Mel14+CD4+ T cells from aged mice display functional and phenotypic characteristics of memory cells

Aging is accompanied by an increased fraction of memory CD4⁺T cells. Despite the fact that human memory cells have beenreported to produce high levels of IL-2, studies in mice andman indicate an age-related decline in IL-2 production. In thepresent study, we examined whether these conflicting resultsdepend on the activation pathway employed in a comparison ofphenotypically distinct CD4⁺ T cells from young and aged mice.Our data indicate an age-related decline in IL-2 productionby CD4⁺ T cells when the cells were stimulated with concanavalinA in the presence of accessory cells or the combination of immobilizedanti-CD3 and soluble anti-CD28. However, when CD4⁺ T cells wereonly stimulated with Immobilized anti-CD3, an age-related increasein IL-2 production was observed. This age-related increase inIL-2 could be attributed to the ability of CD4⁺ T cells fromaged mice to produce IL-4 on this stimulation, since anti-IL-4inhibited the IL-2 production In these cultures to levels foundwith cells from young mice. The addition of exogenous IL-4 greatlyenhanced the IL-2 production of CD4⁺ T cells from young miceto levels far beyond that of the aged counterparts, emphasizingthe dominant role of IL-4 In the induction of IL-2 stimulatedwith immobilized anti-CD3. No differences were observed in theactivation requirements of Mel14^– CD4⁺ T cells from youngand aged mice. However, Mel14⁺ CD4⁺T cells from aged mice werefunctionally and phenotypically more mature than their youngcounterparts, since they were capable of IL-2 and IL-4 productionin response to antl-CD3 without the need of CD28 triggeringand expressed Pgp-1 and ICAM-1 in a higher density. Our dataindicate therefore that Mel14 is not a stable marker for naiveCD4⁺ T cells and might not be appropriate to distinguish thesecells from memory cells.     

Anomalous reactivity of anti-T cell sera on spleen cells from T-cell-deprived mice and on mitogen-stimulated nude mice spleen cells

Recent results have shown that various alloantisera which react in the expected fashion on lymphocyte populations can react anomalously against cultured tumor cell populations because of the presence of contaminating antibodies against murine leukemia virus (MLV) antigens. Since it is now known that activation of MLV can occur in certain types of dividing lymphocyte populations, anti-T cell sera were tested on lymphoid cell populations in which T cells were absent or greatly reduced in numbers, but where activation of MLV in the B cell population would be expected. Whereas normal, freshly harvested, nude mice spleen cells were unreactive, in vitro:stimulation of these cells with the B cell mitogen, lipopolysaccharide, led to a high degree of sensitivity to the cytotoxic effects of anti-T alloantisera or heteroantisera. Spleen cells from adult thymectomized, lethally-irradiated, bone marrow-reconstituted mice also showed unexpected reactivity with the anti-T cell sera. In both cases, reactivity was noted only if absorbed rabbit serum was used as a complement source in the cytotoxic assays. The anomalous anti-B cell activity of the anti-T cell sera could be removed by absorption with relatively small numbers of cells from Thy-negative cultured tumor cell lines, including fibrosarcomas, but not by absorption with thymocytes. Hence, activated or stimulated B cells may react strongly with allo-or hetero- anti-T cell sera under certain conditions, and this anomalous reactivity appears unrelated to the presence of the anti-T cell antibodies in the sera.     

The phenotypic heterogeneity of mouse thymic stromal cells

Sixteen monoclonal antibodies (mAb) were produced against mouse thymic stromal elements. These mAb fell into two groups of reactivity: (i) thymic epithelial markers (screened and presented according to the guidelines proposed in the 1989 Rolduc Thymic Epithelial Workshop); and (ii) non-epithelial thymic markers. Specificities of these mAb included extensive subpopulations of both epithelial and non-epithelial thymic stromal cells, as well as isolated stromal cells, demonstrating some of the complex microspecificities in existence within the thymic microenvironment. Furthermore, six of these mAb demonstrated shared antigenicity between thymocytes and thymic stromal cells, revealing greater similarities than previously recognized between these two components. Three mAb detected antigens illustrating three consecutive layers of the blood-thymus barrier: the vascular endothelium; connective tissue of the capsule and perivascular spaces; and the connective tissue associated with the basal laminae lining these regions. This study illustrates unequivocably that there are indeed complex and varied microenvironments existing within the thymus, and emphasizes the need for reclassification of these cells.     

Strain-dependent disruption of the functional activity of bone marrow stromal cells from mice infected with influenza A]

Infection of mice with influenza A virus disturbs the proliferative activity of bone marrow stromal fibroblasts involved in regulation of bone marrow hemopoiesis. Myelosuppression induced by influenza A virus can be a result of dysfunction of bone marrow stromal tissue. Disorders in multiplication of bone marrow fibroblasts induced by influenza virus are strain-dependent.     

Follicular lymphoma triggers phenotypic and functional remodeling of the human lymphoid stromal cell landscape

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Dendritic cells present an intracellular viral antigen derived from apoptotic cells and induce a T-cell response

We investigated if dendritic cells (DCs) were able to present intracellularly located antigens derived from apoptotic cells to T cells, thereby inducing a CD4+ and a CD8+ response. A transfected cell line with the cytomegalovirus-derived protein pp65 was triggered to go into apoptosis by ultraviolet B (UVB) irradiation, and after the uptake of apoptotic cells by DC, the activation and proliferation of T cells were determined. We found that DC efficiently phagocytosed apoptotic cells and induced a CD4+ and a CD8+ T-cell response specific for the viral protein pp65. This mechanism can be useful for vaccination studies to induce an antiviral immune response.     

Dendritic immune complex trapping cells in the spleen of the snake,

The spleen of the snake , was investigated as to its general histology as well as the presence of immune complex trapping cells both at the light and electron microscopical level. Histological examination revealed that the spleen of this reptile was encapsulated and contained some trabeculae. In the splenic parenchyma two different regions could be distinguished: viz. red and white pulp. The white pulp appeared to be arranged around “central arterioles” and their smaller branches extending towards the periphery of the white pulp. The red pulp was composed of blood sinusoids and cell cords. Electron microscopy revealed at least three types of non-lymphoid cells in the white pulp of the spleen of python: (1) reticulum cells, forming the framework; (2) some macrophages and (3) dendritic cells predominantly located in the periphery of the white pulp. Of these types of non-lymphoid cells, only dendritic cells were able to trap and to retain intravenously injected horseradish peroxidase (HRP)-rabbit-anti-HRP immune complexes on their cell surface as determined by enzymehistochemistry at the light and electron microscopical level. These dendritic cells were frequently found in association with collagen fibres and did not engulf large quantities of carbon particles. These data suggest that dendritic cells in the spleen of the python might be the phylogenetic precursors of the mammalian follicular dendritic cells.     

Dendritic cells derived from bone marrow cells fail to acquire and present major histocompatibility complex antigens from other dendritic cells

Dendritic cells stimulate primary T-cell responses and a major activation route is via presentation of antigens pre-processed by other dendritic cells. This presentation of pre-processed antigens most likely proceeds through transfer of functional major histocompatibility complex (MHC) antigens through exosomes, 'live nibbling' or apoptotic vesicles. We hypothesized that not all dendritic cell populations may both donate MHC antigen to dendritic cells and present antigens acquired from other dendritic cells. All populations tested, including those derived from bone marrow precursor cells stimulated primary, allogeneic T-cell responses and acted as accessory cells for mitogen stimulation. Populations of responder type, splenic dendritic cells promoted allogeneic responses indirectly but those derived from bone marrow cells blocked rather than promoted T-cell proliferation. To identify mechanisms underlying this difference we studied transfer of I-A antigens between cells. Active, two-way transfer of allogeneic I-A occurred between splenic primary antigen presenting cells including CD8alpha+ lymphoid dendritic cells, CD8alpha- myeloid dendritic cells and B220+ cells; all these cell types donated as well as acquired MHC molecules. By contrast, the bone marrow-derived dendritic cells donated I-A antigens but acquired negligible amounts. Thus, dendritic cells derived directly from bone marrow cells may stimulate primary T-cell responses through transferring functional MHC to other dendritic cells but may not be able to acquire and present antigens from other dendritic cells. The evidence suggests that T-cell activation may be blocked by the presence of dendritic cells that have not matured through lymphoid tissues which are unable to acquire and present antigens pre-processed by other dendritic cells.     

Tissue engineering of corneal stromal layer with dermal fibroblasts: phenotypic and functional switch of differentiated cells in cornea

Previously, we successfully engineered a corneal stromal layer using corneal stromal cells. However, the limited source and proliferation potential of corneal stromal cells has driven us to search for alternative cell sources for corneal stroma engineering. Based on the idea that the tissue-specific environment may alter cell fate, we proposed that dermal fibroblasts could switch their phenotype to that of corneal stromal cells in the corneal environment. Thus, dermal fibroblasts were harvested from newborn rabbits, seeded on biodegradable polyglycolic acid (PGA) scaffolds, cultured in vitro for 1 week, and then implanted into adult rabbit corneas. After 8 weeks of implantation, nearly transparent corneal stroma was formed, with a histological structure similar to that of its native counterpart. The existence of cells that had been retrovirally labeled with green fluorescence protein (GFP) demonstrated the survival of implanted cells. In addition, all GFP-positive cells that survived expressed keratocan, a specific marker for corneal stromal cells, and formed fine collagen fibrils with a highly organized pattern similar to that of native stroma. However, neither dermal fibroblast-PGA construct pre-incubated in vitro for 3 weeks nor chondrocyte-PGA construct could form transparent stroma. The results demonstrated that neonatal dermal fibroblasts could switch their phenotype in the new tissue environment under restricted conditions. The functional restoration of corneal transparency using dermal fibroblasts suggests that they could be an alternative cell source for corneal stroma engineering.     

In vitro responses of spleen cells from Trichinella spiralis-infected mice

The in vitro cellular immune responses of spleen cells from mice infected with Trichinella spiralis and immunized with BCG have been investigated. ICR/CD-1 mice were originally infected with 200 T. spiralis larvae 22 days prior to infection with 4 X 10(6) viable or heat-killed mycobacteria. Analysis of the splenic cell populations indicated that significant increases in adherent cells (macrophages) were noted only in groups previously infected with the nematode; the concentration of non-adherent cells (lymphocytes) did not vary insignificantly among any of the experimental groups. Assay of blast cell transformation and 3H-thymidine incorporation demonstrated the ability of T. spiralis infection to potentiate in vitro cellular immune reactions. These findings support earlier in vivo studies concerning nematode-induced immunopotentiation of delayed-type hypersensitivity reactions, and provide additional evidence that infection with this nematode enhances the immune capabilities of both stimulated lymphocytes and nonspecific phagocytic cells.     

Dendritic cells from Peyer's patches and mesenteric lymph nodes differ from spleen dendritic cells in their response to commensal gut bacteria

Commensal gut bacteria have potent effects on the immune system, which are partially mediated by intestinal dendritic cells (DC). Distinct commensals confer different properties to in vitro -generated DC. The aim of the present study was to reveal strain-dependent maturation patterns in primary DC. To this end, we compared the response of mouse Peyer's patch (PP) DC, mesenteric lymph node (MLN) DC and spleen DC to the commensal bacteria, Bifidobacterium longum Q46, Lactobacillus acidophilus X37 and Escherichia coli Nissle 1917. Bacterial maturation of DC occurred independently of tissue origin. Expression of CCR7 and CD103 on the surface of MLN DC, necessary for the induction of gut-homing regulatory T cells, increased with stimulation by Gram-positive commensals. Bacteria-dependent cytokine production (IL-6, IL-10 and TNF-α) was similar in spleen and MLN DC, and contaminant cells in these DC preparations produced IFN-γ in response to L. acidophilus . In contrast, PP DC produced IL-6 only in response to E. coli , little IL-10 and no TNF-α, and this low cytokine production was not due to inhibition by IL-10 or TGF-β. Bifidobacteria downregulate IL-6, TNF-α and IL-12 production induced in in vitro -generated DC by L. acidophilus . Similar inhibition was observed in splenic DC, but not in MLN DC. MLN cells responded to bacterial stimulation with higher IFN-γ production than spleen cells, possibly due to the presence of more responsive natural killer cells. Commensal bacteria therefore play specific roles in the gut immune system distinguishable from the effect they would have if recognized by the systemic immune system.     

Ovarian monocyte progenitor cells: phenotypic and functional characterization

Leukocytes of the macrophage lineage are abundant in the ovarian tissues and have an important function in both follicular development and regression of postovulatory follicles. In this study, we tested the hypothesis that continuous production of macrophages in the ovarian stroma is maintained by a resident population of progenitors. We established a long-term culture of ovarian follicular stromal cells from BALB/c and green fluorescent protein-transgenic (GFP-TG) C57BL/6 mice. Nonadherent cells were collected and tested for hematopoietic function in vitro and in vivo. Histological and ultrastructural analyses revealed a homogenous population of monocyte-like rounded cells. Nonadherent cells continued to proliferate in culture for several months without senescence. When plated at very low density in methylcellulose, these cells formed colonies consisting of monocyte-like cells. Ovarian monocyte-like cells reacted with CD45, CD11b, CD11c, and Ly6-Gr-1 cell surface markers. A distinct CD45low population within these cells reacted with CD117 (C-kit) surface marker, suggestive of a primitive hematopoietic progenitor. Fifty thousand nonadherent cells failed to provide radioprotection to lethally irradiated mice and thus were not considered to be equivalent to pluripotent hematopoietic stem cells. Ovarian nonadherent stromal cells were positive for alkaline phosphatase but lacked embryonic cell antigens stage-specific embryonic antigen (SSEA-1) and Oct-4. We conclude that in the ovaries, a higher requirement for macrophages is provided by a resident stromal population of progenitors whose progeny is restricted to the production of cells of the monocyte-macrophage lineage.     

Dendritic immune complex trapping cells in the spleen of the snake, Python reticulatus

The spleen of the snake Python reticulatus, was investigated as to its general histology as well as the presence of immune complex trapping cells both at the light and electron microscopical level. Histological examination revealed that the spleen of this reptile was encapsulated and contained some trabeculae. In the splenic parenchyma two different regions could be distinguished: viz. red and white pulp. The white pulp appeared to be arranged around "central arterioles" and their smaller branches extending towards the periphery of the white pulp. The red pulp was composed of blood sinusoids and cell cords. Electron microscopy revealed at least three types of non-lymphoid cells in the white pulp of the spleen of python: reticulum cells, forming the framework; some macrophages and dendritic cells predominantly located in the periphery of the white pulp. Of these types of non-lymphoid cells, only dendritic cells were able to trap and to retain intravenously injected horseradish peroxidase (HRP)-rabbit-anti-HRP immune complexes on their cell surface as determined by enzymehistochemistry at the light and electron microscopical level. These dendritic cells were frequently found in association with collagen fibres and did not engulf large quantities of carbon particles. These data suggest that dendritic cells in the spleen of the python might be the phylogenetic precursors of the mammalian follicular dendritic cells.     

Production of alpha and gamma interferons by spleen cells from cytomegalovirus-infected mice.

Antigen-induced lymphocyte proliferation and the production of a murine immune or type II interferon (MuIFN-gamma) by spleen cells in vitro were used to examine the cellular immune response to a cytomegalovirus infection of mice. Lymphocyte blastogenesis was induced by interaction with cytomegalovirus-infected mouse embryo fibroblasts in spleen cells from mice infected at least 6 days previously with cytomegalovirus. The peak of the blastogenic response occurred after 72 hr in culture with antigen. MuIFN was detected in cultures of cytomegalovirus-infected mouse embryo fibroblasts and spleen cells from both normal and infected mice. The MuIFN produced by spleen cells from normal or infected mice early during the course of the infection (days 1 to 2) was predominantly viral or type I interferon (MuIFN-alpha). Peak titers of MuIFN-alpha were present 24 to 48 h after exposure to antigen in vitro and before the peak of the blastogenic response. In contrast, spleen cells from mice infected at least 6 days previously produced both MuIFN-alpha and MuIFN-gamma in culture with the infected mouse fibroblasts. MuIFN-alpha was present early in the culture, before peak blastogenic activity. Peak levels of MuIFN-gamma were detected as lymphocyte blastogenic activity subsided. These results indicate that the cellular immune system of the murine host is capable of responding to cytomegalovirus infection, the afferent limb by antigen recognition and the efferent limb by the production of the lymphokine MuIFN-gamma.     

Enhancement of terminal B lymphocyte differentiation in vitro by fibroblast-like stromal cells from human spleen

Stromal elements are major components of lymphoid tissues contributing to both tissue architecture and function. In this study we report on the phenotype and function of fibroblast-like stromal cells obtained from human spleen. These cells express high levels of CD44 and ICAM-1 and moderate levels of VLA-4, VCAM, CD40 and CD21. They fail to express endothelial, epithelial, lymphocyte and monocyte/macrophage markers. We show that these cells interact with B cell blasts induced in vitro by anti-CD40 and anti-μ stimulation. As a result of these interactions both IL-6 and IgG secretion into culture medium is increased. The enhanced secretion of IgG is partly inhibited by abolishing B cell blast-stromal cell contact or by anti-IL-6, anti-VCAM or anti-CD49d antibodies. Our studies also suggest that the ability of stromal cells to promote B cell survival is most likely the underlying mechanism of the enhanced immunoglobulin secretion. Comparison of stromal cells from different lymphoid and non-lymphoid organs revealed that bone marrow- and spleen-derived stromal cells are the most effective in promoting B cell blast differentiation.