NK Cell Ontogeny Research Articles

Stochastic Modeling of Hematopoietic Stem and Progenitor Cell Barcoding Data from Rhesus Macaques Challenges the Classic Model of Hematopoiesis

The classical model of hematopoiesis states that differentiation proceeds from hematopoietic stem cells (HSC) to mature blood cells via specified multipotent and bipotent progenitors, such as the common myeloid progenitor (CMP), common lymphoid progenitor (CLP), erythrocyte-megakaryoctye progenitor and granulocyte-monocyte progenitor. However, recent studies question this assumption and suggest that these intermediates are neither required nor prevalent. As an example, analyses of binarized data from murine barcoding experiments (Perie et al, Cell Reports, 2014) raise the possibility that hematopoiesis progresses via a random loss of potentials rather than discrete steps. Additionally, Notta et al (Science, 2015) showed that oligopotent progenitor cells form only a negligible component in the hierarchy by studying the distribution of progenitors in human marrow, leading them to infer that HSC and earliest multipotent progenitors differentiate directly into unipotent cells. Although these data challenge fundamental beliefs, the quantitative contributions of HSC and progenitors to cell lineages could not be tracked in individual mice or persons over time.We developed a statistical method to infer the rates and probabilities of cell fate decisions in a class of stochastic branching models and used this to analyze sequence data from a rhesus macaque transplanted with lentivirally barcoded CD34+ HSC and progenitor cells. The macaque's blood granulocytes (Gr), monocytes (Mo), B cells, T cells, and NK cells were tracked over 30 months. Our quantitative framework is based on computing correlations between pairs of observable mature blood cell types across all independently barcoded lineages. The method also accounts for experimental uncertainties intrinsic to blood sampling, cell purification and PCR amplification.Specifically, our approach relies on a loss function estimator that minimizes residuals between empirical pairwise correlations across barcode lineages and analytical model-based correlations derived generally for continuous-time multi-type branching processes. We integrate over sampling distributions accounting for noise in experimental protocol and CBC counts. Candidate models represent possible hematopoietic structures and allow an arbitrary number of progenitor and mature cell types descended from each HSC. We identify best-fitting fate decision rates and initial marking levels with corresponding confidence intervals via nonlinear least squares and can assess whether a given model is statistically consistent with the data. This is the first statistical method to our knowledge for fitting stochastic models of hematopoiesis to lineage barcoding time-series, and together with the rhesus macaque data, enables quantitative analysis of in vivo dynamics in a large animal model.Using this new approach, we confirmed the major finding in Wu et al. (Cell Stem Cell, 2014) of a distinct NK cell ontogeny, i.e., that CD16+ blood NK cells do not overlap in origin with T and B cell lineages. We estimate that 13.9% of HSC and 86.1% of progenitors were initially barcoded, which is consistent with the finding by Wu that the percentage of blood cells expressing GFP stabilized at 13% after 6 months. Additionally, we estimate that HSC self-renew approximately once every 12 weeks, which is consistent with the range estimated in previous primate studies based on telomere studies (Shepherd, Blood, 2007). These initial analyses help validate our method. We then showed that Gr and Mo cells derive from a common precursor in vivo (correlation ρ ≈.9 across time). We also estimated progenitor differentiation rates and showed that Gr and Mo cells are produced up to 10- to 100-fold more rapidly than T, B and NK cells, and that each progenitor committed to the Gr/Mo lineage (i.e., CFUGM) produces thousands of mature cells per day. Importantly, we tested models requiring an ordered differentiation through defined intermediaries and found that they did not suitably fit the data compared to models allowing for non-restricted pathways. Together these analyses challenge the classic model of blood cell differentiation and provide new insights into the structure of hematopoiesis. DisclosuresDunbar:GSK/Novartis: Research Funding.

Insights into Clonal Relationships of Putative Adaptive Natural Killer Cells (NK) in Humans, Via Mapping of Somatic Piga Mutations in Patients with Paroxysmal Nocturna Hemoglobinuria (PNH)

NK cells play a central role in innate immunity, specifically in tumor surveillance and microbial pathogen control. Recent murine models and human studies have identified subsets of NK-cells with apparent memory cell function, strongly linked to CMV infection in humans and termed "adaptive" NK. Our recent clonal tracking studies following autologous hematopoietic stem cells transplantation (HSCT) of genetically-barcoded CD34+ cells in macaques revealed distinct clonal ontogeny of a subset of NK cells within the primate equivalent of the human CD56dim population (Wu et al., Cell Stem Cell, 2014), with little clonal overlap with T-, B-lymphoid or myeloid cells, suggesting a separate precursor pool for this NK subtype. Peripheral blood CD 56bright -NK cells have been previously hypothesized to be precursors for the main population of circulating cytotoxic CD56dim cells. To further investigate NK-cell ontogeny and clonal relationships in humans we took advantage of naturally-occurring somatic mutations in the X-linked phosphatidylinositol glycan class A (PIGA) gene in patients with the hemolytic disorder paroxysmal nocturnal hemoglobinuria. This gene codes for an enzyme required for cell surface localization of glycosylphophatidylinositol (GPI)-anchored proteins, and thus loss of function mutations result in hematopoietic cells lacking GPI-anchored proteins, and red cell hemolysis. PNH patients have not been reported to have immune dysfunction and can have stable disease for many years. Membrane bound GPI anchors can be detected on any cell via flow cytometry using a labeled inactive aerolysin (FLAER), and serves as a marker for the fraction of cells comprising the PIGA mutant (GPI negative) clonal compartment. The PNH clone sizes contributing to peripheral blood cells are variable in but can reach almost 100% in some patients, and can be stable over decades. We selected 9 PNH patients with GPI negative granulocytes ranging from 5% to 98% and a median time from diagnosis of 43.7 months (15-100) for this study. NK cells were defined as CD56+/CD16+/CD3-/CD20- lymphocytes. We observed disproportionally fewer GPI negative NK cells compared to granulocytes (Fig 1), with the discrepancy most marked the major peripheral blood CD56dim population (mean 65% vs 25% GPI negative granulocytes, p = 0.0028, paired t-test), in contrast to 46% GPI negative cells in the CD56bright population (p=0.057). Due to the prolonged life span of memory T and B cells, fewer GPI negative B and particularly T-lymphocytes have been reported in PNH patients. In our cohort 3.4% of CD3+ T-cells and 13.2% of CD20+ B-cells were GPI negative (p=0.0005 and 0.0014, respectively versus granulocytes). Compared to the NK subsets, the CD56bright population showed the most significant differences (p = 0.0063 versus CD3 and p=0.0151 versus CD20). To further characterize the phenotype of the GPI positive versus negative CD56dim cells, we analyzed cells co-expressing either the terminal differential marker CD57, the inhibitory receptor NKG2A, or activating receptor NKG2C for FLAER positivity. Prior studies have suggested that the human CMV-linked adaptive NK subset is CD57+, NKG2A- and NKG2C+. The NKG2C+ CD 56dim population was highly enriched for GPI positive cells (p=0.0024 vs granulocytes, Fig 1). This profile was most prominent in CMV-IgG positive patients who had also significantly more GPI positive CD 56dim/CD57+ cells compared to granulocytes (p=0.008). Interestingly, one CMV positive patient (#5) had a complete lack of NKG2C expression, most likely due to homozygous loss of function mutation, and this patient had almost 100% GPI negative NK cells, matching his neutrophil pattern. Compared to granulocytes, NKGA2A+ or CD57+ positive CD56dim cells were also mostly GPI negative (p=0.0081 and 0.028).Circulating NK cell turnover has been estimated to be about 14 days. The PNH patients studied had documented clonal PIG-A mutations for many years. Our observation that the majority of CD56dim NK cells, specifically the NKG2C subset, are not progeny of the same progenitors producing CD56bright NK cells or myeloid cells based on clonal disparity regarding the PNH clone is suggestive of an independent, very long-lived or self-renewing NK cell progenitor for a CMV-linked CD56dim/CD57+/NKG2C+ memory NK cell compartment. These observations provide novel further insights into the human adaptive NK cell subset. [Display omitted] DisclosuresWinkler:Novartis: Research Funding; GSK: Research Funding. Townsley:Novartis: Research Funding; GSK: Research Funding.

Novel molecular mechanism for generating NK-cell fitness and memory.

The mammalian immune system has been traditionally subdivided into two compartments known as the innate and the adaptive. T cells and B cells, which rearrange their antigen-receptor genes using the RAG recombinase, comprise the adaptive arm of immunity. Meanwhile, every other white blood cell has been grouped together under the broad umbrella of innate immunity, including NK cells. NK cells are considered innate lymphocytes because of their rapid responses to stressed cells and their ability to develop without receptor gene rearrangement (i.e. in RAG-deficient mice). However, new findings implicate a critical function for RAG proteins during NK-cell ontogeny, and suggest a novel mechanism by which controlled DNA breaks during NK-cell development dictate the fitness, function, and longevity of these cells. This review highlights recent work describing how DNA break events can impact cellular differentiation and fitness in a variety of cell types and settings.

Peripheral natural killer cell maturation depends on the transcription factor Aiolos.

Natural killer (NK) cells are an innate lymphoid cell lineage characterized by their capacity to provide rapid effector functions, including cytokine production and cytotoxicity. Here, we identify the Ikaros family member, Aiolos, as a regulator of NK-cell maturation. Aiolos expression is initiated at the point of lineage commitment and maintained throughout NK-cell ontogeny. Analysis of cell surface markers representative of distinct stages of peripheral NK-cell maturation revealed that Aiolos was required for the maturation in the spleen of CD11b(high)CD27(-) NK cells. The differentiation block was intrinsic to the NK-cell lineage and resembled that found in mice lacking either T-bet or Blimp1; however, genetic analysis revealed that Aiolos acted independently of all other known regulators of NK-cell differentiation. NK cells lacking Aiolos were strongly hyper-reactive to a variety of NK-cell-mediated tumor models, yet impaired in controlling viral infection, suggesting a regulatory function for CD27(-) NK cells in balancing these two arms of the immune response. These data place Aiolos in the emerging gene regulatory network controlling NK-cell maturation and function.

Role of Common‐Gamma Chain Cytokines in NK Cell Development and Function: Perspectives for Immunotherapy

NK cells are components of the innate immunity system and play an important role as a first-line defense mechanism against viral infections and in tumor immune surveillance. Their development and their functional activities are controlled by several factors among which cytokines sharing the usage of the common cytokine-receptor gamma chain play a pivotal role. In particular, IL-2, IL-7, IL-15, and IL-21 are the members of this family predominantly involved in NK cell biology. In this paper, we will address their role in NK cell ontogeny, regulation of functional activities, development of specialized cell subsets, and acquisition of memory-like functions. Finally, the potential application of these cytokines as recombinant molecules to NK cell-based immunotherapy approaches will be discussed.

Intestinal intraepithelial NK and NKT cell ontogeny in Lewis rats

Natural killer T (NKT) cells have been described in the liver and spleen of adult rats, but their presence and function in other tissues and in early life remains uncertain. This study was designed to determine the proportion of NK cells and NKT cells among small intestine intraepithelial (IE) lymphocytes in suckling rats and adult animals by flow cytometry. Very few intestinal IE-NKT cells (NKR-P1A+ TCRalphabeta+) were present in adult rats ( approximately 1%), but a high proportion of this population was found during early life ( approximately 40% of IE lymphocytes in 9-day-old rats), with a marked age-decreasing pattern. Most of these cells presented the CD8alphabeta+ phenotype. Intestinal IE-NK cells (NKR-P1A+ TCRalphabeta-) were also present in a relatively high proportion during the suckling period ( approximately 30% of IE lymphocytes). Thus, a predominance of both NK and NKT cell subpopulations in small intestine epithelium is characteristic in the early life of rats and may have a protective role during the suckling period.

Distinct receptor repertoire formation in mouse NK cell subsets regulated by MHC class I expression

The acquisition of inhibitory MHC-specific receptors occurs during NK cell differentiation and has been considered important in regulating NK cell responsiveness. NK cell differentiation has been studied on the basis of cell surface phenotype, function, and proliferative capacity. Together with phenotypically immature Mac-1lo NK cells, the mature Mac-1hi NK cell pool can be dissected further into two functionally distinct CD27hi and CD27lo subsets. Two major inhibitory receptors, CD94/NKG2A and Ly-49, are expressed on mouse NK cells. The acquisition of the CD94/NKG2A receptor seems to be an early event, whereas Ly-49 receptor expression is considered a relatively late event during NK cell ontogeny. In this study, we demonstrated a distinct NK cell inhibitory receptor repertoire formation within mature NK cell populations as defined by Mac-1 and CD27. By analyzing mice deficient in MHC class I expression or NKG2D ligand transgenic mice, we have shown that the inhibitory receptor repertoire can be modulated according to the differentiation/maturation status of NK cells, and the receptor acquisition is imprinted at an early stage of NK cell development by MHC class I interactions.

Limiting γc expression differentially affects signaling via the interleukin (IL)-7 and IL-15 receptors

X-linked severe combined immunodeficiency (SCID-X1) results from mutations in the IL2RG gene, which encodes the common gamma chain (gammac) of the receptors for interleukin (IL)-2, 4, 7, 9, 15, and 21. Affected infants typically lack T and natural killer (NK) cells as a consequence of loss of signaling via the IL-7 receptor (IL-7R) and the IL-15R, respectively. In some infants, however, autologous NK cells are observed despite failure of T-cell ontogeny. The mechanisms by which mutations in gammac differentially impact T- and NK-cell ontogeny remain incompletely understood. We used SCID-X1 patient-derived EBV-transformed B cells to test the hypothesis that the IL-15R-mediated signaling is preferentially retained as gammac expression becomes limiting. Signal transduction via the IL-15R was readily detected in control EBV-transformed B cells, and via the IL-7R when modified to express IL-7Ralpha. Under the same experimental conditions, patient-derived EBV-transformed B cells expressing trace amounts of gammac proved incapable of signal transduction via the IL-7R while retaining the capacity for signal transduction via the IL-15R. An equivalent result was obtained in ED-7R cells modified to express varying levels of gammac. Collectively, these results confirm that signal transduction via the IL-15R, and hence NK ontogeny, is preferentially retained relative to the IL-7R as gammac expression becomes limiting.

Differential and Nonredundant Roles of Phospholipase Cγ2 and Phospholipase Cγ1 in the Terminal Maturation of NK Cells

NK cells play a central role in mediating innate immune responses. Activation of NK cells results in cytotoxicity, cytokine, and chemokine secretions. In this study, we show that in mice with targeted deletion of phospholipase Cgamma (PLCgamma)2, one of the key signal transducers, there are profound effects on the development and terminal maturation of NK cells. Lack of PLCgamma2 significantly impaired the ability of lineage-committed NK precursor cells to acquire subset-specific Ly49 receptors and thereby terminal maturation of NK cells. Overexpression of isozyme, PLCgamma1, in PLCgamma2-deficient NK cells resulted in the successful Ly49 acquisition and terminal maturation of the NK cells; however, it could only partially rescue NKG2D-mediated cytotoxicity with no cytokine production. Furthermore, PLCgamma2-deficient NK cells failed to mediate antitumor cytotoxicity and inflammatory cytokine production, displaying a generalized hyporesponsiveness. Our results strongly demonstrate that PLCgamma1 and PLCgamma2 play nonredundant and obligatory roles in NK cell ontogeny and in its effector functions.

Characterization of a Novel Hematopoietic Marker Expressed from Early Embryonic Hematopoietic Stem Cells to Adult Mature Lineages

ABSTRACT A novel membrane protein has been identified in the course of screening for differentially expressed cDNAs in human embryonic hematopoietic sites. This 37- to 38-kDa molecule, designated KLIP-1 (killer lineage protein), consisting of 350 amino acids and containing five transmembrane domains, is encoded by the 5093-bp KLIP-1 gene, composed of nine exons and located on chromosome 6 (6p21.1–6p21.2). We found the KLIP-1 protein to be expressed by nucleated hematopoietic cells, from early embryonic hematopoietic stem cells through mature adult blood lymphoid lineages, either as membrane or as cytoplasmic molecules. In day-30/32 human embryo sections, KLIP-1 protein expression is restricted to circulating hematopoietic cells at hematopoiesis sites. Membrane KLIP-1 is expressed by fetal and adult GP-A + erythroblasts, the fetal liver CD34 + subset, fetal spleen, and adult bone marrow CD56 + NK and CD19 + B cells. Among mature blood cells, surface KLIP-1 expression is restricted to CD56 + NK cells, indicating KLIP-1 to be a novel marker of this population. Altogether, these results indicate that membrane export of KLIP-1 antigen is developmentally and ontogenetically regulated. The high degree of conservation of the KLIP-1 protein sequence among mammals strongly suggests that it plays an important role during hematopoiesis and may exercise similar functions in human and mouse blood cells. The KLIP-1 molecule may therefore constitute a powerful tool for improving knowledge of both human hematopoiesis and NK cell ontogeny and immune functions.

The biology of natural killer cells in cancer, infection, and pregnancy.

NK cells are important cells of the immune system. They are ultimately derived from pluripotent hematopoietic stem cells. NK cell cytotoxicity and other functions are tightly regulated by numerous activating and inhibitory receptors including newly discovered receptors that selectively recognize major histocompatibility complex class I alleles. Based on their defining function of spontaneous cytotoxicity without prior immunization, NK cells have been thought to play a critical role in immune surveillance and cancer therapy. However, new insights into NK cell biology have suggested major roles for NK cells in infection control and uterine function. The purpose of this review is to provide an update on NK cell function, ontogeny, and biology in order to better understand the role of NK cells in health and disease. In the Medline database, the major subject heading "Natural Killer Cells" was introduced in 1983, identifying 16,848 citations as of December 31, 2000. Since 1986, there have been approximately 1000 citations per year under this subject heading. In this database, 68% of manuscripts are limited to human NK cells; 40% of citations cross with the major sub-heading of cytotoxicity, 40% with cytokines, 36% with neoplasm, 5% with antibody-dependent cellular cytotoxicity, 2.8% with pregnancy, and 1.3% with infection. Of references from the year 2000-2001, 46 were selected to combine with contributions from earlier literature. NK cells should no longer be thought of as direct cytotoxic killers alone as they clearly serve a critical role in cytokine production which may be important to control cancer, infection, and fetal implantation. Understanding mechanisms of NK cell functions may lead to novel therapeutic strategies for the treatment of human disease.

Ontogeny and Expansion of Human Natural Killer Cells: Clinical Implications

Our knowledge of NK cells and their critical role in the innate immune system has increased enormously since their discovery several decades ago. However, it is only within the last 10 years that rational cytokine therapies, such as those utilizing low doses of IL-2, have been successful in expanding NK cells in patients with cancer and/or immunodeficiency. Such experiences in vivo have highlighted the importance of basing immunotherapeutic strategies on the known cellular and molecular properties of the targeted cell population. Recent advances in our understanding of the physiologic factors and events that orchestrate NK cell ontogeny, including IL-15 and receptor tyrosine kinase ligands to c-kit and fit3, provide novel therapeutic possibilities for cytokine therapy. This review summarizes our current understanding of human NK cell ontogeny, and links this knowledge to ongoing and future clinical strategies for the endogenous expansion of NK cells in patients with cancer and/or immunodeficiency.

Diversity of NK Cell Receptor Repertoire in Adult and Neonatal Mice

Murine NK cytotoxicity is regulated by two families of MHC class I-specific receptors, namely Ly49 and CD94/NKG2. We developed a single-cell RT-PCR method to analyze expression of all known Ly49 and NKG2A genes in individual NK cells and determined the receptor repertoires of NK cells from adult and neonatal (1-wk-old) C57BL/6 mice. In adult mouse NK cells, up to six different receptors were coexpressed in random combinations. Of 62 NK cells examined, 42 different patterns of receptor expression were observed. Most of them expressed at least one Ly49, whereas NKG2A was detected in 32% of the cells. Over 75% of them expressed Ly49C, I, or NKG2A, which are thought to recognize self-class I MHC (H-2b). Coexpression of multiple Ly49 receptors and NKG2A was stochastic. In contrast, very few neonatal NK cells expressed any Ly49, but almost 60% of them expressed NKG2A. These results demonstrate that adult NK cells are quite heterogeneous and have diverse receptor repertoires. They also suggest that the expression of NKG2A precedes Ly49 expression in NK cell ontogeny, and NKG2A is a major inhibitory receptor in neonatal NK cells.

Development and function of T lymphocytes and natural killer cells after bone marrow transplantation of severely immunodeficient mice.

Bone marrow (BM) transplantation experiments were performed in a strain of CD3 epsilon-transgenic mice, termed tg epsilon 26, which are completely deficient in T-cell and natural killer (NK) cell development. We found that an interaction of stromal cells and prothymocytes is required for the induction of a cortical thymic microenvironment. This induction takes place in a time window from fetal development to early neonates. Although the thymic environment is not required for NK-cell development, we found that aberrantly educated alpha beta or gamma delta T lymphocytes can influence NK-cell ontogeny. Surprisingly, BM transplantation of tg epsilon 26 fetuses and neonates results in normal T-cell development, but very low levels of NK cells. The poor NK-cell reconstitution in fetal and neonatal stages could be explained by an inefficient migration of hematopoietic progenitor cells to the BM. By contrast, migration of the progenitor cells to the thymus was efficient to initiate T-cell development. BM transplantation of adult tg epsilon 26 mice resulted in abnormal T-cell development which, in turn, caused an inflammatory bowel disease (IBD) in the recipient mice. Studies in these BM chimeras have revealed that both alpha beta and gamma delta T cells can be pathogenic and, further, that Th1-like cytokines produced by these cells are causal factors in the pathogenesis of IBD.

Mouse natural killer subsets defined by their target specificity and their ability to be separately rendered unresponsive in vivo.

NK cells from F1(AxB) hybrid mice are known to reject bone marrow grafts from either parent A or parent B (hybrid resistance). Using cold target competition in an in vitro hybrid resistance assay, we demonstrate in this work that parent A and parent B are killed by different NK subsets. As confirmation of the existence of these subsets, and to determine whether they undergo a selection/education process during NK cell ontogeny, we constructed bone marrow chimeras in which NK1.1-depleted bone marrow cells from F1 mice were allowed to mature in the microenvironment of either parent A or parent B. These F1-reconstituted chimeras were shown to have normal NK cell numbers and lytic ability in terms of YAC-1 killing and Ab-mediated redirected lysis. Using a three-color flow cytometry analysis in an in vivo hybrid resistance assay, we found that A targets, but not B targets, were less effectively removed by the F1 NK cells that develop in an A recipient (F1-->A chimeras) than in normal F1 or F1-->F1 chimeric mice. IL-2-activated killer cultures of cells from these chimeras, however, did not show a significant difference in the anti-parent killing response in an in vitro assay. These results suggest that NK cells exist in subsets and that self-reactive NK subsets can be rendered unresponsive by radioresistant host element(s). However, this unresponsiveness can be broken when the NK cells are removed from the in vivo environment.

Natural killer cell ontogeny in the athymic rat. Relationship between functional maturation and acquired resistance to E1A oncogene-expressing sarcoma cells.

Newborn nude rats are NK cell deficient and highly susceptible to tumor induction by NK-susceptible, E1A oncogene-expressing, BHK-21 sarcoma cells, whereas adults have normal NK activity and are tumor resistant. This model of NK cell-related tumor rejection was used to characterize age-related functional maturation of NK cells during nude rat ontogeny. Small numbers of cytolytic incompetent, NKR-P1+ cells were detected in the spleens of newborns. During the first month of life, there was an age-related increase in the numbers of splenic NK cells, followed by a stepwise maturation in their cytolytic competence. Three functional stages in NK cell cytolytic activity were apparent. A nonlytic newborn stage was followed by a transitional stage, during which absent or relatively weak spontaneous cytolytic activity was augmented by prolonged NK cell exposure to susceptible target cells. In the final stage, both spontaneous and augmented cytolytic activity increased toward the adult level that was attained by 4 wk of age. The data suggest the existence of a transient stage in NK cell ontogeny when triggering through the NKR-P1 molecule is functionally immature, and confirm the importance of a cytolytic competent, NK cell response for nude rat rejection of E1A oncogene-expressing tumor cells.

Monoclonal antibody to a triggering structure expressed on rat natural killer cells and adherent lymphokine-activated killer cells.

To study the cellular structures involved in NK and lymphokine-activated killer (LAK) cell function, we have produced a panel of mAbs that modulate the cytolytic function of a population of cells with LAK activity that derive from large granular lymphocyte (LGL)/NK cells (adherent LAK [A-LAK] cells). In this report, we describe an mAb (3.2.3; IgG1k) that recognizes a triggering structure that is expressed on rat LGL/NK cells and A-LAK cells. This epitope is also expressed on polymorphonuclear leukocytes (PMN). The expression of the epitope identified by mAb 3.2.3 increased progressively on A-LAK cells after culture in the presence of rIL-2. mAb 3.2.3 enhanced the cytolytic activity of NK and A-LAK cells against FcR+ target cells, but not FcR- target cells. However, this effect was not induced by F(ab')2 fragments of 3.2.3. This antibody also induced the release of N-alpha-benzyloxycarbonyl-L-lysine thiobenzy esteresterase by A-LAK cells. These data suggest that the epitope identified by mAb 3.2.3 is on a triggering structure expressed on rat NK cells and A-LAK cells. The expression of the epitope recognized by mAb 3.2.3 on LGL/NK cells and PMN suggests that this structure may be analogous to that identified by the anti-CD16 (-FcR) mAbs. However, the molecule immunoprecipitated by mAb 3.2.3 was a 60-kD dimer composed of two 30-kD chains. These data suggest that mAb 3.2.3 recognizes a unique triggering structure. As mAb 3.2.3 is the first antibody recognizing a determinant with functional significance, selectively expressed on both rat NK cells and A-LAK cells, it will be a useful tool for the study of NK cell ontogeny and function, and the development of cells with LAK activity from the NK cell compartment.

Altered natural killer cell biology in C57BL/6 mice after leukemogenic split-dose irradiation.

Natural killer (NK) cell activity was examined in the spleens of C57BL/6 mice given leukemogenic split-dose irradiation. The radiation protocol resulted in severe depression of spontaneous NK cell activity; this activity was not fully restored after treatment with the interferon inducer poly I:C. In vitro mixing studies provided no evidence for active suppression in vivo as a mechanism for this decrease in activity. In addition, spontaneous activity was restored towards control levels after bone marrow transfusion from nonirradiated mice. Despite the low NK cell activity, there was no difference between control and irradiated mice in the numbers of target-binding cells (TBC). The results are most compatible with the radiation-induced loss of a cell with normal NK activity from spleen and bone marrow after the split-dose radiation protocol. In addition, a population of cells able to competitively block normal NK cell lysis of YAC-1 tumor cells is found in the bone marrow, spleen, and thymus of the irradiated mice lacking NK cell activity. These findings are considered from the perspective of their implications regarding NK cell ontogeny, and the possible role of the NK cell in radiation leukemogenesis.