Pax5 determines B- versus T-cell fate and does not block early myeloid-lineage development

Progenitor B cells deficient in Pax5 are developmentally multipotent, suggesting that Pax5 is necessary to maintain commitment to the B-cell lineage. Commitment may be mediated, in part, by Pax5 repression of myeloid-specific genes. To determine whether Pax5 expression in multipotential cells is sufficient to restrict development to the B-cell lineage in vivo, we enforced expression of Pax5 in hematopoietic stem cells using a retroviral vector. Peripheral blood analysis of all animals reconstituted with Pax5-expressing cells indicated that more than 90% of Pax5-expressing cells were B220+ mature B cells that were not malignant. Further analysis showed that Pax5 completely blocked T-lineage development in the thymus but did not inhibit myelopoiesis or natural killer (NK) cell development in bone marrow. These results implicate Pax5 as a critical regulator of B- versus T-cell developmental fate and suggest that Pax5 may promote commitment to the B-cell lineage by mechanisms that are independent of myeloid gene repression.     

Production of interferons by dendritic cells, plasmacytoid cells, natural killer cells, and interferon-producing killer dendritic cells.

The capacity of mouse spleen conventional dendritic cells (cDCs) and plasmacytoid dendritic cells (pDCs) to produce interferon-gamma (IFN-gamma) or IFN-alpha was assessed, and compared with that of natural killer (NK) cells and the recently identified interferon-producing killer dendritic cells (IKDCs), both of which are frequent contaminants in DC preparations. Fully developed cDCs or pDCs, if free of NK cells or IKDCs, showed little capacity for IFN-gamma production. However, an early developmental form of the CD4-8+ cDC subtype, and the Ly6C- Ly49Q- pDC subtype, both were able to produce moderate amounts of IFN-gamma, although less than IKDCs. In response to toll-like receptor 9 stimuli, both the Ly6C+ Ly49Q+ and the Ly6C- Ly49Q- pDC subtypes were effective producers of IFN-alpha. However, IKDCs, which efficiently produced IFN-gamma and showed immediate cytotoxicity on NK target cells, did not produce IFN-alpha under these conditions.     

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.     

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.     

Anti-myeloma activity of endogenous and adoptively transferred activated natural killer cells in experimental multiple myeloma model

OBJECTIVE: Despite advances in autologous stem cell transplantation and chemotherapy, multiple myeloma (MM) remains an incurable disease. Due to the role of natural killer (NK) cells in host resistance against several tumors, it is of interest to explore the anti-MM activity of NK cells. For this reason, we aimed to determine if NK cells provide anti-MM activity following interleukin-2 (IL-2) administration, and if ex vivo activated and intravenously administered NK cells prolong survival in MM-bearing C57BL/KaLwRij mice. METHODS: The anti-MM effect of IL-2 was tested by intraperitoneal injection into the 5T33MM-inoculated mice. Subsequently, in vivo effector cell depletions were performed by administration of anti-NK1.1 or anti-CD8 monoclonal antibodies. Finally, magnetically separated and activated NK cells from splenocytes of C57BL/KaLwRij mice were adoptively transferred to tumor-bearing mice in conjunction with IL-2 treatment. RESULTS: IL-2 administration into MM-bearing mice significantly prolonged their survival. This effect was diminished by in vivo depletion of NK cells. Adoptive transfer of activated NK cells showed a significant in vivo anti-MM effect that was dependent on cell dose. Biodistribution of the marked adoptively transferred NK cells correlated with MM cells' homing sites. CONCLUSION: These data suggest that activated NK cells have a promising potential in adoptive immunotherapy for MM.     

Molecular cytogenetics of stem cells: target cells of chromosome aberrations as revealed by the application of fluorescence in situ hybridization to fluorescence-activated cell sorting

The lineage involvement in stem cell disorders, such as chronic myeloid leukemia (CML) and myelodysplastic syndrome (MDS), remains unclear. To explore this issue, we used fluorescence in situ hybridization for cells sorted by fluorescence-activated cell sorting (FACS) from 12 patients with chronic-phase CML. Philadelphia chromosome (Ph) was found in pluripotent stem cells (CD34+Thy-1+), B cells (CD34+CD19+), and T/natural killer (NK) progenitor cells (CD34+CD7+) collected by FACS from bone marrow cells. B (CD19+), T (CD3+), and NK (CD3-CD56+) cells showed a marked decrease in Ph+ cells between progenitor cells and mature cells The Ph+ T and NK cells decreased to below background levels. These data suggest that Ph+ lymphocytes either do not differentiate or are eliminated during their maturation process Among 7 MDS patients associated with trisomy 8, sorted lymphocytes from peripheral blood did not have +8. CD34+ subpopulations from bone marrow including B,T/NK progenitors, and pluripotent progenitor cells also did not have +8.Trisomy 8 was identified from the level of multipotent colony-forming units (CD34+CD33+), and the lymphoid lineage was not involved. Thus, MDS with trisomy 8 conceivably arises from nonlymphoid progenitor cells, sparing T, B, or NK cells. Further studies using molecular cytogenetics may clarify the mechanism of leukemia happening at the level of stem cells.     

Differential effects of dehydroepiandrosterone (DHEA) on murine lymphopoiesis and myelopoiesis.

Dehydroepiandrosterone (DHEA) inhibits murine lymphopoiesis, especially after sublethal irradiation, without affecting mature lymphocyte function. We demonstrate here that dietary DHEA differentially affected myelopoiesis and lymphopoiesis in sublethally irradiated mice. Erythropoietic progenitor cell and stem cell function was not affected by DHEA although the magnitude of granulopoiesis was slightly reduced. Regeneration of marrow B220+ B cells, natural killer (NK) function, and thymus repopulation were significantly delayed in DHEA-fed mice. Interleukin 2 (IL-2) failed to restore NK activity of DHEA-fed mice to normal levels. Marrow from DHEA-fed mice contained competent thymocyte progenitors capable of repopulating thymi of irradiated hosts fed a control diet but not those fed the DHEA diet. Thus DHEA inhibits lymphopoiesis while sparing myelopoiesis, affecting the growth and maturation of T, B, and NK cells by a mechanism other than the inhibition of IL-2 production.     

Activated natural killer cells and interleukin-2 promote granulocytic and megakaryocytic reconstitution after syngeneic bone marrow transplantation in mice

Purified populations of natural killer (NK) cells were obtained from mice with severe combined immune deficiency (SCID). SCID spleen cells were cultured and activated with recombinant human interleukin-2 (rhIL- 2) in vitro. The activated NK cells were then transferred with syngeneic BALB/c bone marrow cells (BMC) and rhIL-2 into lethally irradiated syngeneic recipients to determine their effect on long-term hematopoietic reconstitution. On analysis, the transfer of rhIL-2- activated NK cells along with BMC resulted in significant increases in splenic and BM hematopoietic progenitor cells when compared with those for mice not receiving NK cells. Histologic and flow cytometric analysis showed a marked increase in granulocytic and megakaryocytic lineage cells present in the spleens of the mice receiving activated NK cells. Analysis of the peripheral blood indicated that the transfer of activated NK cells with BMC also significantly improved platelet and total white blood cell counts, with increases in segmented neutrophils. Erythroid recovery was not affected. Finally, lethally irradiated mice receiving activated NK cells and rhIL-2 along with limiting numbers of syngeneic BMC showed a marked increase in survival rate. These results show that the use of populations enriched for activated NK cells after syngeneic BM transplantation (BMT) has a profound enhancing effect on engraftment primarily affecting megakaryocytic and granulocytic cell reconstitution. Therefore, the transfer of activated NK cells and rhIL- 2 may be of clinical use to promote hematopoietic reconstitution after BMT.     

The receptor tyrosine kinase c-kit provides a critical signal for survival, expansion, and maturation of mouse natural killer cells

Fetal liver kinase ligands (flk2L/flt3L) and stem cell factor (SCF) have been shown to promote natural killer (NK) cell differentiation from hematopoietic stem cell (HSC) precursors in vitro. However, the contribution of signaling through the receptors for these growth factors for in vivo NK cell development remains ill-defined. We have analyzed the role of the SCF receptor c-kit in NK cell differentiation by reconstituting NK-deficient mice with fetal liver (FL) HSCs of c-kit(-/-) (W/W) mice. Although c-kit(-/-)NK cells were generated in W/W chimeras, they were reduced in number, contained a lower percentage of CD45R (B220)(+) cells, and were poorly cytolytic. In vitro experiments showed that generation of NK cells from FL precursors was reduced in the absence of c-kit signaling and that SCF promoted the survival of peripheral c-kit(+) NK cells. We conclude that c-kit/SCF interactions in vivo are dispensable for the commitment of HSC to the NK lineage, but they provide essential signals for generating normal numbers of fully mature NK cells.     

Lymphokine activated killer (LAK) cells in patients with gastrointestinal cancer.

Lymphokine activated killer (LAK) cells are a recently described cellular immune phenomenon with exciting potential for the treatment of tumours arising from solid organs. A comparison of some aspects of LAK cell precursors and LAK cell function was undertaken in 44 control subjects and 44 preoperative patients suffering from gastrointestinal cancer (20 localised and 24 advanced). Lymphokine activated killer cell precursor (natural killer (NK) cell) activity was significantly diminished in patients with advanced tumours (p less than 0.02) as was fully mature LAK cell activity against an NK resistant target cell (p less than 0.012). T-lymphocyte responses were not significantly different between the three groups. The reduced LAK cell generation was associated with a significantly diminished proliferative response of LAK precursors to stimulation with high dose IL-2 in vitro (p less than 0.012). Impaired LAK cell generation may explain the failure of adoptive cellular immunotherapy with LAK cells in some patients with advanced gastrointestinal cancer and prompts the search for means of augmenting this activity in such patients.     

Expansion of spleen myeloid suppressor cells represses NK cell cytotoxicity in tumor-bearing host

Tumor growth promotes the expansion of myeloid suppressor cells. An inverse correlation between natural killer (NK) cell activation and myeloid suppressor cell (MSC) expansion in tumor-bearing patients and mice prompted us to investigate the role of MSCs in controlling NK antitumor cytotocixity. After adoptive transfer to naive recipients, CD11b(+)Gr-1(+) MSCs freshly isolated from spleens of tumor-bearing mice but not naive mice were able to inhibit NK cell cytotoxicity. An in vivo imaging analysis indicates that the removal of tumors resulted in a significant increased ability (P < .05) in NK cell cytotoxicity to eliminate injected YAC-1 cells from the lungs. Fluorescence-activated cell sorter (FACS) analysis of the composition of lung leukocytes further indicates that the removal of tumors also leads to the reduction of MSCs accumulated in the lung. These data suggest that MSCs suppress NK cell cytotoxicity. The inhibition of NK cell cytotoxicity is cell-cell contact dependent. Inhibition of perforin but not granzyme B production was responsible for MSC-mediated inhibition of NK cytotoxicity. Western blot analyses further suggests that MSCs suppress IL-2-mediated NK cell cytotoxicity by affecting the activity of Stat5.     

Leukemia and lymphoma of natural killer lineage cells

Natural killer (NK) cells are lymphocytes with a large granular lymphocyte morphology, a CD3- CD56+ phenotype, a non-major histocompatibility complex-restricted cytotoxicity, and germline configuration T-cell receptor genes. NK cell lineage tumors originate from either precursor NK cells or mature NK cells. Tumors originating conceivably from precursor NK cells include myeloid/NK cell precursor acute leukemia, precursor NK cell acute lymphoblastic leukemia, and blastic NK cell lymphoma. However, because the developmental pathway of normal NK cells and the characteristics of these NK precursors are not fully understood, the definition and characterization of the tumors are only provisional. Tumors of mature NK cell origin include aggressive NK cell leukemia/lymphoma, nasal-type NK cell lymphoma, and chronic NK lymphocytosis, but the last disorder seems to be reactive in most cases. Because NK cell tumors are rare and difficult to manage, vigorous studies are required for their understanding and management.     

Differential effect of 4-hydroperoxycyclophosphamide and antimyeloid monoclonal antibodies on T and natural killer cells during bone marrow purging

Autologous bone marrow (BM) transplantation after high dose therapy is widely used to treat acute leukemia, lymphoma, and selected solid tumors. In studies of BM purging with chemical agents, monoclonal antibodies (MoAbs), or other agents, the emphasis has been on the efficacy of tumor cell removal and sparing of hematopoietic progenitor cells. Two commonly used methods of BM purging for patients with acute myeloid leukemia have been the drug 4-hydroperoxycyclophosphamide (4- HC) and (MoAbs) directed to myeloid antigens such as CD14, CD15, and CD33. Although both methods of BM purging have potent activity against leukemia cells, 4-HC is also quite toxic to normal hematopoietic progenitor cells in the same concentrations that are used to deplete leukemia cells. To further characterize the cellular composition of BM after purging, we examined the effects of MoAbs plus complement and 4- HC on cells of the lymphoid lineage in the BM. 4-HC exerted a concentration-dependent cytotoxicity on clonogenic T lymphocytes, natural killer (NK) cells, and lymphokine (interleukin-2)-activated killer (LAK) cells, whereas the anti-CD14 and anti-CD15 MoAbs had little effect. At a concentration of 4-HC commonly used for BM purging (60 micrograms/mL), there were 4 to 5 logs of T-cell depletion and almost complete elimination of NK- and LAK-cell activity. In contrast, 4-HC at low concentrations (eg, 3 micrograms/mL) spared the majority of lymphoid cells suggesting that low concentration 4-HC combined with MoAb purging may be a desirable alternative to higher concentration 4- HC. These data indicate that purging with antimyeloid MoAbs, but not with 4-HC, spares the function of mature graft lymphocytes. Infusion of viable lymphocytes may be important for the transfer of immune memory against microbial and neoplastic antigens and may hasten immune reconstitution. In addition, mature graft lymphocytes may also be selectively activated and expanded in conjunction with interleukin-2 administration after BM transplantation.     

Clinical scale isolation of T cell-depleted CD56+ donor lymphocytes in children

We present a clinical scale method for immunomagnetic separation of CD56+ donor natural killer cells for adoptive immunotherapy of pediatric leukemias after allogeneic transplantation. This time-saving and partially automated procedure employed CD56+ selection followed by CD3+ depletion, resulting in a median purity of 98.6% NK cells and a four-log depletion of T cells. The enriched NK cells demonstrated high cytotoxic activity against K562 target cells and fresh leukemic blasts with low HLA class I expression, which could be further enhanced by IL-2 stimulation. Lysis of NK-insensitive leukemic cells with high HLA class I expression could also be demonstrated via ADCC. Due to the high degree of T cell depletion, alloreactive proliferation in mixed lymphocyte cultures and response to T cell-specific mitogen stimulation was profoundly decreased. Our results suggest that, even in the case of mismatched donors, infusions of donor NK cells with extremely low T cell content may be a promising treatment option for leukemic minimal residual disease after allogeneic transplantation without risk of inducing severe GVHD.     

Engineering lymph node homing of ex vivo-expanded human natural killer cells via trogocytosis of the chemokine receptor CCR7

Natural killer (NK) cells have gained significant attention in adoptive immunotherapy for cancer. Consequently, novel methods of clinical-grade expansion of NK cells have emerged. Subsets of NK cells express a variety of chemokine receptors. However, to expand the scope of adoptively transferred NK cell homing to various malignancies, expression of corresponding chemokine receptors on NK cells is essential. Here, we have explored the use of trogocytosis as a tool to transiently express the chemokine receptor CCR7 on expanded human NK cells with the aim to enhance their homing to lymph nodes. We generated a K562-based "donor" cell line expressing CCR7, Clone9.CCR7, to transfer CCR7 onto NK cells via trogocytosis. CCR7 expression occurred in 80% of expanded NK cells within 1 hour after coculture with Clone9.CCR7. After removal of the donor cells from the coculture, the CCR7 expression on NK cells steadily declined to baseline levels by 72 hours. The acquired CCR7 receptors mediated in vitro migration of NK cells toward CCL19 and CCL21 and increased the lymph node homing by 144% in athymic nude mice. This is the first report on exploiting trogocytosis to rapidly and transiently modify lymphocytes, without direct genetic intervention, for adoptive transfer.     

Identification of the earliest natural killer cell-committed progenitor in murine bone marrow

Natural killer (NK) cells develop in the bone marrow and are known to gradually acquire the ability to eliminate infected and malignant cells, yet the cellular stages of NK lineage commitment and maturation are incompletely understood. Using 12-color flow cytometry, we identified a novel NK-committed progenitor (pre-NKP) that is a developmental intermediate between the upstream common lymphoid progenitor and the downstream NKP, previously assumed to represent the first stage of NK lineage commitment. Our analysis also refined the purity of NKPs (rNKP) by 6-fold such that 50% of both pre-NKP and rNKP cells gave rise to NKp46+ NK cells at the single-cell level. On transplantation into unconditioned Rag2-/-Il2rγc-/- recipients, both pre-NKPs and rNKPs generated mature NK cells expressing a repertoire of Ly49 family members that degranulated on stimulation ex vivo. Intrathymic injection of these progenitors, however, yielded no NK cells, suggesting a separate origin of thymic NK cells. Unlike the rNKP, the pre-NKP does not express IL-2Rβ (CD122), yet it is lineage committed toward the NK cell fate, adding support to the theory that IL-15 signaling is not required for NK commitment. Taken together, our data provide a high-resolution in vivo analysis of the earliest steps of NK cell commitment and maturation.     

In vitro expansion and analysis of cloned cytotoxic T cells derived from patients with chronic T gamma lymphoproliferative disorders

Patients with T gamma lymphocytosis are a heterogeneous group, clearly distinguishable from other patients with chronic T lymphoproliferative disorders and usually without proven malignancy. We have attempted in vitro cloning of lymphocytes from three patients with an expansion of phenotypically and functionally different types of T gamma cells. One had T3+ B73.1+ T4 T8+ OKM1+ T gamma cells exerting antibody-dependent cellular cytotoxicity (ADCC) and natural killer (NK) cell cytotoxicity; another had T3+B73.1-T4-T8+OKM1-ADCC+NK- and a third had T3-B73.1+T4-T8- OKM1+ADCC+NK+ cells. On morphological characterization, most of the mononuclear cells of these patients resembled large granular lymphocytes (LGLs). Although lymphocytes of these patients showed almost no proliferative response capacity after stimulation with mitogens, they shared the capacity to proliferate after stimulation with Epstein-Barr virus-transformed lymphoblastoid B (B-LCL) feeder cells. Stable clones were established by this procedure. Clones from patient 3 exerted cytolytic activity against a broad spectrum of tumor cell lines, including fresh biopsy specimens of melanoma tumor target cells. All of these clones (termed activated killer [AK] cells) had the surface phenotype T3-, T4-, T8- or +, HNK1-, OKM1-, Lyt3+, WT1+ and showed ADCC in addition to AK cell cytotoxicity. Most of them were B73.1+ and expressed IgG-Fc receptors. They most likely belong to the T cell lineage, since they express IL2 receptors as recognized by the Tac antibody and did not bind monoclonal antibodies directed against monocytes or granulocytes. Thus lymphocytes with the functional and phenotypical characteristics of T gamma cells can be cloned and expanded in vitro from the peripheral lymphocytes of these patients by using the appropriate stimulus. Our results indicate that, of the heterogeneous population of NK cells, the T3- cells are more rapidly expanded than T3+ subsets. It is discussed whether or not our culture system might selectively induce proliferation in "normal" T cells rather than aberrant ones.     

TRAIL identifies immature natural killer cells in newborn mice and adult mouse liver

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a key effector molecule expressed by natural killer (NK) cells and has been shown to prevent tumor initiation, growth, and metastasis. Here we demonstrate that TRAIL is the dominant cytotoxic effector molecule expressed by NK cells in fetal mice. On birth and with age, NK cells develop full functional capacity, including the ability to secrete interferon gamma (IFN-gamma) and interleukin 13 (IL-13) and mediate perforin- and Fas ligand-mediated cytotoxicity. However, interestingly, a phenotypically immature TRAIL+ NK cell subpopulation is retained in the liver of adult mice, and its retention is dependent on IFN-gamma but not dependent on host IL-12, IL-18, or endogenous host pathogens. Adoptive transfer of either adult liver or neonatal TRAIL+ NK cells resulted in the appearance of TRAIL- NK cells with a mature phenotype, suggesting that these TRAIL+ NK cells were indeed a precursor. Although inducers of IFN-gamma stimulated TRAIL expression on mature NK cells, our data indicated that constitutive TRAIL expression was a hallmark of immature cytotoxic NK cells. This study is the first to describe the concomitant maturation of NK cell effector function with surface phenotype in vivo and implies an important defense role for NK cell TRAIL in the developing immune system.