The functional maturation of dendritic cells (DC) and other antigen-presenting cells is believed to reflect the upregulation of cell surface major histocompatibility complex (MHC) class II and other T cell costimulatory molecules, especially the CD28 ligands B7-1 (CD80) and B7-2 (CD86). In this study, we propagated cells exhibiting characteristics of DC precursors from the bone marrow (BM) of BIO mice (H_2 b ; I-A +) in response to granulocyte-macrophage colony stimulating factor (GM-CSF). The methods used were similar to those employed previously to propagate DC progenitors from normal mouse liver. Cells expressing DC lineage markers (NLDC 145+, 33Dl +, N418+) harvested from 8-tO-day GM-CSF stimulated BM cell cultures were CD45+, heat-stable antigen+, CD54+, CD44+, MHC class 11+, B7_1 dbn but B7-2-(costimulatory molecule-deficient). Supplementation of cultures with interleukin-4 (IL-4) in addition to GM-CSF however, resulted in marked upregulation of MHC class II and B7-2 expression. These latter cells exhibited potent allostimulatory activity in primary mixed leukocyte cultures. In contrast, the cells stimulated with GM-CSF alone were relatively weak stimulators and induced alloantigen-specific hyporesponsiveness in allogeneic T cells (C3H; H_2k; I-E+) detected upon restimulation in secondary MLR. This was associated with blockade of IL-2 production. Reactivity to thirdparty stimulators was intact. The hyporesponsiveness induced by the GM-CSF stimulated, costimulatory molecule-deficient cells was prevented by incorporation of anti-CD28 monoclonal antibody in the primary MLR and was reversed by addition of IL-2 to restimulated T cells. The findings show that MHC class 11+ B7-2-cells with a DC precursor phenotype can induce alloantigen-specific hyporesponsiveness in vitro. Under the appropriate conditions, such costimulatory molecule-deficient cells could contribute to the induction of donor-specific unresponsiveness in vivo.
Allografts of the liver, which has a comparatively heavy leukocyte content compared with other vascularized organs, are accepted permanently across major histocompatibility complex barriers in many murine strain combinations without immunosuppressive therapy. It has been postulated that this inherent tolerogenicity of the liver may be a consequence of the migration and perpetuation within host lymphoid tissues of potentially tolerogenic donor-derived ("chimeric") leukocytes, in particular, the precursors of chimeric dendritic cells (DC). In this study, we have used granulocyte/macrophage colony-stimulating factor to induce the propagation of progenitors that give rise to DC (CD45+, CD11c+, 33D1+, nonlymphoid dendritic cell 145+, major histocompatibility complex class II+, B7-1+) in liquid cultures of murine bone marrow cells. Using this technique, together with immunocytochemical and molecular methods, we show that, in addition to cells expressing female host (C3H) phenotype (H-2Kk+; I-E+; Y chromosome-), a minor population of male donor (B10)-derived cells (H-2Kb+; I-A+; Y chromosome+) can also be grown in 10-d DC cultures from the bone marrow of liver allograft recipients 14 d after transplant. Highly purified nonlymphoid dendritic cell 145+ DC sorted from these bone marrow-derived cell cultures were shown to comprise approximately 1-10% cells of donor origin (Y chromosome+) by polymerase chain reaction analysis. In addition, sorted DC stimulated naive, recipient strain T lymphocytes in primary mixed leukocyte cultures. Evidence was also obtained for the growth of donor-derived cells from the spleen but not the thymus. In contrast, donor cells could not be propagated from the bone marrow or other lymphoid tissues of nonimmunosuppressed C3H mice rejecting cardiac allografts from the same donor strain (B10). These findings provide a basis for the establishment and perpetuation of cell chimerism after organ transplantation.
In this report, we present data on heterogeneity of rat NK cells utilizing a combination of antibody and lectin-binding characteristics. Among NKR-P1bright NK cells, two discrete populations characterized as Lycopersicon esculentum lectin (L.E.)bright (60 to 80%) and L.E.dim (20 to 40%) were identified by flow cytometry. Comparison of the morphology of sorted NKR-P1bright/L.E.bright and NKR-P1bright/L.E.dim cells indicated that both were greater than 90% LGL. An analysis of the functional capabilities of the sub-populations indicated that NKR-P1bright/L.E.bright NK cells were more efficient in lysis of YAC-1 target cells (1743 LU20/10(7) cells) than were NKR-P1bright/L.E.dim cells (504 LU20/10(7) cells). Conversely, NKR-P1bright/L.E.dim NK cells were much more efficient at lysis of antibody-sensitized erythrocytes (antibody-dependent cellular cytotoxicity (ADCC)) (1412 LU20/10(7) cells) than were NKR-P1bright/L.E.bright cells (165 LU20/10(7) cells). Lysis of antibody sensitized P815 target cells yielded similar results as NKR-P1bright/L.E.dim cells and NKR-P1bright/L.E.bright cells had 905 LU20/10(7) and 189 LU20/10(7), respectively. Additional experiments indicated that NKR-P1bright/L.E.bright NK cells had the capacity to trigger lytic activity via NKR-P1 whereas NKR-P1bright/L.E.dim NK cells did not. NKR-P1bright/L.E.bright sorted cells had a greater capacity to form conjugates with YAC-1 target cells than did NKR-P1bright/L.E.dim sorted cells. Conversely, NKR-P1bright/L.E.dim NK cells were demonstrated to form E-A rosettes whereas the NKR-P1bright/L.E.bright NK cells were not. Additional experiments indicated that tomato lectin itself was not responsible for the differences in reverse ADCC activity or ADCC activity among the subsets. However, lysis of YAC-1 target cells was modulated to some degree by the lectin. These data indicate that NKR-P1bright/L.E.bright and NKR-P1bright/L.E.dim subpopulations of rat NK cells have different capacities for: 1) triggering through NKR-P1; and 2) E-A rosette formation and lysis of antibody-sensitized target cells by ADCC.
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