Unique progenitors in mouse lymph node develop into CD127+ NK cells: thymus-dependent and thymus-independent pathways

A subset of natural killer (NK) cells in normal mouse lymph node (LN) expresses CD127 (IL-7 receptor-α chain) and is thought to derive from the thymus. However, CD127(+) NK cells are found in the LN of athymic mice. Therefore, the origin of CD127(+) NK cells in the LN is unclear. Here, we have identified unique NK-cell progenitors (NKPs) in the LN that express the pan-NK cell marker CD49b and CD127 but lack CD122 and lineage markers. The LN NKPs develop in vitro into CD127(+) NK cells that display natural cytotoxicity and cytokine production capacity. They also become CD127(+) NK cells in lymphopenic mice that received a transplant. LN NKPs can be divided into stem cell antigen-1 (Sca-1)(hi) and Sca-1(lo) subsets. The latter comprise ～ 60% of LN NKPs in normal mouse and < 10% of athymic mouse LN NKPs. Whereas both Sca-1(hi) and Sca-1(lo) NKPs develop into CD127(+) NK cells in vitro, only those derived from Sca-1(lo) LN NKPs have rearranged TCRγ genes. Thus, CD127(+) NK cells in the LN seem to be generated, at least in part, from both thymus-dependent Sca-1(lo) and thymus-independent Sca-1(hi) LN NKPs.     

Differentiation of mouse embryonic stem cells into hepatocytes in vitro and in vivo

OBJECTIVE: Embryonic stem (ES) cells have a pluripotent ability to differentiate into a variety of cell lineages. Cell‐to‐cell contact is important for cell differentiation. Mouse ES cells were cocultured with mouse fetal liver cells and the green fluorescent protein (GFP) positive ES cells were transplanted into rats liver through the portal vein in order to investigate their potential to differentiate into hepatocytes. METHODS: Mouse ES cells were cocultured with the mouse fetal liver cell line, BNL.CL₂. They did not make direct contact; instead the culture media was exchanged freely. After coculture for 48 h, albumin, transthyretin, glucose 6 phosphates, hepatic nuclear factor 4 and SEK1 mRNA were assayed by RT‐PCR, and alpha‐fetoprotein by immunohistochemistry. The morphology was investigated by microscopy. After transplantion of the GFP‐positive ES cells, the whole liver was removed from a rat every four days. The liver slices were examined under a fluorescent microscope to detect the GFP‐positive cells. Albumin was detected on the same slices by immunohistochemistry. RESULTS: After coculture with BNL.CL₂ cells, the differentiated ES cells had the same morphology as the BNL.CL₂ cells, and albumin, transthyretin, glucose 6 phosphates and SEK‐1 mRNA were found by RT‐PCR, and alpha‐fetoprotein was detected immuno­histochemically. The transplanted GFP‐positive ES cells were found in the rats’ liver slices by GFP fluorescence, and development of teratomas was not observed. The immunohistochemistry results indicated that the transplanted GFP‐positive ES cells retained an albumin‐producing ability. CONCLUSIONS: Cell‐to‐cell contact is important for the differentiation of ES cells. Mouse embryonic stem cells can differentiate into hepatocytes directly either in vitro or in vivo.     

Role of T and NK cells and IL7/IL7r interactions during neonatal maturation of lymph nodes

Lymph node (LN) development depends on prenatal interactions occurring between LN inducer and LN organizer cells. We have distinguished defects in LN formation due to failure in embryonic development (aly/aly) from defects in postnatal maturation (Il2rgamma(-/-)Rag2(-/-)). Both mutant strains form normal primordial LNs with differing fate. In aly/aly mice, the LN primordium dissipates irreversibly late in gestation; in contrast, Il2rgamma(-/-)Rag2(-/-) LN anlage persists for a week after birth but disperses subsequently, a process reversible by neonatal transfer of WT IL7r(+) TCR(+) T or natural killer (NK) cells, suggesting a role for IL7/IL7r interactions. Thus, we reveal a unique stage of postnatal LN development during which mature lymphocytes and IL7/IL7r interactions may play an important role.     

Lymphoid cells in lymph nodes and peripheral blood of patients with squamous cell carcinoma of the head and neck

Summary Fifty-five patients with squamous cell carcinoma of the head and neck were evaluated immunologically by measuring the level of T cells (E-RFC) and high affinity subset T cells (E-29) in the peripheral blood and peritumorous lymph nodes. A significant decrease (p<0.05) in mean percentage of E-29 was observed in cancer patient peripheral blood. In peritumorous lymph nodes, there was no difference in terms of total T cells or of high affinity subset T cells, as compared to non-malignant lymph nodes, or between tumor-free and metastatic lymph nodes. Macrophage content was much higher in metastatic than in tumor-free lymph nodes (p<0.05) and these macrophages frequently appeared to be more active when tested in phagocytosis of sheep red blood cells sensitized with IgG or IgM+C.Supported in part by grant no. 79-7-0670 from the DGRST     

Human virus-specific effector-type T cells accumulate in blood but not in lymph nodes

It is believed that the size of the CD8(+) T-cell pool is fixed and that with every new viral challenge, the size of the pre-existing memory-cell population shrinks to make way for the new virus-specific cells. CMV-seropositive individuals have high numbers of CMV-specific resting-effector type CD8(+) T cells in their peripheral blood (PB). This prompted us to investigate whether CMV infection limits immunologic space at sites where immune reactions are initiated, such as in the lymph nodes (LNs). LN and paired PB samples were analyzed for CMV-, EBV-, and influenza-specific CD8(+) T cells. In marked contrast to blood, LNs contained significantly lower numbers of CX3CR1-expressing effector-type CD8(+) T cells, whereas the CMV-specific cells that were found in the LNs resembled polyfunctional memory-type cells. In contrast, EBV- and influenza-specific CD8(+) T cells were highly similar between PB and LNs both in number and function. Therefore, it is unlikely that CMV-specific CD8(+) T cells in the LNs restrain the immunologic space of other virus-specific cells.     

Lymphoid subsets in acute myeloid leukemias: Increased number of cells with NK phenotype and normal T-cell distribution

Summary Natural killer (NK) and T subsets were analyzed with appropriate dual labeling by flow cytometry in peripheral blood (PB) (66 cases) and bone marrow (BM) (55 cases) from patients with de novo AML in order to determine: (a) their distribution at diagnosis, (b) the correlation between PB and BM in NK subpopulations, (c) their relationship with the clinical and hematological disease characteristics, and (d) the changes occurring upon achieving complete remission (CR). NK cells defined by the expression of CD56 in the absence of CD3 were significantly increased at diagnosis and their levels in PB correlated with those of BM. By contrast, NK subsets defined by CD16 expression (CD16+ CD2+ and CD16+ CD2– NK-cell subsets) as well as T lymphocytes with NK activity (CD56+ CD3+), although increased in PB, displayed normal levels in BM. An additional observation of interest was the expansion of an immature NK population lacking CD16 Ag expression (CD56+CD16–). AML cases were divided into two groups according to the absolute number of NK cells in PB; patients with the highest levels showed an increased proportion of blast cells in PB (p=0.01), monocytic subtypes (p=0.03), and expression of CD11b, CD14, and CD4 antigens (p=0.05). Infections at diagnosis were not related to the level of NK cells. In 19 patients who achieved complete remission the number of CD56+CD3– cells tended to be reduced to within the normal range. Other T-cell populations, including the CD4 naive and memory cells, were also explored, their distribution being normal in the PB of AML patients. By contrast, the cytotoxic subset CD8+/CD57+was significantly increased (p< 0.001). These data point to the existence of marked alterations of NK cells in AML patients, possibly reflecting a host-tumor immunological interaction.     

Myoinjury transiently activates muscle antigen–specific CD8+ T cells in lymph nodes in a mouse model

Objective

To investigate the influence of myoinjury on antigen presentation to T cells in draining lymph nodes (LNs).

Methods

Muscle crush was performed in mice injected with exogenous ovalbumin (OVA) and in transgenic SM‐OVA mice expressing OVA as a muscle‐specific self antigen. Antigen exposure and the resulting stimulation of T cell proliferation in draining LNs was assessed by transferring carboxyfluorescein succinimidyl ester (CFSE)–labeled OVA‐specific CD8+ and CD4+ T cells from OT‐I and OT‐II mice and by measuring the dilution of CFSE, which directly reflects their proliferation. The role of monocyte‐derived dendritic cells (DCs) in T cell priming was assessed using pharmacologic blockade of DC migration. Immunofluorescence was used to detect CD8+ T cells, inflammatory monocyte‐derived DCs, and type I major histocompatibility complex (MHC)–expressing myofibers in crushed muscle, and to assess expression of perforin, interferon‐γ (IFNγ), interleukin‐2 (IL‐2), IL‐10, and transforming growth factor β1 (TGFβ1).

Results

OVA injection into intact muscle induced strong proliferation of CD4+ and CD8+ T cells, indicating efficient exposure of soluble antigens in draining LNs. OVA‐specific CD8+ T cell proliferation in draining LNs of SM‐OVA mice required myoinjury and was unaffected by pharmacologic inhibition of monocyte‐derived DC migration. On day 7 postinjury, activated CD8+ T cells expressing perforin, IFNγ and IL‐2 were transiently detected in crushed muscle, and these cells were in close contact with class I MHC–positive regenerating myofibers. Beginning on day 7, the immunosuppressive cytokines IL‐10 and TGFβ1 were conspicuously expressed by CD11b+ cells, and CD8+ T cells rapidly disappeared from the healing muscle.

Conclusion

Myofiber damage induces an episode of muscle antigen–specific CD8+ T cell proliferation in draining LNs. Activated CD8+ T cells transiently infiltrate the injured muscle, with prompt control by immunosuppressive cues. Inadequate control might favor sustained autoimmune myositis.

     

Identification of expandable human hepatic progenitors which differentiate into mature hepatic cells in vivo

BACKGROUND: Liver diseases include a wide spectrum of both acute and chronic conditions which are associated with significant morbidity and mortality worldwide. Hepatocyte transplantation has therapeutic potential in the treatment of liver diseases, but its clinical use is hampered by the lack of donor tissue. Generation of hepatocytes in vitro from adult or fetal liver cell progenitors or, alternatively, identification of a progenitor population which in vivo can generate mature liver cells could solve this problem. METHODS: CD117+/CD34+/Lin- human fetal liver cells were isolated by magnetic cell sorting and expanded in culture. Both freshly isolated and in vitro expanded cells in various passages were studied for their ability to be functional in hepatic parenchyma following d-galactosamine (GalN) induced injury in nude C57 black mice. RESULTS: Freshly isolated and in vitro expanded CD117+/CD34+/Lin- cells, when transplanted intrasplenically into GalN treated mice, morphologically and functionally differentiated into hepatocytes and cholangiocytes. Human specific albumin, alpha fetoprotein, cytokeratin 19, and antitrypsin mRNA were expressed in mouse liver. In addition, the human progenitor cells expressed glucose-6-phosphatase, glycogen, albumin, gamma glutamyl transpeptidase, and dipeptidyl peptidase IV after transplantation. Expanded cells in various passages maintained their capacity to differentiate into functional liver cells. CONCLUSIONS: Fetal liver CD117+/CD34+/Lin- progenitors and their progeny proliferated in vitro and also functionally differentiated into mature hepatic cells in an acute liver injury model. Successful in vitro expansion of liver progenitor cells provides a basis for developing cell therapy strategies, metabolic and toxicity testing systems, and may serve as a vehicle for gene therapy.     

食管癌贲门癌患者T淋巴细胞、NK细胞免疫功能检测及意义

目的本研究检测食管癌、贲门癌患者的免疫功能,寻找肿瘤患者的病情量化指标,用来预测和判定临床治疗效果。方法采集本院胸外科手术前的食管癌贲门癌患者115例及359例正常体检者的血液标本,应用流式细胞术检测外周血T淋巴细胞表面CD3、CD4、CD8及NK细胞(CD56)的表达。结果食管癌、贲门癌组与健康对照组相比较CD3、CD4有明显下降(P0.05),而CD8、CD56有明显的增高(P0.05)。将癌症组分为淋巴结转移组和无淋巴转移组,两组间CD3、CD4、CD8、CD56的差异均无统计学意义(P0.05)。结论食管癌外周血T细胞免疫检测有助于了解疾病的发展进程。     

Identification, activation, and selective in vivo ablation of mouse NK cells via NKp46

Natural killer (NK) cells contribute to a variety of innate immune responses to viruses, tumors and allogeneic cells. However, our understanding of NK cell biology is severely limited by the lack of consensus phenotypic definition of these cells across species, by the lack of specific marker to visualize them in situ, and by the lack of a genetic model where NK cells may be selectively ablated. NKp46/CD335 is an Ig-like superfamily cell surface receptor involved in human NK cell activation. In addition to human, we show here that NKp46 is expressed by NK cells in all mouse strains analyzed, as well as in three common monkey species, prompting a unifying phenotypic definition of NK cells across species based on NKp46 cell surface expression. Mouse NKp46 triggers NK cell effector function and allows the detection of NK cells in situ. NKp46 expression parallels cell engagement into NK differentiation programs because it is detected on all NK cells from the immature CD122(+)NK1.1(+)DX5(-) stage and on a minute fraction of NK-like T cells, but not on CD1d-restricted NKT cells. Moreover, human NKp46 promoter drives NK cell selective expression both in vitro and in vivo. Using NKp46 promoter, we generated transgenic mice expressing EGFP and the diphtheria toxin (DT) receptor in NK cells. DT injection in these mice leads to a complete and selective NK cell ablation. This model paves a way for the in vivo characterization and preclinical assessment of NK cell biological function.     

Superior growth of glycophosphatidy linositol-anchored protein-deficient progenitor cells in vitro is due to the higher apoptotic rate of progenitors with normal phenotype in vivo

OBJECTIVE: Recently, phenotypically normal CD34 cells from the marrow of patients with paroxysmal nocturnal hemoglobinuria (PNH) were reported to show impaired growth and elevated Fas receptor expression as compared to glycophosphatidylinositol-anchored protein (GPI-AP)-deficient CD34 cells and CD34 cells from normal individuals. These results are consistent with the theory that PNH cells have an intrinsic growth advantage, but their superior expansion in vitro could also be the outcome of selective extrinsic pressure in vivo. MATERIAL AND METHODS: Growth characteristics, competitive features, and susceptibility to apoptosis of sorted normal or GPI-AP-deficient CD34(+) cells derived from PNH patients were assessed in suspension and methylcellulose cultures. RESULTS: When we directly compared the growth of patients' CD34 cells, separated based on expression of GPI-AP CD55 and CD59, in most of the patients studied, mutant CD34 cells showed higher progeny production and outgrew phenotypically normal CD34 cells derived from PNH patients in mixing experiments. However, their proliferation rate did not exceed that of control CD34 cells. To determine whether deficient growth of phenotypically normal CD34 cells in PNH was secondary to a pre-existing in vivo insult, we determined the fraction of apoptotic cells within fresh normal and PNH CD34 cells. Normal CD34 cells from PNH patients showed a high proportion of apoptotic cells and higher Fas expression, while GPI-AP-deficient and control CD34 cells showed similar, low rates of apoptosis. After correction for pre-existing apoptosis, the proliferation potential of normal and PNH CD34 cells was similar. CONCLUSIONS: These results strongly suggest that clonal expansion of GPI-AP-deficient progenitor cells from PNH patients is due to their selection in the hostile marrow environment of the patient.