SOCS5 is expressed in primary B and T lymphoid cells but is dispensable for lymphocyte production and function

Suppressors of cytokine signaling (SOCSs) are key regulators of cytokine-induced responses in hematopoietic as well as nonhematopoietic cells. SOCS1 and SOCS3 have been shown to modulate T-cell responses, whereas the roles of other SOCS family members in the regulation of lymphocyte function are less clear. Here, we report the generation of mice with a targeted disruption of the Socs5 gene. Socs5^−/− mice were born in a normal Mendelian ratio and were healthy and fertile. We found that SOCS5 is expressed in primary B and T cells in wild-type mice. However, no abnormalities in the lymphocyte compartment were seen in SOCS5-deficient mice. We examined antigen- and cytokine-induced proliferative responses in B and T cells in the absence of SOCS5 and found no deviations from the responses seen in wild-type cells. Because SOCS5 has been implicated in Th1 differentiation, we also investigated the importance of SOCS5 in T helper cell responses. Unexpectedly, SOCS5-deficient CD4 T cells showed no abnormalities in Th1/Th2 differentiation and Socs5^−/− mice showed normal resistance to infection with Leishmania major. Therefore, although SOCS5 is expressed in primary B and T cells, it appears to be dispensable for the regulation of lymphocyte function.     

Mycolactone diffuses from Mycobacterium ulcerans-infected tissues and targets mononuclear cells in peripheral blood and lymphoid organs

Background

Buruli ulcer (BU) is a progressive disease of subcutaneous tissues caused by Mycobacterium ulcerans. The pathology of BU lesions is associated with the local production of a diffusible substance, mycolactone, with cytocidal and immunosuppressive properties. The defective inflammatory responses in BU lesions reflect these biological properties of the toxin. However, whether mycolactone diffuses from infected tissues and suppresses IFN-γ responses in BU patients remains unclear.

Methodology/Principal Findings

Here we have investigated the pharmacodistribution of mycolactone following injection in animal models by tracing a radiolabeled form of the toxin, and by directly quantifying mycolactone in lipid extracts from internal organs and cell subpopulations. We show that subcutaneously delivered mycolactone diffused into mouse peripheral blood and accumulated in internal organs with a particular tropism for the spleen. When mice were infected subcutaneously with M. ulcerans, this led to a comparable pattern of distribution of mycolactone. No evidence that mycolactone circulated in blood serum during infection could be demonstrated. However, structurally intact toxin was identified in the mononuclear cells of blood, lymph nodes and spleen several weeks before ulcerative lesions appear. Importantly, diffusion of mycolactone into the blood of M. ulcerans–infected mice coincided with alterations in the functions of circulating lymphocytes.

Conclusion

In addition to providing the first evidence that mycolactone diffuses beyond the site of M. ulcerans infection, our results support the hypothesis that the toxin exerts immunosuppressive effects at the systemic level. Furthermore, they suggest that assays based on mycolactone detection in circulating blood cells may be considered for diagnostic tests of early disease.     

Differential expression of apoptosis-related Fas antigen on lymphocyte subpopulations in human peripheral blood.

The Fas Ag is a newly defined cell-surface molecule that may mediate apoptosis. The antibody against Fas Ag can induce the apoptotic cell death in cell lines expressing this Ag. PBL subpopulations at various ages were here examined for Fas expression by two-or three-color flow-cytometric analyses using anti-Fas mAb. It was found that Fas Ag was appreciably detected on a proportion of T and B cells, whereas its expression was absent for NK cells. For CD4+ and CD8+ T cells, Fas Ag was expressed preferentially on CD45RO+ (memory or previously activated) populations, but not on CD45RO- naive ones. TCR-gamma/delta+ T cells, especially their CD45RO+ subsets, also expressed Fas Ag. Expectably, neonatal T cell subpopulations, most of which had the naive (CD45RO-) phenotype, expressed little Fas Ag. Fas-expressing B cells dominated in surface(s) IgD- populations, but neonatal B cells as well as adult sIgD+ B cells had little Fas Ag. The Fas Ag was inducible after in vitro mitogenic stimulation of naive T and B cells from neonatal blood. These observations suggested that expression of Fas Ag on T and B cells in the peripheral blood might reflect their in vivo Ag-activated status. In contrast to Fas-expressing cultured cell lines, however, viability of in vitro stimulated T and B cells as well as freshly isolated CD45RO+ T cells was not significantly changed after the treatment with anti-Fas mAb, indicating that additional cellular conditions to Fas expression might be required for anti-Fas-induced cell death.     

Murine embryonic antigen (SSEA-1) is expressed on human cells and structurally related human blood group antigen I is expressed on mouse embryos

The stage-specific embryonic antigen (SSEA-1), present on embryonal carcinoma cells and on murine preimplantation embryos, is defined by a monoclonal antibody. The antigenic determinant of SSEA-1 is a carbohydrate structurally related to the human blood group antigen I. Since it has been suggested that the I antigen might represent a precursor or SSEA-1, we used antibodies to SSEA-1 and to I to analyze their expression on mouse preimplantation embryos. Both are expressed on mouse embryos; moreover, I is expressed on earlier embryos than SSEA-1. The I antigen is defined by its expression on human erythrocytes; accordingly, we examined expression of I and SSEA-1 on human peripheral blood elements. We find SSEA-1 to be expressed exclusively on human granulocytes while I is found only on erythrocytes. These results suggest that these closely related antigens can be independently expressed. Analysis of the expression of I and SSEA-1 was then extended to a series of mouse and human cell lines; some express both, some express only one, and some express neither of these antigens. The activation of specific glycosyltransferases and/or glycosidases during development and differentiation appears to be the biochemical mechanism regulating expression of these antigens.     

Immunohistochemical analysis of human peripheral blood and lymphoid tissues using monoclonal antibodies immunoreactive with non-lymphoid cells

Summary Immunoperoxidase methods were used to study human peripheral blood and lymphoid tissues using a panel of monoclonal antibodies to non-lymphoid cells. The majority of peripheral blood monocytes were immunoreactive for LeuM1, LeuM2, LeuM3 and LeuM5. Rare peripheral blood monocytes were immunoreactive for R4/23. The different antibodies showed characteristic patterns of immunoreactivity in peripheral lymphoid tissues. LeuM1 was immunoreactive with scattered cells in the paracortex of lymph node and tonsil and with many cells in the marginal zone of the spleen. LeuM2 was immunoreactive with endothelial cells in lymph node and tonsil. A few cells in the red pulp of the spleen were immunoreactive for LeuM2. LeuM3 and R4/23 showed distinctive immunoreactivity in germinal centers of secondary follicles, giving a lacy pattern. LeuM3 was also immunoreactive with endothelium in lymph node and tonsil and with sinus lining cells in lymph node. LeuM5 was immunoreactive with macropages in the germinal center, fibroblastic reticulum cells in the mantle zone and interdigitating reticulum cells in the paracortex of lymph node and tonsil.     

Immunohistochemical analysis of human peripheral blood and lymphoid tissues using monoclonal antibodies immunoreactive with non-lymphoid cells

Immunoperoxidase methods were used to study human peripheral blood and lymphoid tissues using a panel of monoclonal antibodies to non-lymphoid cells. The majority of peripheral blood monocytes were immunoreactive for LeuM1, LeuM2, LeuM3 and LeuM5. Rare peripheral blood monocytes were immunoreactive for R4/23. The different antibodies showed characteristic patterns of immunoreactivity in peripheral lymphoid tissues. LeuM1 was immunoreactive with scattered cells in the paracortex of lymph node and tonsil and with many cells in the marginal zone of the spleen. LeuM2 was immunoreactive with endothelial cells in lymph node and tonsil. A few cells in the red pulp of the spleen were immunoreactive for LeuM2. LeuM3 and R4/23 showed distinctive immunoreactivity in germinal centers of secondary follicles, giving a "lacy" pattern. LeuM3 was also immunoreactive with endothelium in lymph node and tonsil and with sinus lining cells in lymph node. LeuM5 was immunoreactive with macrophages in the germinal center, fibroblastic reticulum cells in the mantle zone and interdigitating reticulum cells in the paracortex of lymph node and tonsil.     

Studies of complement receptors on cytotoxic effector cells in human peripheral blood

The question of whether cells bearing complement receptors (CR) mediate cytotoxicity in vitro against allogeneic Chang liver cell targets was investigated by assessing peripheral blood mononuclear cells (PBMC) from normal humans for cell surface characteristics and cytotoxic capacity before and after depletion of CR+ cells capable of forming rosettes with sheep erythrocytes coated with 19S antibody and mouse complement (EAC) and depletion of Fc receptor-bearing cells capable of forming rosettes with human O+ erythrocytes coated with Ripley antibody (EA-Ripley). PBMC depleted of CR+ cells by density centrifugation contained markedly reduced proportions of phagocytes and sIg + cells and increased proportions of both sIg ?, FcR+ cells as well as cells forming rosettes with sheep erythrocytes (E). PBMC depleted of CR+ cells mediated cytotoxicity to an extent equal to or greater than that mediated by unfractionated PBMC in assays of spontaneous cell-mediated cytotoxicity (SCMC), antibody-dependent cellular cytotoxicity (ADCC), and mitogen-induced cellular cytotoxicity (MICC). Cells harvested from the EAC-rosette enriched pellet mediated cytotoxicity 5- to 10-fold less than unfractionated PBMC; however, the cytotoxic activity of the pellet could not be attributed to CR + effector cells since similar cytotoxic activity was present in cell pellets obtained by density centrifugation of PBMC which had been incubated with E coated with 19S antibody or E alone. PBMC depleted of EA-Ripley rosette-forming cells contained decreased proportions of sIg?, FcR+ cells and increased proportions of CR+ cells; PBMC so depleted contained virtually no SCMC and ADCC effector cell activity. These findings indicate that at least the majority of effector cells which mediate SCMC, ADCC, and MICC do not bear CR.     

Dynamic differentiation of activated human peripheral blood CD8+ and CD4+ effector memory T cells

Two functionally different memory T cell subsets were originally defined based on their different CCR7 expression profile, but the lineage relationship between these subsets referred to as central memory T cells (T(CM)) and effector memory T cells (T(EM)), is not resolved. A prevalent model proposes a linear progressive differentiation from T(CM) to T(EM). Our results demonstrate that on activation, human CCR7-CD62L- peripheral blood CD8+ and CD4+ T(EM) cells exhibit a dynamic differentiation, involving transient as well as stable changes to T(CM) phenotype and properties. Whereas the larger fraction of T(EM) cells increases expression of effector molecules, such as perforin or IFN-gamma, a smaller fraction first acquires CCR7 expression. We demonstrate that this acquisition of lymph node homing potential is associated with strong proliferation similar to that of activated T(CM) cells. After proliferation, most of these cells lose CCR7 expression again and acquire effector functions (e.g., perforin production). A small proportion (approximately 6%), however, maintain phenotypic and functional T(CM) properties over a long time interval. These results suggest that T(EM) cells provide immediate effector function by a fraction of cells as well as self-renewal by others through up-regulation of CCR7 followed by either secondary peripheral effector function or long term maintenance of T(CM)-like properties.     

B lymphocyte colony-forming cells in the SJL/J mouse.

Mercatoethanol-induced B lymphocyte cloning in semi-solid agar has been used to study lymphocyte colony formation by cells from the SJL/J mouse thymus. From the 3rd month of life, the SJL/J mouse thymus. From the 3rd month of life, the SJL thymus develops an increasing frequency of cells forming B lymphocyte colonies in agar. The peak frequency in 6- to 12-month-old mice was one colony per 1000 to 2000 cultured thymus cells. In contrast, 10 to 100 times lower frequencies were found in the thymus of five other inbred mouse strains. The rise in B lymphocyte colony-forming cells correlated well with the age-related rise in Ig-positive cells and approximately 50% of the colony cells reacted with anti-micron-serum indicating the B lymphocyte nature of the colony cells. Colony-forming cells from the thymus showed higher sensitivity than colony-forming spleen cells to cortisol and irradiation. Cell transfer experiments and thymus grafting suggested that the increased frequency of colony-forming cells in the thymus is caused by development of special thymus-seeking B lymphocytes in ageing SJL/J mice. Finally, B lymphocyte colony-forming cells were found to be more frequent in the thymus, spleen, and lymph nodes from healthy aged mice than in lymphoid organs from mice with spontaneous reticulum cell tumors.     

Migration of antigen-presenting B cells from peripheral to mucosal lymphoid tissues may induce intestinal antigen-specific IgA following parenteral immunization.

Parenterally administered immunizations have long been used to induce protection from mucosal pathogens such as Bordetella pertussis and influenza virus. We previously found that i.m. inoculation of mice with the intestinal pathogen, rotavirus, induced virus-specific Ab production by intestinal lymphocytes. We have now used adoptive transfer studies to identify the cell types responsible for the generation of virus-specific Ab production by gut-associated lymphoid tissue (GALT) after i.m. immunization. Three days after i.m. immunization with rotavirus, cells obtained from the draining peripheral lymph nodes of donor mice were transferred into naive recipient mice. We found that intestinal lymphocytes produced rotavirus-specific Igs (IgM, IgA, and IgG) 2 wk after transfer of either unfractionated cells, or unfractionated cells rendered incapable of cellular division by mitomycin C treatment. Additional studies demonstrated that rotavirus-specific IgA, but not IgG, was produced by intestinal lymphocytes after transfer of purified B cells. Ig allotype analysis revealed that rotavirus-specific IgA was produced by intestinal B cells of recipient origin, suggesting that migration of Ag-presenting B cells from peripheral lymphoid tissues to GALT may contribute to the generation of mucosal IgA responses after parenteral immunization. Strategies that promote Ag uptake and presentation by B cells may enhance mucosal IgA production following parenteral immunization.     

A novel antigen is common to the dome epithelium of gut- and bronchus-associated lymphoid tissues

Summary The dome epithelium (DE) covering bronchus- and gut-associated lymphoid tissues (BALT and GALT) is composed of columnar cells, groups of lymphocytes, M cells, and pre-M cells. Although the cell biology and immunologic processes of this tissue are likely important in the afferent arm of secretory immune responses, virtually nothing is known about biochemical constituents of the DE. Therefore, a monoclonal antibody, 30E5, was used to study the distribution of a novel antigen, common to dome epithelia of GALT and BALT. 30E5 was secreted by a hybridoma, prepared by fusing murine splenocytes, immunized against dome epithelial cells, with P3×68/Ag8 myeloma cells. Reactivity of antigens was defined by indirect immunocytochemistry on sections of rabbit tissues or with dissociated epithelial cells. In situ, 30E5-reactive antigen circumscribed each group of dome epithelial lymphocytes, most or all of which were T cells, in rabbit appendix, sacculus rotundus, cecal patch, Peyer's patch, and BALT. In the DE this antigen was associated with the apical surface and the supranuclear or perinuclear regions of epithelial cells, but it was not associated with epithelial cells of villi, epithelium, or with individual lymphocytes. In peripheral lymph nodes, spleen, and in domes and follicles of GALT or BALT, 30E5-reactive antigen was visualized in linear wisps, primarily in regions populated by thymocytes. In other adult tissues, 30E5-reactive antigen was associated with involuntary muscle, myoepithelial cells of lactating mammary gland and with what appeared to be neural dendrites; but it was not found in epithelia other than DE. In neonatal rabbit appendix, this antigen first appeared in the upper dome epithelium two days after birth, a period coinciding with T cell infiltration and M cell maturation. The histologic distribution of 30E5-reactive antigen suggested that it might be a contractile filament, a receptor, or a differentiation antigen. Since 30E5 was associated with DE of both GALT and BALT, results support the concept of a molecule common to all mucosa-associated lymphoid tissues.In conducting the research described in this report, the investigators adhered to standards set forth in the Guide for the Care and Use of Laboratory Animals (NIH Publication 85-23) as promulgated by the Committee on Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources, National Research Council, USALimited quantities of ascites containing monoclonal antibody 30E5 will be distributed to interested investigators until such time as the hybridoma is available from American Type Culture CollectionAbbreviations ABC avidin-biotin-horseradish peroxidase complex - BALT bronchus-associated lymphoid tissues - DMEM Dulbecco's modified Eagle medium - GALT gut-associated lymphoid tissues - DE dome epithelium - DEL dome epithelial lymphocytes - MAb monoclonal antibody - MALT mucosal-associated lymphoid tissues The views of the authors expressed here do not purport to reflect the position of the Department of the Army or the Department of Defense Send offprint requests to: Department of Experimental Pathology, Division of Pathology, Walter Reed Army Institute of Research, Washington, D.C. 20307-5100, USA     

The activatory receptor 2B4 is expressed in vivo by human CD8+ effector alpha beta T cells.

The membrane receptor 2B4 is a CD2 family member that is involved in lymphocyte activation. A fraction of human CD8+ alphabeta T cells up-regulate 2B4 in vivo, and here we demonstrate that this correlates with the acquisition of effector cell properties such as granzyme B and perforin expression, rapid IFN-gamma production, and down-regulation of the lymph node homing chemokine receptor CCR7. In PBLs from healthy donors, cytomegalovirus-specific effector T cells were 2B4 positive, whereas naive melanoma Ag (Melan-A/melanoma Ag recognized by T cells-1)-specific T cells were 2B4 negative. In melanoma patients, Melan-A-specific T cells up-regulated 2B4 in parallel with in vivo differentiation. This occurred in PBLs after vaccination with Melan-A peptides and in tumor-infiltrated lymph nodes, likely through disease-associated activation of Melan-A-specific T cells. Thus, 2B4 expression correlates with CD8+ T cell differentiation in vivo.     

CD5+ B cells predominate in peripheral tissues of rabbit.

Restricted usage of VH genes is observed in rabbit B lymphocytes and in human and murine CD5 B lymphocytes. This observation raised the possibility that most rabbit B lymphocytes were CD5+. To investigate this we cloned the CD5 gene from a rabbit cosmid library, using a probe derived from human CD5 cDNA. The rabbit CD5 gene was transfected into a murine T cell line and then we used the transfectants to develop anti-rabbit CD5 mAb. By Western blot analysis, the mAb reacted with a 67-kDa protein in lysates prepared from mesenteric lymph node and spleen cells. We determined the frequency of CD5+ B lymphocytes in peripheral lymphoid tissues of adult rabbits by two-color immunofluorescence analysis using anti-CD5 mAb and anti-L chain antibodies. The analysis showed that essentially all peripheral B lymphocytes in adult rabbits express CD5. The observation that CD5 is expressed on nearly all rabbit B lymphocytes contrasts markedly to mouse and human, where only a small number of B lymphocytes express CD5. We propose that most peripheral B lymphocytes in rabbit, as in chicken, develop early in ontogeny and are maintained throughout life by a self-renewing process.     

Mitochondria connects the antigen receptor to effector caspases during B cell receptor-induced apoptosis in normal human B cells.

We have previously reported that CD40 stimulation sensitizes human memory B cells to undergo apoptosis upon subsequent B cell receptor (BCR) ligation. We have proposed that activation stimuli connect the BCR to an apoptotic pathway in mature B cells and that BCR-induced apoptosis of activated B cells could serve a similar function as activation-induced cell death in the mature T cell compartment. Although it has been reported that caspases are activated during this process, the early molecular events that link the Ag receptor to these apoptosis effectors are largely unknown. In this study, we report that acquisition of susceptibility to BCR-induced apoptosis requires entry of memory B cells into the S phase of the cell cycle. We also show that transduction of the death signal via the BCR sequentially proceeds through a caspase-independent and a caspase-dependent phase, which take place upstream and downstream of the mitochondria, respectively. Furthermore, our data indicate that the BCR-induced alterations of the mitochondrial functions are involved in activation of the caspase cascade. We have found both caspases-3 and -9, but not caspase-8, to be involved in the BCR apoptotic pathway, thus supporting the notion that initiation of the caspase cascade could be under the control of the caspase-9/Apaf-1/cytochrome c multimolecular complex. Altogether, our findings establish the mitochondria as the connection point through which the Ag receptor can trigger the executioners of apoptotic cell death in mature B lymphocytes.     

EBI2 guides serial movements of activated B cells and ligand activity is detectable in lymphoid and nonlymphoid tissues

EBV-induced gene 2 (EBI2) was recently shown to direct the delayed movement of activated B cells to interfollicular and outer follicular regions of secondary lymphoid organs and to be required for mounting a normal T-dependent Ab response. In this study, we show that EBI2 promotes an early wave of Ag-activated B cell migration to the outer follicle in mice. Later, when B cells have moved to the T zone in a CCR7-dependent manner, EBI2 helps distribute the cells along the B zone-T zone boundary. Subsequent EBI2-dependent movement to the outer follicle coincides with CCR7 downregulation and is promoted by CD40 engagement. Using a bioassay, we identify a proteinase K-resistant, hydrophobic EBI2 ligand activity in lymphoid and nonlymphoid tissues. Production of EBI2 ligand activity by a cell line is sensitive to statins, suggesting production in a 3-hydroxy-3-methyl-glutaryl-CoA reductase-dependent manner. CD40-activated B cells show sustained EBI2-dependent responsiveness to the bioactivity. These findings establish a role for EBI2 in helping control B cell position at multiple stages during the Ab response and they suggest that EBI2 responds to a broadly distributed lipid ligand.     

The anti-apoptotic protein lifeguard is expressed in breast cancer cells and tissues

Lifeguard (LFG) is an anti-apoptotic protein that inhibits Fas-mediated death in tumour cells. However, the molecular function of human LFG in the carcinogenesis of human breast cells is uncertain. We studied the expression and function of endogenous LFG in four breast cancer cell lines (MCF-7, MDA-MB-231, T-47D and HS 578T), a human breast epithelial cell line (HS 578Bst), and in healthy and cancerous breast tissues. Molecular (Western blot and RT-PCR) and immunohistochemical techniques were used to investigate the LFG expression. To investigate the breast cancer cell proliferation in the presence of Fas, we performed fluorescent cell viability assays. The possible association of Fas with LFG was analyzed by immunofluorescence microscopy. In this paper, we provide convincing evidence that LFG is overexpressed in several human breast cancer cell lines. More importantly, we found that the LFG expression correlates with high tumour grades in primary breast tumours. Finally, we demonstrated that Fas sensitivity is reduced in breast cancer cell lines expressing LFG. Our results indicated that LFG is strongly expressed in breast cancer epithelial cells. Moreover, the overexpression of LFG correlated with tumour grade and reduced Fas sensitivity. Our findings support the idea that LFG may have a role in the downregulation of apoptosis in breast cancer cells.     

Selective transformation of primitive lymphoid cells by the BCR/ABL oncogene expressed in long-term lymphoid or myeloid cultures.

The BCR/ABL gene, formed by the Philadelphia chromosome translocation (Ph1) of human chronic myelogenous leukemia, encodes an altered ABL gene product, P210. P210 is strongly implicated in the malignant process of chronic myelogenous leukemia, but it precise role is unknown. Infection of long-term bone marrow cultures enriched for B-lymphoid cell types with a Moloney murine leukemia virus retroviral vector containing the BCR/ABL cDNA resulted in clonal outgrowths of immature B-lymphoid cells which expressed abundant P210 kinase activity. Surprisingly, infection of long-term myeloid lineage-enriched cultures also resulted in clonal outgrowths of immature B-lymphoid cells. The P210-expressing lymphoid cell lines resulting from either type of culture were resistant to the lethal effects of corticosteroids. These findings indicate that high levels of P210 expressed from a Moloney murine leukemia virus long terminal repeat preferentially stimulate the growth of immature B-lineage cells, and this effect is apparent even in myeloid lineage-enriched cultures, in which few if any lymphoid cells can be detected prior to infection.