Lymphocyte-stromal cell interaction induces IL-7 expression by interferon regulatory factors.

The interaction between lymphocytes and stromal cells plays important roles in coordinated development of early lymphocytes. IL-7 is an essential cytokine for early lymphocyte development produced by stromal cells in the thymus and bone marrow. Although IL-7 is induced by interaction of early lymphocytes and stromal cells, its molecular basis is still unknown. To address this question, we employed co-culture system with an IL-7-dependent pre-B cell line, DW34, and a thymic stromal cell line, TSt-4. Co-culture with DW34 cells enhanced the levels of IL-7 transcripts in TSt-4 cells. Interestingly, the co-culture also induced transcripts of IFN-α and IFN-ß but not of IFN-γ. In addition, exogenous IFN-ß stimulation increased the levels of IL-7 transcripts in TSt-4 cells. Next, to elucidate the molecular mechanism of IL-7 induction, we analyzed the IL-7 promoter activity by reporter assay. The IL-7 promoter showed specific transcriptional activity in TSt-4 cells. An interferon-stimulated response element (ISRE) in the IL-7 promoter was essential for the induction of IL-7 transcription by both co-culture and IFN-ß stimulation. Finally, overexpression of wild-type and dominant-negative forms of interferon regulatory factors (IRFs) activated and repressed, respectively, the IL-7 promoter in TSt-4 cells. Collectively, these results suggested that IRFs activated by lymphocyte adhesion induce IL-7 transcription through ISRE in stromal cells and that type I IFNs may be involved in the activation of IRFs. Thus, this study implied a physiological function of the IFN/IRF signal during lymphocyte development.

Attenuation of murine graft-versus-host disease by a tea polyphenol.

Since donor T-cells' allorecognition of host antigens is a prerequisite for the onset of graft-versus-host disease (GVHD), blocking their cellular signaling pathways can decrease the severity of GVHD. We hypothesized that epigallocatechin-3-gallate (EGCG), due to its strong affinity to macromolecules, would adhere to surface molecules of donor T cells, inhibit their allorecognition, and attenuate GVHD in the recipient. We tested the hypothesis by treating donor splenocytes with EGCG in both in vitro and in vivo murine GVHD models. EGCG treatment decreased the proliferation of donor cells in MLR cultures and secretion of IL-2 and INF-γ. It also reduced the epitope detection of CD3ɛ, CD4, and CD28 but did not downregulate the protein expression of these molecules, suggesting blockage of cell surface stimulatory signals. Similarly, EGCG treatment did not decrease mRNA expression for some of these molecules but decreased mitogen-induced cell proliferation, indicating that EGCG did not interfere the transcription of these genes but affected cell proliferation pathways. Furthermore, EGCG-treated donor splenocytes, when transplanted into immunocompromized recipient mice, decreased of proliferation, and the treatment extended the recipients' survival at least during the early stage of GVHD. These results strongly suggest that EGCG attenuates GVHD by both blocking specific cell surface molecules and affecting the donor T-cell proliferation pathways.

Forced expression of Id2 in fetal thymic T cell progenitors allows some of their progeny to adopt NK cell fate.

The E proteins are indispensable for early T cell development. On the other hand, we previously demonstrated that their inhibitor Id2 is essential for NK lineage commitment from bipotent progenitors generating both T and NK cells (p-T/NK). To shed more light on the role of E proteins and Id2 in the development of early intrathymic progenitors, we performed a clonal analysis: individual fetal thymic CD4(-)CD8(-)CD44(+)CD25(-)CD122(-) (DN1CD122(-)) cells were retrovirally transduced with an Id2-internal ribosomal entry site (IRES)-green fluorescent protein (GFP) (Id2-GFP) gene or a control IRES-GFP (GFP) gene, and cultured in a modified fetal thymus organ culture able to support T and NK cell development. After the culture, both T and NK cells, T cells and no NK cells, NK cells and no T cells, or completely no cells were generated from single cells in each lobe. Hence, the seeded cells were regarded as p-T/NK, unipotent progenitors generating T cells (p-T), unipotent NK progenitors, or cells without progenitor activity, respectively. With Id2-GFP transduction, p-T disappeared and more p-T/NK emerged than with GFP transduction. This increase corresponded to the number of p-T that was counted when the vector-transduced-DN1CD122(-) cells of the same number were examined. Additionally, a fraction of GFP(-) NK cells obtained after Id2-GFP transduction underwent TCRbeta D-J rearrangement. Our data strongly suggest that forced expression of Id2 allows some progeny of p-T to adopt an NK cell fate, and that p-T retain a program for NK lineage development that can be implemented by inhibiting the function of E proteins.

Tea polyphenol inhibits allostimulation in mixed lymphocyte culture.

Green tea polyphenols are known to protect allogenic donor tissues from acute rejection by their recipients. This immunosuppressive effect may be generated by a unique chemical property of the major component, epigallocatechin-o-gallate (EGCG), which can block specific cell surface molecules of the donor tissues. To test this hypothesis, we examined the effects of EGCG on the murine mixed lymphocyte reactions. EGCG treatment of stimulator cells significantly attenuated the proliferation of responder T cells. The proliferation did not recover upon the secondary stimulations by fresh untreated cells or exogenous IL-2. Flow cytometric analyses showed that EGCG treatment decreased the staining intensities of various cell surface molecules including MHC II, which plays a major role in antigen presentation, and B7.1, B7.2, and their ligand, CD28, which are required for costimulatory signals in T-cell activation. These results suggest that an anergic state of alloreactive T cells may be induced by either weakening of antigen signaling or blockage of costimulatory signals with EGCG. Other possible mechanisms behind the immunosuppressive effect and a potential use of EGCG treatment of donor tissues in transplantation medicine are discussed.

Glycophorin A requirement for expression of O-linked antigens on the erythrocyte membrane.

BACKGROUND: Glycophorin A (GPA) has a large number of sialic acid-containing oligosaccharide chains. GPA is highly conserved among vertebrates, mice with a GPA deletion have not been reported and GPA's physiologic role remains uncertain. RESULTS: GPA-/- homozygotes were obtained by intercrossing GPA+/- heterozygotes based on Mendelian genetics. The amount of O-linked oligosaccharide chains in the erythrocyte membrane of GPA-/- mice decreased to 60% compared to that of the wild-type mice. Flow cytometry and Western blot analysis revealed that the TER antigen that is associated with GPA on the erythrocyte membrane was totally abrogated from the cell surface in GPA-/- mice. Several glycoproteins that were detected with peanut agglutinin (PNA), a lectin that recognizes O-linked oligosaccharide chains, were absent from the GPA-/- erythrocyte membrane. Erythrocytes lacking GPA were more sensitive to hypo-osmotic stress than wild-type erythrocyte. CONCLUSIONS: GPA-/- mice show apparently normal phenotypes at least during the early generations. The disappearance of many glycoproteins recognized by PNA lectin on the GPA-/- erythrocyte membrane proteins suggests that GPA has an essential role in the expression of O-linked antigens on the erythrocyte membrane protein. These interactions of GPA and other glycoproteins may contribute to maintaining the physical strength of the erythrocyte membrane.

Selective increase of autoimmune epitope expression on aged erythrocytes in mice: implications in anti-erythrocyte autoimmune responses.

We investigated the impact of changes occurring during red blood cell (RBC) ageing on the RBC-binding activity of pathogenic anti-erythrocyte monoclonal antibodies derived from autoimmune-prone New Zealand black (NZB) mice. As assessed by flow cytometric analysis on in vivo biotinylated RBCs, all five NZB-derived anti-RBC mAb exhibited more efficient binding to aged RBCs than to young RBCs, and resulted in a selective elimination of more aged RBCs from the circulating blood. In addition, treatment of RBCs with proteases markedly enhanced the binding of all five anti-RBC mAb, raising the possibility that increased exposure of autoimmune epitopes on aged RBCs may be in part, a result of contacts with proteolytic enzymes during the lifetime of circulating RBCs. In marked contrast, the binding activity of mAb raised in non-autoimmune animals against antigens expressed on RBCs, such as CD44, CD47, CD147 and TER-119, was either decreased or unchanged with RBC ageing, and these epitopes, except for that recognized by anti-CD47 mAb, were highly sensitive to mild treatment with proteases. Our data unravel the unique molecular feature of RBC epitopes involved in autoimmune haemolytic anaemia, suggesting that membrane alterations in aged RBCs might play a significant role in the development of the autoantibody response to RBCs.