursors were higher in postpartum animals whose litters were removed at birth, suggesting that lactation may prolong regeneration of lymphocyte production.(ABSTRACT TRUNCATED AT 400 WORDS)

Tight conjugate formation is not always required for natural killer cell-mediated lysis.

We have utilized several clonal cell lines, derived from the murine lymphoma ASL1w, to investigate the early events in NK-mediated lysis. The studies described here examine the relationship between NK recognition, NK cell:tumor cell conjugate formation, and NK-mediated lysis. The AW4F and AW4D tumor lines were susceptible to NK-mediated lysis and efficiently inhibited NK recognition in competitive inhibition assays, whereas the AW5J tumor, which is relatively resistant to NK-mediated lysis, did not. In contrast, the AW5E tumor was NK resistant but inhibited NK recognition almost as well as the NK-sensitive tumors, suggesting that it was deficient in a postbinding event required for NK-mediated lysis. These findings demonstrate a correlation, with one exception, between the susceptibility of the ASL1w-derived tumor lines to NK-mediated lysis and their ability to inhibit NK recognition. In contrast, there was no apparent correlation between tight conjugate formation, as assessed in three independent target binding assays, and the susceptibility of these tumors to NK-mediated lysis, showing that tight conjugate formation is not required for either efficient NK recognition or lysis.

In vitro growth of bone marrow-resident T cell precursors supported by mast cell growth factor and IL-3.

The growth requirements of bone marrow-resident cells that are able to differentiate along the T cell lineage (pre-T cells) have not been well established. We recently have shown that the T cell-derived lymphokine IL-3 is able to maintain pre-T cells in vitro for at least 2 weeks. However, in our initial studies, we were not able to ascertain whether IL-3 induced pre-T cell growth during culture, or whether IL-3 simply maintained the viability of these progenitors. To address this issue, we used a multiple dose assay system to assess the level of pre-T cell activity (thymic repopulation) in a selected population of bone marrow cells (CD3-, Thy-1.2+) both before and after culture in IL-3. In addition, we tested the potential role of mast cell growth factor (MGF) in the growth and maintenance of pre-T cells in vitro. The results of these studies showed that IL-3 produced a modest, but consistent increase in the pre-T cell activity during culture. Culture of CD3-, Thy-1.2+ bone marrow cells in MGF also resulted in an increase in the total amount of detectable pre-T cell activity among the cultured cells. The most dramatic increases in pre-T cell activity, however, were induced by the culture of the selected marrow cells in both MGF and IL-3. Cultures supplemented with both cytokines produced net increases in pre-T cell activity of 40- to 75-fold after 10 days of culture. Because the increases in pre-T cell activity were not accompanied by observable increases in the size of thymic colonies produced by the pre-T cells, the increased levels of pre-T cell activity appeared to result from increases in pre-T cell numbers during culture. Thus, in addition to the other activities ascribed to MGF, this cytokine displays pre-T cell growth factor activity and can synergize with IL-3 in that capacity. The use of MGF in conjunction with IL-3 provides the best system described to date for the propagation of pre-T cells in primary bone marrow cell cultures.

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.