Rabbit lymphocyte subpopulations. I. Separation of Ig+ and Ig- cells and their interaction in cultures stimulated by mitogens.

Rabbit lymph node cells (Ig⁺Ig^?) were rosetted with anti-Ig antibody-coated erythrocytes and the rosetted Ig⁺ cells (B cells) were separated from unrosetted Ig^? cells (T cells) by centrifugation through Ficoll-Hypaque medium. The Ig^? cells were recovered from the top and the Ig⁺ cells from the bottom of the Ficoll-Hypaque layer. Some of the purified Ig⁺ cells lost their ability to form rosettes when cultured with the mitogen associated with streptolysin O. This suggested that the Ig⁺ population might contain two distinct subpopulations. The response of Ig⁺Ig^?, Ig⁺, and Ig^? cells to various mitogens was studied. The Ig^? cells incorporated more ³H-TdR when they were incubated by themselves than when they were cultured with Ig⁺ cells in an Ig⁺Ig^? culture. On the other hand, the Ig⁺ cells incorporated less ³H-TdR when they were incubated by themselves than when they were incubated with Ig^? cells in an Ig⁺Ig^? culture. Thus, Ig⁺ cells suppressed the response of Ig^? cells whereas Ig^? cells enhanced the response of Ig⁺ cells. We conclude that rabbit Ig⁺ cells (B cells) and Ig^? cells (T cells) interact with a feedback pattern of regulation.     

Rabbit lymphocyte subpopulations. II. Separation and characterization of two Ig+ cell subpopulations.

Rabbit lymph node cells (Ig⁺Ig^?) were separated into Ig⁺ and Ig^? populations by rosette formation with anti-Ig antibody-coated erythrocytes and centrifugation on Ficoll-Hypaque. Subpopulations of Ig⁺ cells were obtained by treating rosetted cells with autologous serum which dissociated approximately half of the rosettes. The stable rosetted cells (Ig⁺ S) were separated from the labile unrosetted cells (Ig⁺L) by centrifugation on Ficoll-Hypaque. The Ig⁺S population contained most of the Ig-secreting cells and responded poorly to mitogens. The Ig⁺L population contained few Ig-secreting cells and responded well to mitogens. Approximately 50% of Ig⁺L cells became Ig⁺S when cultured with Ig^? cells but this transition did not occur if Ig⁺S cells were added to the culture at the start of the incubation period. Purified Ig⁺ L cells lost their ability to form rosettes when cultured by themselves but retained their ability to form rosettes when cultured wih Ig^? cells. The data indicate that the Ig⁺S and Ig⁺L populations are at different stages in the differentiation of Ig⁺ cells (B cells) and that the Ig⁺L cells are subject to the regulatory influences of both Ig^? and Ig⁺S cells.     

Hematopoietic reconstitution of CD34+ cells derived from short-term cultured cord blood mononuclear cells

Ex vivo expansion of hematopoietic stem cells (HSCs) is very important for clinical applications of cord blood (CB). With the aim to find proper culture duration for ex vivo expansion, mononuclear cells (MNC) was applied as starting culture cells to expand HSCs and the repopulating potential of seven-day and fourteen-day cultured CD34⁺ cells were compared. The average expansion of total cells and CD34⁺ cells cultured for 7 days were higher than those cultured for 14 days. The results of phenotypic analysis of fresh and cultured cells showed that the percentage of CD3⁺ cells declined and the percentage of CD33⁺ cells increased during culture. The engraftment levels of fourteen-day cultured CD34⁺ cells were higher than those of fresh and seven-day cultured CD34⁺ cells. Fourteen-day cultured CD34⁺ cells also showed better multilineage reconstitution ability than fresh and seven-day cultured CD34⁺ cells. The results of the present study demonstrated that prolonged culture could preserve the hematopoietic reconstitution ability of ex vivo cultured CB cells and improve the engraftment level in NOD/SCID mice.     

CD45-positive cells are not an essential component in cardiosphere formation

The cardiosphere (CS) is composed of a heterogeneous population of cells, including CD45⁺ cells that are bone marrow (BM)-derived. However, whether the CD45⁺ cells are an essential cell component in CS formation is unknown. The current study was undertaken to address this question. Cardiospheres (CSs) were harvested from 1-week post-myocardial infarction (MI) or non-MI hearts of C57BL/6 J mice. The process of CS formation was observed by timelapse photography. To analyze the role of BM-derived CD45⁺ cells in CS formation, CD45⁺ cells were depleted from populations of CS-forming cells by immunomagnetic beads. We recorded the number of CSs formed in culture from the same amount (10⁵) of intact CS-forming cells, from CD45⁺-cell-depleted CS-forming cells and from CD45⁺ cells alone (n=6–9/cell type). CS-forming cells selectively aggregated together to form CSs by 35 h after plating. The depletion of CD45⁺ cells from CS-forming cells actually increased the formation of CSs (67±10 CSs/10⁵ cells) compared with non-depleted CS-forming cells (51±6 CSs/10⁵ cells, P<0.0001). Purified CD45⁺ cells from CS-forming cells did not form CSs in culture. Thus, BM-derived CD45⁺ cells including BM progenitors are neither necessary nor sufficient for CS formation.     

Immunological analysis and characterization of lymphocyte subsets in specimens of human hepatocellular carcinomas and metastatic liver cancers

Summary The distribution and number of CD2 (Coulter T11)⁺ cells, CD16 (Leu 11b)⁺ cells, Leu 7⁺ cells, CD8 (OKT 8)⁺ cells, CD11 (Leu 15)⁺ cells, CD4 (Leu 3a+3b)⁺ cells and Leu 10⁺ or Leu 14⁺ cells in the liver of patients with hepatocellular carcinoma (HCC) and metastatic liver cancer (MLC) were investigated using monoclonal antibodies and immunohistological methods. In the majority of those with HCC and MLC, CD8 (OKT 8)⁺, Leu 7⁺ and CD16 (Leu 11b)⁺ cells were present both in the tumor and non-tumor tissues. The CD8 (OKT 8)⁺ cells were more numerous than Leu 7⁺ and CD16 (Leu 11b)⁺ cells. No significant difference was observed in the distribution and number of Leu 7⁺ and CD16 (Leu 11b)⁺ cells, in any area, in both groups. The number of CD8 (OKT 8)⁺ cells predominated in the non-tumor area, in both groups. CD11 (Leu 15)⁺ cells and CD8 (OKT 8)⁺ cells were present in the ratio of 1:3 or 1:4. The number of CD4 (Leu 3a+3b)⁺ cells was less than that of CD8 (OKT 8)⁺ cells in both groups, especially in the tumor area. A few Leu 10⁺ or Leu 14⁺ cells were present in all areas, in both groups. In most cases of MLC, the CD8 (OKT 8)⁺ cells were absent in the tumor area. There was no correlation between the distribution and number of these cells and anti-tumor chemotherapy or non-specific immunotherapy.     

Alkaline phosphatase activity and the regulation of growth in transformed mammalian cells

The relationship between alkaline phosphatase activity and cell growth has been studied in hamster cells transformed by different carcinogens. About 90% of normal hamster embryo cells were constitutively positive for alkaline phosphatase activity (AP⁺). However, there were no AP⁺ cells in cell lines transformed after treatment with the chemical carcinogens dimethylnitrosamine or 4-nitro-quinoline-N-oxide and 0.02% and 4% AP⁺ cells in cell lines transformed by polyoma virus or Simian virus 40. The glucocorticoid hormone, prednisolone, induced alkaline phosphatase activity in 12% and 44% of the enzyme-negative (AP^?) cells in cell lines transformed by polyoma or Simian virus 40, but this hormone did not induce alkaline phosphatase activity in AP^? cells from cell lines transformed after treatment with the chemical carcinogens. Treatment of polyoma transformed AP^? cells with the mutagen N-methyl-N′-nitro-N-nitro-soguanidine produced AP⁺ cells, whereas no AP⁺ cells were found after mutagen treatment of AP^? cells from the chemically transformed cell lines. Studies on spontaneous segregation in the polyoma transformed cell line has shown that AP⁺ cells segregated AP^? cells both in vitro and in vivo, although no spontaneous segregation was observed from AP^? to AP⁺ cells. AP⁺ cells, compared to AP^? cells, showed a decrease in DNA synthesis, cell multiplication, the ability to form colonies in soft agar and tumorogenicity in animals. AP^? cells induced for alkaline phosphatase activity by prednisolone, showed the same growth properties in vitro as uninduced AP^? cells. The decreased cell growth found in AP⁺ cells which were constitutive for alkaline phosphatase activity was therefore not found in the hormone induced AP^? cells. The results indicate that constitutive alkaline phosphatase activity appears to be related to the regulation of cell growth and that AP^? cells have a selective advantage over AP⁺ cells.     

Comparative effects of adenine analogs upon metabolic cooperation between Chinese hamster cells with different levels of adenine phosphoribosyltransferase activity

Colony formation by variant Chinese hamster cells highly resistant to adenine analogs and deficient in adenine phosphoribosyltransferase (APRT) activity was measured after co-cultivation with APRT⁺, CHO-K1 cells in medium containing one of three different adenine analogs. Depending upon the density of APRT⁺ cells and the specific adenine analog, large differences in the recovery of APRT^? colonies were observed. The particular adenine analog and APRT⁺ cell density were more significant factors in the recovery of APRT^? colonies than the concentration of the analog or the level of APRT activity. The number of wild-type cells (CHO-K1) required to inhibit formation of APRT^? colonies by 50% (mean lethal density; MLD₅₀) with 65 μg/ml 8-aza-adenine (AzA) as the selective drug was 8.0 × 10⁵ cells/100 mm dish (1.5 × 10⁴/cm²). With 100 μg/ml 2,6-diaminopurine (DAP) the MLD₅₀ for CHO-K1 was 4.0 × 10⁵ cells/100 mm dish (7.3 × 10³/cm²). The MLD₅₀ for CHO-K1 when the DAP concentration was decreased to 50 μg/ml was only slightly higher, 5 × 10⁵ cells/100 mm dish (9.1 × 10³/cm²). The most toxic effect was observed with 2-fluoroadenine (FA). The MLD₅₀ for CHO-K1 in 2 μg/ml FA was 4.5 × 10⁴ cells/100 mm dish (8.2 × 10²/cm²), a cell density which permits minimal direct contact between APRT⁺ and APRT^? cells. The toxic effects of FA on individually resistant, APRT^? cells were found to be mediated by metabolites released into the medium by dying APRT⁺ cells. This metabolite toxicity to APRT^? cells was also demonstrated in mixtures with cells having only 8% of wild-type APRT activity. The MLD₅₀ for these APRT⁺ (8%) cells in 2 μg/ml FA was 7.5 × 10⁴ cells/100 dish (1.4 × 10³/cm²), a small difference from the MLD₅₀ for cells with wild-type levels of APRT activity. The differences in the recovery of APRT^? colonies from mixtures with APRT⁺ cells in these three adenine analogs are critical to the design of procedures for the selection of APRT^? cells from populations of APRT⁺ cells and emphasize the importance of establishing the parameters of metabolic cooperation, not only in terms of cell density but also with regard to the particular selective agent, in any experiment designed to determine precise mutation rates or to test putative mutagens upon mammalian cells in culture.     

Identification and characterization of autoantibody-producing B220 B (B-1) cells appearing in malarial infection

Mice with malaria showed unique immunological responses, including the expansion of NK1.1⁻TCR^int cells (extrathymic T cells). Since TCR^int cells with autoreactivity and autoantibody-producing B cells (B-1 cells) are often simultaneously activated under autoimmune conditions, it was examined whether B-1 cells were activated in the course of malarial infection. From days 14 after infection, B220^low B-1 cells appeared in the liver and spleen. The number of B220^low B cells was highest at day 14, but the ratio was highest at days 28-35. In parallel with the appearance of B220^low cells, autoantibodies against HEp-2 cells and double-stranded DNA were detected in sera. These B220^low cells had phenotypes of CD44^high, CD23⁻ and CD62L⁻. In sharp contrast, conventional B220^high B cells (B-2 cells) were CD44^low, CD23⁺ and CD62L⁺. These results suggested that malaria immune responses were not mediated by conventional T and B cells but resembled the responses during autoimmune diseases.     

Class II MHC-expressing cells in the rat adrenal gland defined by monoclonal antibodies

 The detailed distribution and heterogeneity of various immunocompetent cells were characterized in the normal adrenal gland of the rat, with special emphasis on major histocompatibility complex (MHC) class II-expressing cells and macrophages. All adrenals contained at least two different populations of cells reactive with the dendritic cell or the macrophage antibodies. These cells were clearly distinguished from adrenal parenchymal cells by their morphology and location. The majority of dendritic cells were immunoreactive for the MHC class II (Ia) antigen (MRC OX6) and/or the dendritic cell antibodies (MRC OX62), and negative for the macrophage antibodies (ED1, ED2, and/or MRC OX42), whereas the main population of macrophages was immunonegative for the former antibodies and positive for the latter. The OX62-positive cells and the OX42-labeled cells occurred exclusively throughout the medulla. The cellular density of dendritic cells in the adrenal cortex was significantly higher than that of macrophages. Double-immunoperoxidase staining for ED1 and OX6 revealed that positively stained cells could be classified into the following categories: ED1⁺OX6⁺, ED1⁺OX6⁻, and ED1⁻OX6⁺. More then 40% of OX6⁺ cells were immunoreactive for ED1 in the zona glomerulosa, while approximately 15%, 20%, and 30% of OX6⁺ cells were positive for ED1 in the zona fasciculata, zona reticularis and medulla, respectively. ED1⁺ED2⁻ cells were more frequently detected in the zona glomerulosa than in other adrenal zones. Only a few ED1⁻ED2⁺ cells were located in the zona glomerulosa, whereas a large number of them were found in the zona fasciculata. In the zona reticularis and medulla, ED1⁺ED2⁺, ED1⁺ED2⁻, and ED1⁻ED2⁺ cells were detected in the ratio 2:1:3. Our rsults suggest that dendritic cells and macrophages mature during their migration within the adrenal gland. These immunocompetent cells may contribute to a paracrine regulation of adrenal function under physiological conditions. Accepted: 3 November 1997     

Immunological properties of Fc receptor on lymphocytes. 2. Differentiation from FcR- to FcR+ cells and their functional differences in in vitro antibody response.

In in vitro plaque-forming cell (PFC) response to particulate as well as to soluble antigen, the functional difference between Fc receptor-bearing (FcR⁺) and nonbearing (FcR^?) murine splenic lymphocytes was analyzed using the EA rosetting method. In the secondary anti-horse red blood cell (HRBC) response of C3H mice, FcR^? cells showed higher IgM and IgG responses than did FcR⁺ cells. When nylon wool (NW)-purified T cells primed with keyhole limpet hemocyanin (KLH) were fractionated into FcR^? and FcR⁺ T cells, helper activity was proven in the former subset in the cooperation with syngeneic spleen cells primed with dinitrophenylated ascaris extract (DNP-Asc). FcR⁺ T cells showed essentially no helper activity. When FcR^? cells were cultured, neogenesis of FcR⁺ cells was observed on Days 3 to 5. The conversion from FcR^? to FcR⁺ cells was prominent in B cells (40 to 50%), whereas NW-purified nonadherent FcR^? T cells converted poorly (15 to 20%). The converting process was accelerated slightly by mitogens, but was least affected by antigens. To examine the possible contribution of neogeneic FcR⁺ T cells in the helper activity, KLH-primed FcR^? T cells were precultured for 7 days with homologous antigen. The specific helper activity of the cultured T cells proved to be unaffected by the depletion of neogeneic FcR⁺ T cells by EA rosetting. The neogeneic FcR⁺ T cells had no helper activity. It was thus suggested that helper T cells remain in the FcR^? cell fraction and do not convert to the FcR⁺ state during the cooperating process.     

Use of positively selected Lyt-2+ mouse splenocytes to examine interleukin-2 secretion in responses to alloantigens and to TNP-modified syngeneic cells

Current models for T-cell interactions in the generation of cytotoxic T lymphocytes have encountered a technical problem, since it has until recently been impossible to purify the peripheral Lyt-1⁺2⁺ subset from the Lyt-1⁺2^? helper cell set. Reports that the helper factor Interleukin-2 (IL-2) can be synthesized by Lyt-2⁺ spleen cells have suggested that the peripheral Lyt-2⁺ set, unlike Lyt-2⁺ thymocytes, might not depend on help from Lyt-1⁺2^? cells. To clarify this question, we have produced spleen Lyt-2⁺ cells, and the complementary Lyt-2^? set, by a positive selection method. The Lyt-2⁺ cells were able to produce high levels of anti-hapten CTL only if supplemented with either Lyt-2^? cells or with semi-purified IL-2. Although IL-2 synthesis from Lyt-2⁺ cells, or from unseparated T cells, could be induced by H-2I region-disparate stimuli, Lyt-2⁺ cells produced very little IL-2 in response to H-2I or to H-2K region-disparate cells. IL-2 synthesis in hapten-stimulated cultures was found not to depend on the presence of the hapten per se, and probably represents a response to components of the fetal calf serum supplementation. Lyt-2⁺ cells were also much less able to generate IL-2 than Lyt-2^? cells in response to these stimuli. Cell mixing experiments provided no evidence that Lyt-2⁺ cells could suppress IL-2 secretion by Lyt-2^? cells. We conclude that generation of CTL from splenic Lyt-2⁺ cells requires IL-2 produced by Lyt-2^? cells, because Lyt-2⁺ cells do not produce high levels of IL-2 themselves, even when stimulated across an H-2K difference alone.     

Immunological properties of Fc receptor on lymphocytes. 1. Functional differences between Fc receptor-positive and negative lymphocytes in humoral immune responses.

Using an EA rosetting system, it was observed that Fc receptors (FcR) were present on the surface of T cells as well as B cells, and that functional differences existed between FcR-positive (FcR⁺) and FcR-negative (FcR^?) cells in both T and B cells in in vivo humoral immune responses. Approximately 15% of splenic T cells obtained by nylon wool passage are FcR⁺. The number of surface immunoglobulinbearing cells as detected by immunofluorescent staining accounted for less than 10% of these FcR⁺ cells. FcR⁺ and FcR^? T+B-cell populations obtained from spleens contain 60 and 20% of surface immunoglobulin-positive cells, respectively. In the adoptive primary response in which horse RBC and dinitrophenyl-conjugated dextran (DNP-DE) were used as T-dependent and T-independent antigens, respectively, the majority of precursor B cells were FcR^?. In the secondary response using hapten-primed B cells and carrier-primed T cells, the majority of memory B cells for a haptenic determinant were also FcR^?. Furthermore, the majority of functional cells exerting helper activity in the same hapten-carrier system are FcR^? cells, and FcR⁺ T cells collaborate much less effectively with either memory B cells or helper FcR^? T cells.     

The Lyt phenotype of the T cells responsible for in vivo tumor rejection in syngeneic mice

Summary Spleen cells of BALB/c mice hyperimmunized with a transplantable methylcholanthrene-induced sarcoma Meth A (Meth A-Im-SPL) inhibited the growth of Meth A tumor in vivo in a tumor neutralizing test. Meth A-Im-SPL did not neutralize another antienically distinct sarcoma, Meth 1, indicating that the antitumor activity is tumor specific. Lyt-1⁺2^– cells of Meth A-Im-SPL (Im-Lyt-1⁺2^–) were the effectors since in vitro treatment of Meth A-Im-SPL with anti-Thy 1.2 or anti-Lyt 1.2 antibody plus complement completely abrogated their neutralizing activity, whereas treatment with anti-Lyt 2.2 plus complement did not. To further confirm the effector activity of Im-Lyt-1⁺2^– cells, T cell subpopulations were separated from Meth A-Im-SPL by the panning method. The purified Im-Lyt-1⁺2^–, but not Im-Lyt-1⁺2⁺ cells neutralized the tumor in athymic nu/nu mice as efficiently as in +/+ mice, suggesting that the donor Im-Lyt-1⁺2^– cells but not recipient T cells were primarily responsible for neutralizing the tumor. The present study, however, did not exclude the possible contribution of recipient T cells to the tumor neutralization and this is open to further investigation.Abbreviations Meth A-Im-SPL Meth A-immune mouse spleen cells - Meth 1-Im-SPL Meth 1-immune mouse spleen cells - sIg⁺ cells surface immunoglobulin positive cells - moAb monoclonal antibody     

CD4+CD62L+ Central Memory T Cells Can Be Converted to Foxp3+ T Cells

The peripheral Foxp3⁺ Treg pool consists of naturally arising Treg (nTreg) and adaptive Treg cells (iTreg). It is well known that naive CD4⁺ T cells can be readily converted to Foxp3⁺ iTreg in vitro, and memory CD4⁺ T cells are resistant to conversion. In this study, we investigated the induction of Foxp3⁺ T cells from various CD4⁺ T-cell subsets in human peripheral blood. Though naive CD4⁺ T cells were readily converted to Foxp3⁺ T cells with TGF-β and IL-2 treatment in vitro, such Foxp3⁺ T cells did not express the memory marker CD45RO as do Foxp3⁺ T cells induced in the peripheral blood of Hepatitis B Virus (HBV) patients. Interestingly, a subset of human memory CD4⁺ T cells, defined as CD62L⁺ central memory T cells, could be induced by TGF-β to differentiate into Foxp3⁺ T cells. It is well known that Foxp3⁺ T cells derived from human CD4⁺CD25^- T cells in vitro are lack suppressive functions. Our data about the suppressive functions of CD4⁺CD62L⁺ central memory T cell-derived Foxp3⁺ T cells support this conception, and an epigenetic analysis of these cells showed a similar methylation pattern in the FOXP3 Treg-specific demethylated region as the naive CD4⁺ T cell-derived Foxp3⁺ T cells. But further research showed that mouse CD4⁺ central memory T cells also could be induced to differentiate into Foxp3⁺ T cells, such Foxp3⁺ T cells could suppress the proliferation of effector T cells. Thus, our study identified CD4⁺CD62L⁺ central memory T cells as a novel potential source of iTreg.     

Human Natural Killer Cell Maturation Defect Supports In Vivo CD56bright to CD56dim Lineage Development

Two populations of human natural killer (NK) cells can be identified in peripheral blood. The majority are CD3⁻CD56^dim cells while the minority exhibits a CD3⁻CD56^bright phenotype. In vitro evidence indicates that CD56^bright cells are precursors of CD56^dim cells, but in vivo evidence is lacking. Here, we studied NK cells from a patient that suffered from a melanoma and opportunistic fungal infection during childhood. The patient exhibited a stable phenotype characterized by a reduction in the frequency of peripheral blood CD3⁻CD56^dim NK cells, accompanied by an overt increase in the frequency and absolute number of CD3⁻CD56^bright cells. These NK cells exhibited similar expression of perforin, CD57 and CD158, the major activating receptors CD16, NKp46, NKG2D, DNAM-1, and 2B4, as well as the inhibitory receptor CD94/NKG2A, on both CD56^bright and CD56^dim NK cells as healthy controls. Also, both NK cell subpopulations produced IFN-γ upon stimulation with cytokines, and CD3⁻CD56^dim NK cells degranulated in response to cytokines or K562 cells. However, upon stimulation with cytokines, a substantial fraction of CD56^dim cells failed to up-regulate CD57 and CD158, showed a reduction in the percentage of CD16⁺ cells, and CD56^bright cells did not down-regulate CD62L, suggesting that CD56^dim cells could not acquire a terminally differentiated phenotype and that CD56^bright cells exhibit a maturation defect that might result in a potential altered migration pattern. These observations, support the notion that NK cells of this patient display a maturation/activation defect that precludes the generation of mature NK cells at a normal rate accompanied by CD56^dim NK cells that cannot completely acquire a terminally differentiated phenotype. Thus, our results provide evidence that support the concept that in vivo CD56^bright NK cells differentiate into CD56^dim NK cells, and contribute to further understand human NK cell ontogeny.     

Activation of Glioma Cells Generates Immune Tolerant NKT Cells

Therapeutic outcomes of glioma are currently not encouraging. Tumor tolerance plays an important role in the pathogenesis of glioma. It is reported that micro RNAs (miR) are associated with tumor development. This study aims to investigate the role of miR-92a in the development of tolerant natural killer T (NKT) cells. In this study, U87 cells (a human glioma cell line) and primary glioma cells were prepared. The assessment of miR-92a was performed by real time RT-PCR. The expression of interleukin (IL)-10 and IL-6 in NKT cells was evaluated by flow cytometry. Results showed that abundant IL-6⁺ IL-10⁺ NKT cells were detected in glioma tissue. Cultures of glioma cells and NKT cells induced the expression of IL-6 and IL-10 in NKT cells. Glioma cells expressed miR-92a; the latter played a critical role in the induction of IL-6 and IL-10 expression in NKT cells. The expression of the antitumor molecules, including perforin, Fas ligand, and interferon-γ, was significantly attenuated compared with control NKT cells. The IL-6⁺ IL-10⁺ NKT cells showed less capability in the induction of apoptosis in glioma cells, but showed the immune suppressor functions on CD8⁺ T cell activities. We conclude that glioma-derived miR-92a induces IL-6⁺ IL-10⁺ NKT cells; this fraction of NKT cells can suppress cytotoxic CD8⁺ T cells.     

Optimal population of FoxP3+ T cells in tumors requires an antigen priming-dependent trafficking receptor switch

FoxP3⁺ T cells populate tumors and regulate anti-tumor immunity. The requirement for optimal population of FoxP3⁺ regulatory T cells in tumors remains unclear. We investigated the migration requirement and stability of tumor-associated FoxP3⁺ T cells. We found that only memory, but not naïve, FoxP3⁺ T cells are highly enriched in tumors. Almost all of the tumor-infiltrating FoxP3⁺ T cells express Helios, an antigen associated either with thymus-generated FoxP3⁺ T cells or activated T cells in the periphery. The tumor-infiltrating FoxP3⁺ T cells largely lack CD62L and CCR7, two trafficking receptors required for T cell migration into secondary lymphoid tissues. Instead, the tumor infiltrating FoxP3⁺ T cells highly express memory/tumor-associated CCR8 and CXCR4. Antigen priming is required for induction of this trafficking receptor phenotype in FoxP3⁺ T cells and only antigen primed, but not antigen-inexperienced naive, FoxP3⁺ T cells can efficiently migrate into tumors. While the migration of FoxP3⁺ T cells into tumors was a readily detectable event, generation of induced FoxP3⁺ T cells within tumors was unexpectedly inefficient. Genetic marking of current and ex-FoxP3⁺ T cells revealed that tumor-infiltrating FoxP3⁺ T cells are highly stable and do not readily convert back to FoxP3⁻ T cells. Taken together, our results indicate that population of tumors with thymus-generated FoxP3⁺ T cells requires an antigen priming-dependent trafficking receptor switch in lymphoid tissues.     

外周血Treg细胞、T淋巴细胞及其亚群与早期宫颈癌的关系及对淋巴结转移的预测价值研究

摘要 目的：分析外周血Treg细胞、T淋巴细胞及其亚群与早期宫颈癌的关系及对淋巴结转移的预测价值。方法：选择我院自2017年1月至2020年12月接诊的60例接受子宫颈癌根治术及盆腔淋巴清扫术的早期宫颈癌患者作为观察组,另选同期的60例健康体检者作为对照组。比较两组外周血Treg细胞、T淋巴细胞及其亚群水平,使用受试者工作特征曲线（ROC）下面积（AUC）评价外周血Treg细胞、T淋巴细胞及其亚群对淋巴结转移的预测效能。结果：观察组外周血Treg细胞、CD8⁺T细胞水平高于对照组,CD3⁺T细胞、CD4⁺T细胞、CD4⁺/CD8⁺比值均低于对照组（P＜0.05）;观察组术后外周血Treg细胞、CD8⁺T细胞水平较术前降低,CD3⁺T细胞、CD4⁺T细胞、CD4⁺/CD8⁺比值均较术前升高（P＜0.05）;在60例早期宫颈癌患者中,发生淋巴结转移12例;淋巴结转移组术前外周血Treg细胞水平、CD8⁺T细胞高于非淋巴结转移组,CD3⁺T细胞、CD4⁺T细胞、CD4⁺/CD8⁺比值均低于非淋巴结转移组（P＜0.05）;经多因素Logistic回归分析,外周血Treg细胞、CD3⁺T细胞、CD4⁺/CD8⁺比值均是早期宫颈癌患者发生淋巴结转移的独立预测因素（P＜0.05）;经ROC曲线分析,外周血Treg细胞、CD3⁺T细胞联合CD4⁺/CD8⁺比值预测早期宫颈癌患者发生淋巴结转移的AUC为0.910。结论：外周血Treg细胞、T淋巴细胞及其亚群水平与早期宫颈癌的病情演变有关,其中外周血Treg细胞、CD3⁺T细胞联合CD4⁺/CD8⁺比值预测淋巴结转移的效能较好,值得进一步研究应用。     

Human amniotic fluid-derived c-kit(+) and c-kit (-) stem cells: growth characteristics and some differentiation potential capacities comparison

Amniotic fluid (AF) contains heterogeneous and multipotential cell types. A pure mesenchymal stem cells group can be sorted from AF using flow cytometry. In order to evaluate a possible therapeutic application of these cells, the human AF-derived c-kit⁺ stem cells (c-kit⁺ AFS) were compared with the c-kit⁻ (unselected) stem cells (c-kit⁻ AFS). Our findings revealed that the optimal period to obtain c-kit⁺ AFS cells was between 16 and 22 weeks of gestation. Following cell sorting, c-kit⁺ AFS cells shared similar morphological and proliferative characteristics as the c-kit⁻ AFS cells. Both c-kit⁺ and c-kit⁻ AFS cells had the characteristics of mesenchymal stem cells through surface marker identification by flow cytometric and immunocytochemical analysis. Both c-kit⁺ and c-kit⁻ AFS cells could differentiate along adipogenic and osteogenic lineages. However, the myocardial differentiation capacity was enhanced in c-kit⁺ AFS cells by detecting GATA-4, cTnT, α-actin, Cx43 mRNA and protein expression after myocardial induction; whereas induced c-kit⁻ AFS cells were only detected with GATA-4 mRNA and protein expression. The c-kit⁺ AFS cells could have potential clinical application for myogenesis in cardiac regenerative therapy.