FIGURE 7 - uploaded by Masakazu Hattori
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Histological abnormalities in human IL-2/IL-2-R L chain transgenic mice. (A) Histology of pneumonia in the transgenic mice. (B) Normal lung histology. (C) Histology of the lymphocyte depletion in the cortex of the transgenic thymus. (D) Normal thymus histology. (E) Histology of the lymphocyte depletion in the transgenic spleen. (F) Normal spleen histology. (G) Histology of the selective loss of Purkinje cells in the cerebellum of the transgenic mice. (H) Normal cerebellum histology. In C and D, c and m indicate cortex and medulla, respectively. Mice analyzed were 3 wk old (140x, A and B; 225x, C, D, G, and H; and 110x, E and F) .
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Transgenic mice expressing both human IL-2 and the L chain of IL-2-R constitutively had an unusual expansion of Thy-1+/CD3-4-8- large granular lymphocytes, which bore the elevated NK activity. Unexpectedly, the transgenic mice had neither T cell expansion nor autoreactive antibodies. The increase in number and activity of NK cells seems to be respo...
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... elucidate the primary cause of the early death of the hybrid mice, we analyzed their tissues histologically. The 3-wk-old hybrid mice showed severe interstitial pneu- monia with focal infiltration of lymphocytes and other inflammatory cells (Fig. 7, A and B) . The majority of infiltrating lymphocytes in the lung of the hybrid mice were probably expanded Thy-1 +/CD3" LGL because cells collected from the col- lagenase-treated lungs of the hybrid mice contained a large number of LGL (Fig. 2 D). The thymus and spleen of the 3-wk-old hybrid mice were far smaller than those of the ...
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... expanded Thy-1 +/CD3" LGL because cells collected from the col- lagenase-treated lungs of the hybrid mice contained a large number of LGL (Fig. 2 D). The thymus and spleen of the 3-wk-old hybrid mice were far smaller than those of the control mice, and the profound lymphocyte depletions were found histo- logically in the cortex of the thymus (Fig . 7, C and D) and in the red as well as white pulp ofthe spleen (Fig. 7, E and F). To our surprise, the 3-wk-old hybrid mice selec- tively lost 40-7017o ofPurkinje cells in the cerebellum without any sign of inflamma- tion (Fig. 7, G and H). This change might be associated with the ataxic gait ofthe mice ...
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... lagenase-treated lungs of the hybrid mice contained a large number of LGL (Fig. 2 D). The thymus and spleen of the 3-wk-old hybrid mice were far smaller than those of the control mice, and the profound lymphocyte depletions were found histo- logically in the cortex of the thymus (Fig . 7, C and D) and in the red as well as white pulp ofthe spleen (Fig. 7, E and F). To our surprise, the 3-wk-old hybrid mice selec- tively lost 40-7017o ofPurkinje cells in the cerebellum without any sign of inflamma- tion (Fig. 7, G and H). This change might be associated with the ataxic gait ofthe mice ...
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... of the control mice, and the profound lymphocyte depletions were found histo- logically in the cortex of the thymus (Fig . 7, C and D) and in the red as well as white pulp ofthe spleen (Fig. 7, E and F). To our surprise, the 3-wk-old hybrid mice selec- tively lost 40-7017o ofPurkinje cells in the cerebellum without any sign of inflamma- tion (Fig. 7, G and H). This change might be associated with the ataxic gait ofthe mice ...
Citations
... In addition, low levels of IL-2Rβ were also found in neonatal ELCs of both mouse models (Fig. 8a,b). Interleukin 2 (IL-2) is a major growth factor for mature NK cells 61,62 . Freshly isolated NK cells preferentially express IL-2Rβ , through which IL-2 plays a pivotal role in proliferation and induction of cytolytic activity 63 . ...
... After stimulation with PMA plus ionomycin, around 20% of ELCs from both WT and Rag1 −/− expressed IFN-γ (Fig. 8c,d). Similar percentage of ELCs expressed IL-2 as well, a cytokine crucial for NK differentiation and function 61,62 . Production of IL-4, IL-5, IL-9, IL-10, IL-13, IL-17, IL-22, TNF-α , granzyme and perforin were also analysed, revealing that ELCs did not produce any of them with or without stimuli (data not shown). ...
T cell progenitors are known to arise from the foetal liver in embryos and the bone marrow in adults; however different studies have shown that a pool of T cell progenitors may also exist in the periphery. Here, we identified a lymphoid population resembling peripheral T cell progenitors which transiently seed the epidermis during late embryogenesis in both wild-type and T cell-deficient mice. We named these cells ELCs (Epidermal Lymphoid Cells). ELCs expressed Thy1 and CD2, but lacked CD3 and TCRαβ/γδ at their surface, reminiscent of the phenotype of extra- or intra- thymic T cell progenitors. Similarly to Dendritic Epidermal T Cells (DETCs), ELCs were radioresistant and capable of self-renewal. However, despite their progenitor-like phenotype and expression of T cell lineage markers within the population, ELCs did not differentiate into conventional T cells or DETCs in in vitro, ex vivo or in vivo differentiation assays. Finally, we show that ELC expressed NK markers and secreted IFN-γ upon stimulation. Therefore we report the discovery of a unique population of lymphoid cells within the murine epidermis that appears related to NK cells with as-yet-unidentified functions.
... The deregulated expression of soluble lymphokines has also been linked to T-cell transformation, as mice expressing IL-2 or IL-15 develop NK lymphoproliferative disorders (147,148). Moreover, IL-7 transgenic mice display progressive cutaneous disorders, involving a dermal lymphoid infiltrate, which can be successfully transplanted horizontally (149). ...
T-cell lymphoproliferative disorders are a heterogeneous group of neoplasms with distinct clinical-biological properties. The normal cellular counterpart of these processes has been postulated based on functional and immunophenotypic analyses. However, T lymphocytes have been proven to be remarkably capable of modulating their properties, adapting their function in relationship with multiple stimuli and to the microenvironment. This impressive plasticity is determined by the equilibrium among a pool of transcription factors and by DNA chromatin regulators. It is now proven that the acquisition of specific genomic defects leads to the enforcement/activation of distinct pathways, which ultimately alter the preferential activation of defined regulators, forcing the neoplastic cells to acquire features and phenotypes distant from their original fate. Thus, dissecting the landscape of the genetic defects and their functional consequences in T-cell neoplasms is critical not only to pinpoint the origin of these tumors but also to define innovative mechanisms to re-adjust an unbalanced state to which the tumor cells have become addicted and make them vulnerable to therapies and targetable by the immune system. In our review, we briefly describe the pathological and clinical aspects of the T-cell lymphoma subtypes as well as NK-cell lymphomas and then focus on the current understanding of their pathogenesis and the implications on diagnosis and treatment.
... It bas long been known tbat IL-2 is a potent activator of NK cells, and it causes their expansion and increases their lydc activity (42,43). Unhke T cells, which express the higb-affmity IL-2R (a, P, y) upon activadon, NK cells consdtudvely express the low affmity IL-2R (p, y). ...
In the last few years, the routine development of knockout and transgenic mice and the ease with which rare progenitor populations can be isolated from hematopoietic organs and cultured in vitro has facilitated significant advances in understanding the lineage and development of natural killer (NK) cells. Fluorescence-activated cell sorter analyses have identified a common lymphoid progenitor capable of giving rise to NK, T, and B cells, confirming the lymphoid origin of NK cells. Knockout and transgenic mouse models have pointed to an absolutely critical role for signals sent through the interleukin (IL)-2/lS receptor β (CD 122) chain and common (c) chain for NK development. Such signals are likely relayed inside the cell by the tyrosine kinase Jak3, which associates with c. Recently developed IL-15 and IL-15 receptor a knockout mice have pinpointed IL-15 as the mediator of this signal. Other mouse models have indicated an unexpected role for flt3 ligand in early NK-cell development as well as minor roles for stem cell factor and IL-7 in expanding NK-cell progenitor numbers. Finally, in vitro culture systems have proven useful in identifying the point in NK development at which each of these signals is critical.
... 19 IL-15 transgenic mice that overexpress a highly secreted form of IL-15 show polyclonal expansions of memory CD8 ϩ T cells, 20 while IL-2 transgenic mice demonstrate normal T-cell populations. 21 Our results here show that administration of stoichiometrically equivalent doses of rhIL-2 and rhIL-15 result in significantly different survival rates of hu-PBL-SCID mice and extend the dichotomy of IL-2 and IL-15 function to a model of human GVHD. ...
Interleukin-2 (IL-2) and IL-15 are structurally related cytokines that share receptor components but display markedly different effects in multiple in vivo model systems. Here we demonstrate that IL-15 but not IL-2 exacerbates xenogeneic graft-versus-host disease (X-GVHD) in severe combined immunodeficient murine recipients of human peripheral-blood lymphocytes (hu-PBL-SCID). Treatment of hu-PBL-SCID mice with IL-15 resulted in rapid fatality, lymphocytic infiltrations in the liver, lung, and spleen consistent with X-GVHD, and a marked expansion of human CD4+ and CD8+ T cells compared with controls. Depletion of human T cells in vivo abrogated the lethality of IL-15 treatment. To our knowledge, these data are the first to demonstrate in vivo activation and expansion of human T lymphocytes in response to IL-15 with concomitant exacerbation of human T-cell-mediated X-GVHD.
... Single-cell suspensions were stained with appropriate antibodies and analyzed by FACScan (Becton Dickinson, Mountain View, CA) after exclusion of dead cells by propidium iodide gating. Two-color staining of mononuclear cells with anti-B220 and anti-IgM, anti-Mac-1 and anti-IgM, and anti-CD8 and anti-CD4 antibodies were done as described previously (26,32,33). ...
Activation of peritoneal B-1 cells triggers autoimmune anemia in anti-erythrocyte Ig transgenic mice (HL mice). Numbers of peritoneal B-1 cells and Ig-producing cells were negligible in the T cell-deficient HL mice generated by the cross with RAG-2 ‐/‐ mice (RAG-2 ‐/‐ H HL mice). Proliferation and activation of B-1 cells in RAG-2 ‐/‐ H HL mice were recovered by fetal thymus transfer, indicating involvement of T cells in B-1 cell-mediated autoimmune hemolytic anemia. Involvement of T cells in proliferation and activation of B-1 cells could be by-passed by administration of lipopolysaccharide (LPS), IL-5 or IL-10 to RAG-2 ‐/‐ H HL mice. Administration of LPS elevated the serum IL-10 level in HL, RAG-2 ‐/‐ H HL and normal mice. Proliferation and activation of B-1 cells were blocked by an anti-IL-10 antibody in conventionally bred as well as LPS-treated HL mice. Taken together, IL-10 plays a pivotal role in activation of peritoneal B-1 cells.
... NK cells are known to be activated by some cytokines, including IL-2, IFN-y, and IL-12. For instance, an increased number of NK cells was observed in transgenic mice for both huIL-2 and huIL-2R (42). However, gene expression of IL-2, IFN-y, or IL-12 in whole spleen cells of CD45 -1-mice showed no difference in CD45 +I+ and -/-mice by quantitative reverse-transcription PCR analysis (data not shown). ...
CD45 is a cell membrane-type protein tyrosine phosphatase that is essential for Ag receptor-mediated signaling in both T and B lymphocytes. To characterize roles of CD45 molecules in murine NK cells, we analyzed the development and the cytotoxic functions of NK cells in mice lacking CD45 exon 6 (CD45 -/-). A markedly increased number of NK cells was observed in spleens of the CD45 -/- mice, despite no CD45 surface expression on the NK cells. From the results of mixed bone marrow chimera experiments, it was demonstrated that the expansion of NK cells in CD45 -/- mice was due to the influence of disappeared expression of CD45 in the NK cells per se, but not to the modulation of environmental factors. The NK cells in the CD45 -/- mice had normal cytotoxic activities, including NK and Ab-dependent cell-mediated cytotoxicity activities comparable with those in normal mice (CD45 +/+). Additionally, the CD45 -/- NK cells could functionally differentiate to lymphokine-activated killer cells by culturing with a high dose of IL-2, despite a lack of induced expression of B220. Therefore, these results suggest that CD45 is involved in NK cell development, but is not essential for cytotoxic activities and Fc gamma R-mediated signaling in NK cells.
... In contrast, Kishimoto reported that IL-6 is one of the essential molecules in the mitogen-induced Ig production of B cells (21). Moreover, recent studies with human IL-2 gene and human IL-6 gene transgenic mice suggested that IL-6 but not IL-2 is involved in in vivo B cell responses, because polyclonal hypergammaglobulinemia is noted in IL-6 transgenic mice, whereas IL-2 transgenic mice lack evidence of abnormal B cell maturation and function (43)(44)(45)(46). It should be noted, however, that mouse B cells but not human B cells responded against human lymphokines in the experiments using transgenic mice. ...
Excessive B cell function including autoantibody production is a common feature of SLE and considered to be intimately associated with spontaneous lymphokine secretion by themselves. To clarify roles of IL-6/IL-6 receptor autocrine activation pathway in autoantibody production observed in patients with SLE, we studied expression and function of IL-6 receptors in comparison with those of IL-2 receptors, Tac on SLE B cells. IL-6 receptors and IL-2 receptors have been detected on B cells in the blood without any in vitro stimuli in most patients with SLE. The introduction of anti-IL-6 receptor antibody, which inhibits binding to the receptors of IL-6, and anti-IL-2 receptor antibody, anti-Tac to the cultures of SLE B cells resulted in potent inhibition of spontaneous production of polyclonal Ig and anti-DNA autoantibodies. In addition, fresh SLE B cells secreted high levels of IL-6 without any in vitro stimuli. These results indicate that constitutive expression of IL-6 receptors on B cells in conjunction with spontaneous IL-6 production by B cells induces autocrine B cell activation, which may lead to B cell hyperactivity and autoantibody secretion in SLE patients. Dysregulation of B cell activity observed in patients with SLE could thus be, at least in part, independent of T cell help.
... IL-2 is a major growth factor for mature NK cells (4,5). Freshly isolated NK cells preferentially express IL-2RB, through which IL-2 plays a pivotal role in proliferation and induction of cytolytic activity (6). ...
The interleukin 2 receptor beta chain (IL-2R beta) is preferentially expressed in natural killer (NK) cells, but is not detected in a majority of resting T and B cells. We recently established a novel monoclonal antibody (mAb) to murine IL-2R beta and examined in vivo the effect of the mAb in mice. We found that intraperitoneal injection of the anti-IL-2R beta mAb into adult mice resulted in a selective in vivo elimination of splenic NK function in various mouse strains. The reduction of NK cell function is associated with complete disappearance of NK1.1+ cells in C57BL/6 mice. Other lymphocyte subsets in the thymus and spleen were uncompromised. T cell function was not affected by the mAb treatment as judged by allogeneic cytotoxic T cell induction. The single injection of anti-IL-2R beta mAb caused a long-term elimination of splenic NK cells, lasting for at least 5 wk. We also found that NK and/or NK precursor cells become susceptible to the mAb treatment only after birth, suggesting that functional maturation of NK cells in terms of IL-2R beta expression is a later event in the course of NK cell development. The use of the anti-IL-2R beta mAb will be useful in defining the physiological role of NK cells in host defense as well as dissecting their developmental pathway in vivo.
... Specific mAb for the IL-2 R H peptide are needed to detect this population in early thymus. Ishida et al. (32,37) observed the expansion of LGL only in the IL-2/IL-2-R L chain hybrid mice and not in the IL-2 transgenic mice. In the presence of high concentrations of IL-2 that were added to in vitro cultures, the LGL could be activated without the preexisting expression of IL-2 R L chain (33)(34)(35)(36). ...
These experiments were designed to evaluate the role of cytokines in early T cell development within the thymus. By using a thymic organ culture model, we have studied the influence of high dose of IL-2 (10 to 1000 IU/ml) on the cell populations that are generated during 12 days starting from a thymic rudiment of 14-day-old mouse embryo. The IL-2 treatment resulted in the expansion of Thy-1+/-, CD4-, CD8-, CD3-, Fc gamma RII+, CD5 (Lyt-1)-, HSA-, Pgp- 1+, Mel-14- population. These cells had the morphology of large granular lymphocytes and displayed broad cytotoxic activity. In addition, IL-2-treated organ cultures had a dramatic decrease in CD4+CD8+ thymocytes, a marked reduction in TCR-alpha beta+ thymocytes--even more pronounced in the TCR-V beta 6+ and TCR-V beta 8+ thymocytes--and no significant changes in the number of TCR-gamma delta+ as compared to control organ cultures.
Natural killer (NK) cells can detect and kill tumor cells and infusion of NK cells to cancer
patients may be a promising option to treat cancer. In this context, ex vivo expansion is used to
produce large quantities of activated NK cells, because sufficient numbers of these effector cells
are essential for successful NK cell based adoptive cancer immunotherapy. The development of
efficient NK cell expansion protocols and the transfer of these protocols to clinically applicable
methods represent a major challenge. To overcome this issue, the aim of my project was to
develop a clinically applicable method that yields large numbers of highly functional NK cells.
First, a fully automated technical process was developed to activate and expand NK cells with
(interleukin) IL-2 and irradiated clinical-grade feeder cells (EBV-LCL). In comparison to the
manual procedure, the automated process yielded similar NK cells in terms of cell numbers,
surface marker profile, gene expression and in vitro effector functions. Upon expansion, NK
cells up-regulated functional surface molecules, such as TRAIL, FasL, NKG2D and DNAM-1,
they increased the production of interferon (IFN)-g and tumor necrosis factor (TNF)-a and they
became more cytotoxic against tumor cell lines. Next, because in the used protocol NK cell
expansion was restricted to a period of 2-4 weeks, a more efficient protocol for long-term
expansion was developed. Manual NK cell expansion with EBV-LCL and IL-2 induced a 22–
fold mean NK cell expansion after one week that was significantly increased to 53–fold by
addition of IL-21. Furthermore, repeated stimulation with irradiated EBV-LCL and IL-2 and
addition of IL-21 at the initiation of the culture allowed sustained NK cell proliferation with
1011–fold NK cell expansion after six weeks, which is an unprecedented high expansion rate not
achieved by any other method so far. Most importantly, adoptive transfer of NK cells expanded
with this optimized protocol led to significant inhibition of tumor growth in a melanoma
xenograft mouse model, proofing the therapeutic efficacy of the ex vivo generated NK cells.
This anti-tumor efficacy was superior over that from conventionally IL-2 activated NK cells,
demonstrating that the improved NK cell expansion method enhanced not only the quantity but
also the therapeutic quality of NK cells.
In conclusion, the outcome of this project is a fully automated process for ex vivo production of
NK cells and an optimized protocol for NK cell expansion with unparalleled efficacy. The
expanded NK cells possess potent anti-tumor features and showed therapeutic efficacy in a
preclinical melanoma xenograft model. Thereby, the project serves clinical needs and makes it
possible to generate high cell doses of functional NK cells for the use in cancer immunotherapy.
