Limits...
CD40 ligation on human cord blood CD34+ hematopoietic progenitors induces their proliferation and differentiation into functional dendritic cells.

Flores-Romo L, Björck P, Duvert V, van Kooten C, Saeland S, Banchereau J - J. Exp. Med. (1997)

Bottom Line: CD40-activated cord blood CD34HPC were found to proliferate and differentiate independently of granulocyte/macrophage colony-stimulating factor, into a cell population with prominent dendritic cell (DC) attributes including priming of allogeneic naive T cells.These features were shared by a dendritic population identified in situ in tonsillar T cell areas.Taken together, the present data demonstrate that CD40 is functional on CD34HPC and its cross-linking by CD40L+ cells results in the generation of DC that may prime immune reactions during antigen-driven responses to pathogenic invasion, thus providing a link between hematopoiesis, innate, and adaptive immunity.

View Article: PubMed Central - PubMed

Affiliation: Schering-Plough, Laboratory for Immunological Research, Dardilly, France.

ABSTRACT
Human CD34+ multilineage progenitor cells (CD34HPC) from cord blood and bone marrow express CD40, a member of the tumor necrosis factor-receptor family present on various hematopoietic and nonhematopoietic cells. As hyper-IgM patients with mutated CD40 ligand (CD40L) exhibit neutropenia, no B cell memory, and altered T cell functions leading to severe infections, we investigated the potential role of CD40 on CD34HPC development. CD40-activated cord blood CD34HPC were found to proliferate and differentiate independently of granulocyte/macrophage colony-stimulating factor, into a cell population with prominent dendritic cell (DC) attributes including priming of allogeneic naive T cells. DC generated via the CD40 pathway displayed strong major histocompatibility complex class II DR but lacked detectable CD1a and CD40 expression. These features were shared by a dendritic population identified in situ in tonsillar T cell areas. Taken together, the present data demonstrate that CD40 is functional on CD34HPC and its cross-linking by CD40L+ cells results in the generation of DC that may prime immune reactions during antigen-driven responses to pathogenic invasion, thus providing a link between hematopoiesis, innate, and adaptive immunity.

Show MeSH

Related in: MedlinePlus

CD40 ligation induces proliferation of human CD34+ progenitor cells. (A) Kinetics, (B, E) specificity, (C, D) cell cycle analysis.  CD34HPC cultured as described in Materials and Methods were either  pulsed overnight with 1 μCi of [3H]thymidine (A, B) or counted by Trypan blue exclusion (E). Experiments shown are representative of three experiments and data are presented as mean cpm ± SD (A and B) and cell  numbers per ml ± SD (E), as determined in triplicate wells. For quantitating recovery of viable cells, 105 progenitor cells (indicated by horizontal line in medium control bar) were seeded in 1.0 ml of culture medium  onto 105 CD40L+ cells or control CD32 cells. Results were evaluated at  days 4, 8, and 15 (A) or at day 8 only (B, E). Where indicated, cultures  were supplemented with mAbs to CD40 (mAb 89), CD40L (mAb LL48),  or isotype-matched control antibodies. (C, D) Cell cycle analysis.  CD34HPC freshly isolated (data not shown) or cultured either on CD32L  (C) or CD40L-transfected fibroblasts (D) for 3 d were resuspended and  incubated with Hoechst 33342. To quantitate specifically CD34HPC entering into cycle, Hoechst-stained cells were subsequently labeled with a  mAb to CD34, washed, and analyzed with a FACSTAR+® flow cytometer.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2196112&req=5

Figure 1: CD40 ligation induces proliferation of human CD34+ progenitor cells. (A) Kinetics, (B, E) specificity, (C, D) cell cycle analysis. CD34HPC cultured as described in Materials and Methods were either pulsed overnight with 1 μCi of [3H]thymidine (A, B) or counted by Trypan blue exclusion (E). Experiments shown are representative of three experiments and data are presented as mean cpm ± SD (A and B) and cell numbers per ml ± SD (E), as determined in triplicate wells. For quantitating recovery of viable cells, 105 progenitor cells (indicated by horizontal line in medium control bar) were seeded in 1.0 ml of culture medium onto 105 CD40L+ cells or control CD32 cells. Results were evaluated at days 4, 8, and 15 (A) or at day 8 only (B, E). Where indicated, cultures were supplemented with mAbs to CD40 (mAb 89), CD40L (mAb LL48), or isotype-matched control antibodies. (C, D) Cell cycle analysis. CD34HPC freshly isolated (data not shown) or cultured either on CD32L (C) or CD40L-transfected fibroblasts (D) for 3 d were resuspended and incubated with Hoechst 33342. To quantitate specifically CD34HPC entering into cycle, Hoechst-stained cells were subsequently labeled with a mAb to CD34, washed, and analyzed with a FACSTAR+® flow cytometer.

Mentions: To investigate the potential role of the CD40 molecule on CD34HPC, progenitor cells were cultured over CD40Ltransfected fibroblasts (CD40L cells/CD40L system), (10, 14) or on CD32-transfected cells as control. CD40L-activated CD34HPC entered into DNA synthesis, as measured by tritiated thymidine incorporation that peaked around day 8 and declined by day 15 (Fig. 1 A). CD40-triggered proliferation of progenitor cells was specific, because it was prevented by antibodies to CD40 and CD40L but not by isotype-matched control antibodies (Fig. 1 B and E ). The observed DNA synthesis reflected actual cell proliferation (Fig. 1 E) with a mean 10-fold increase in viable cells observed at day 8 (four independent experiments). In contrast, there was no or only marginal increase in proliferation of CD34HPC cultured over control CD32-transfected cells (CD32 L cells; Fig. 1 A, B, E). Double labeling with Hoechst 33342 and CD34 MoAb to assess the cell cycle of CD34HPC confirmed the quiescent status of these cells when freshly isolated (data not shown). After 3 d of culture over control CD32 L cells, few CD34HPC were cycling (Fig. 1 C ). By contrast, a significant proportion of CD34HPC were found in cycle when stimulated for 3 d by CD40L (Fig. 1 D). However, at 24 h (data not shown), the percentage of cycling cells in CD40L cultures was only marginally higher than that of control cultures, indicating that CD40L probably recruits a small proportion of CD34+ cells whose cycling progeny accumulates with time.


CD40 ligation on human cord blood CD34+ hematopoietic progenitors induces their proliferation and differentiation into functional dendritic cells.

Flores-Romo L, Björck P, Duvert V, van Kooten C, Saeland S, Banchereau J - J. Exp. Med. (1997)

CD40 ligation induces proliferation of human CD34+ progenitor cells. (A) Kinetics, (B, E) specificity, (C, D) cell cycle analysis.  CD34HPC cultured as described in Materials and Methods were either  pulsed overnight with 1 μCi of [3H]thymidine (A, B) or counted by Trypan blue exclusion (E). Experiments shown are representative of three experiments and data are presented as mean cpm ± SD (A and B) and cell  numbers per ml ± SD (E), as determined in triplicate wells. For quantitating recovery of viable cells, 105 progenitor cells (indicated by horizontal line in medium control bar) were seeded in 1.0 ml of culture medium  onto 105 CD40L+ cells or control CD32 cells. Results were evaluated at  days 4, 8, and 15 (A) or at day 8 only (B, E). Where indicated, cultures  were supplemented with mAbs to CD40 (mAb 89), CD40L (mAb LL48),  or isotype-matched control antibodies. (C, D) Cell cycle analysis.  CD34HPC freshly isolated (data not shown) or cultured either on CD32L  (C) or CD40L-transfected fibroblasts (D) for 3 d were resuspended and  incubated with Hoechst 33342. To quantitate specifically CD34HPC entering into cycle, Hoechst-stained cells were subsequently labeled with a  mAb to CD34, washed, and analyzed with a FACSTAR+® flow cytometer.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196112&req=5

Figure 1: CD40 ligation induces proliferation of human CD34+ progenitor cells. (A) Kinetics, (B, E) specificity, (C, D) cell cycle analysis. CD34HPC cultured as described in Materials and Methods were either pulsed overnight with 1 μCi of [3H]thymidine (A, B) or counted by Trypan blue exclusion (E). Experiments shown are representative of three experiments and data are presented as mean cpm ± SD (A and B) and cell numbers per ml ± SD (E), as determined in triplicate wells. For quantitating recovery of viable cells, 105 progenitor cells (indicated by horizontal line in medium control bar) were seeded in 1.0 ml of culture medium onto 105 CD40L+ cells or control CD32 cells. Results were evaluated at days 4, 8, and 15 (A) or at day 8 only (B, E). Where indicated, cultures were supplemented with mAbs to CD40 (mAb 89), CD40L (mAb LL48), or isotype-matched control antibodies. (C, D) Cell cycle analysis. CD34HPC freshly isolated (data not shown) or cultured either on CD32L (C) or CD40L-transfected fibroblasts (D) for 3 d were resuspended and incubated with Hoechst 33342. To quantitate specifically CD34HPC entering into cycle, Hoechst-stained cells were subsequently labeled with a mAb to CD34, washed, and analyzed with a FACSTAR+® flow cytometer.
Mentions: To investigate the potential role of the CD40 molecule on CD34HPC, progenitor cells were cultured over CD40Ltransfected fibroblasts (CD40L cells/CD40L system), (10, 14) or on CD32-transfected cells as control. CD40L-activated CD34HPC entered into DNA synthesis, as measured by tritiated thymidine incorporation that peaked around day 8 and declined by day 15 (Fig. 1 A). CD40-triggered proliferation of progenitor cells was specific, because it was prevented by antibodies to CD40 and CD40L but not by isotype-matched control antibodies (Fig. 1 B and E ). The observed DNA synthesis reflected actual cell proliferation (Fig. 1 E) with a mean 10-fold increase in viable cells observed at day 8 (four independent experiments). In contrast, there was no or only marginal increase in proliferation of CD34HPC cultured over control CD32-transfected cells (CD32 L cells; Fig. 1 A, B, E). Double labeling with Hoechst 33342 and CD34 MoAb to assess the cell cycle of CD34HPC confirmed the quiescent status of these cells when freshly isolated (data not shown). After 3 d of culture over control CD32 L cells, few CD34HPC were cycling (Fig. 1 C ). By contrast, a significant proportion of CD34HPC were found in cycle when stimulated for 3 d by CD40L (Fig. 1 D). However, at 24 h (data not shown), the percentage of cycling cells in CD40L cultures was only marginally higher than that of control cultures, indicating that CD40L probably recruits a small proportion of CD34+ cells whose cycling progeny accumulates with time.

Bottom Line: CD40-activated cord blood CD34HPC were found to proliferate and differentiate independently of granulocyte/macrophage colony-stimulating factor, into a cell population with prominent dendritic cell (DC) attributes including priming of allogeneic naive T cells.These features were shared by a dendritic population identified in situ in tonsillar T cell areas.Taken together, the present data demonstrate that CD40 is functional on CD34HPC and its cross-linking by CD40L+ cells results in the generation of DC that may prime immune reactions during antigen-driven responses to pathogenic invasion, thus providing a link between hematopoiesis, innate, and adaptive immunity.

View Article: PubMed Central - PubMed

Affiliation: Schering-Plough, Laboratory for Immunological Research, Dardilly, France.

ABSTRACT
Human CD34+ multilineage progenitor cells (CD34HPC) from cord blood and bone marrow express CD40, a member of the tumor necrosis factor-receptor family present on various hematopoietic and nonhematopoietic cells. As hyper-IgM patients with mutated CD40 ligand (CD40L) exhibit neutropenia, no B cell memory, and altered T cell functions leading to severe infections, we investigated the potential role of CD40 on CD34HPC development. CD40-activated cord blood CD34HPC were found to proliferate and differentiate independently of granulocyte/macrophage colony-stimulating factor, into a cell population with prominent dendritic cell (DC) attributes including priming of allogeneic naive T cells. DC generated via the CD40 pathway displayed strong major histocompatibility complex class II DR but lacked detectable CD1a and CD40 expression. These features were shared by a dendritic population identified in situ in tonsillar T cell areas. Taken together, the present data demonstrate that CD40 is functional on CD34HPC and its cross-linking by CD40L+ cells results in the generation of DC that may prime immune reactions during antigen-driven responses to pathogenic invasion, thus providing a link between hematopoiesis, innate, and adaptive immunity.

Show MeSH
Related in: MedlinePlus