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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: 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.

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.

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Morphology and phenotype of CD40-activated CD34+HPC and in situ localization of CD1a−/CD40−, DR+ DC. CD34+ HPC prepared  as described were seeded onto control (CD32L cells) cultures (a) or onto CD40L (CD40L-L cells) cultures (b–i) and evaluated at days 8 (a and b) and days  14 (c–i). Photomicrographs were taken from cells in culture wells (a–d) at ×200 (a and b) and ×400 (c and d) magnification, and from cytospin preparations (e) or from BioRad slides (  f–i ) at ×1,000 magnification. In (c) and (d), the same field was taken at a 15-s interval to show dendrite motility. (e) May– Grünwald staining of DC. (  f   ) HIV–gp120 binding. (g) shows a CD26+ DC. (h) A rel-B+ DC together with a rel-B−, nondendritic cell. (i) MHC class II  DR+ DC. (  j and k) Tonsil sections stained for CD1a and CD40 (both in blue) and MHC class II DR (red) showing single DR+, CD1a− and CD40− DC  scattered in the T cell area. (  j ) ×400 magnification and (k) ×1,000 magnification.
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Figure 2: Morphology and phenotype of CD40-activated CD34+HPC and in situ localization of CD1a−/CD40−, DR+ DC. CD34+ HPC prepared as described were seeded onto control (CD32L cells) cultures (a) or onto CD40L (CD40L-L cells) cultures (b–i) and evaluated at days 8 (a and b) and days 14 (c–i). Photomicrographs were taken from cells in culture wells (a–d) at ×200 (a and b) and ×400 (c and d) magnification, and from cytospin preparations (e) or from BioRad slides (  f–i ) at ×1,000 magnification. In (c) and (d), the same field was taken at a 15-s interval to show dendrite motility. (e) May– Grünwald staining of DC. (  f   ) HIV–gp120 binding. (g) shows a CD26+ DC. (h) A rel-B+ DC together with a rel-B−, nondendritic cell. (i) MHC class II DR+ DC. (  j and k) Tonsil sections stained for CD1a and CD40 (both in blue) and MHC class II DR (red) showing single DR+, CD1a− and CD40− DC scattered in the T cell area. (  j ) ×400 magnification and (k) ×1,000 magnification.

Mentions: 8-d cultures of CD34HPC on CD32L cells yielded small round mononuclear cells morphologically comparable to fresh CD34HPC (Fig. 2 a), whereas CD34HPC cultured over CD40L L cells revealed small clusters of 10–50 cells displaying long cytoplasmic projections (Fig. 2 b). These clusters grew progressively in size releasing individual cells (Fig. 2, c and d ) that, by day 14, exhibited long delicate and highly motile cytoplasmic processes (Fig. 2, c and d, 15-s interval). Cytocentrifugation and Giemsa staining of CD40L-cultured CD34HPC revealed that a proportion of cells displayed numerous and long dendrites (Fig. 2 e). A detailed immunophenotypic analysis of CD40-generated DC at day 14 showed high levels of MHC class II DR antigen (Fig. 2 i), and the costimulatory molecules B7-1 (CD80) and B7-2 (CD86) (Table 1). At variance with GM–CSF/TNF-derived DC, CD40-derived DC were negative for CD1a and CD40 (Table 1). Interestingly, CD40generated DC expressed high levels of the transactivating factor rel-B (Fig. 2 h), associated with mature DC but absent from dendritic Langerhans cells (26). A small proportion of CD40-derived DC could bind recombinant HIVgp120 (Fig. 2 f  ) and expressed CD26 (Fig. 2 g). Occasional DC exhibited beaded dendrites reminiscent of those observed on follicular dendritic cells associated with iccosome release (Fig. 2 g) (27). The nondendritic population from CD40L cultures were nongranulocytic monocyte-like cells as determined by May–Grünwald–Giemsa staining (data not shown). Independent analysis of five different CD34HPC samples cultured 14 d over CD40L cells demonstrated 29.9% ± 1.4% motile cells by phase contrast microscopy, 37.4% ± 3.7% cells with dendrites by Giemsa staining, and 39.6% ± 3.6% DC with strong HLA DR expression (data not shown). Taken together, these results demonstrate that a population of CD34HPC can differentiate into DC after CD40 triggering.


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)

Morphology and phenotype of CD40-activated CD34+HPC and in situ localization of CD1a−/CD40−, DR+ DC. CD34+ HPC prepared  as described were seeded onto control (CD32L cells) cultures (a) or onto CD40L (CD40L-L cells) cultures (b–i) and evaluated at days 8 (a and b) and days  14 (c–i). Photomicrographs were taken from cells in culture wells (a–d) at ×200 (a and b) and ×400 (c and d) magnification, and from cytospin preparations (e) or from BioRad slides (  f–i ) at ×1,000 magnification. In (c) and (d), the same field was taken at a 15-s interval to show dendrite motility. (e) May– Grünwald staining of DC. (  f   ) HIV–gp120 binding. (g) shows a CD26+ DC. (h) A rel-B+ DC together with a rel-B−, nondendritic cell. (i) MHC class II  DR+ DC. (  j and k) Tonsil sections stained for CD1a and CD40 (both in blue) and MHC class II DR (red) showing single DR+, CD1a− and CD40− DC  scattered in the T cell area. (  j ) ×400 magnification and (k) ×1,000 magnification.
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Figure 2: Morphology and phenotype of CD40-activated CD34+HPC and in situ localization of CD1a−/CD40−, DR+ DC. CD34+ HPC prepared as described were seeded onto control (CD32L cells) cultures (a) or onto CD40L (CD40L-L cells) cultures (b–i) and evaluated at days 8 (a and b) and days 14 (c–i). Photomicrographs were taken from cells in culture wells (a–d) at ×200 (a and b) and ×400 (c and d) magnification, and from cytospin preparations (e) or from BioRad slides (  f–i ) at ×1,000 magnification. In (c) and (d), the same field was taken at a 15-s interval to show dendrite motility. (e) May– Grünwald staining of DC. (  f   ) HIV–gp120 binding. (g) shows a CD26+ DC. (h) A rel-B+ DC together with a rel-B−, nondendritic cell. (i) MHC class II DR+ DC. (  j and k) Tonsil sections stained for CD1a and CD40 (both in blue) and MHC class II DR (red) showing single DR+, CD1a− and CD40− DC scattered in the T cell area. (  j ) ×400 magnification and (k) ×1,000 magnification.
Mentions: 8-d cultures of CD34HPC on CD32L cells yielded small round mononuclear cells morphologically comparable to fresh CD34HPC (Fig. 2 a), whereas CD34HPC cultured over CD40L L cells revealed small clusters of 10–50 cells displaying long cytoplasmic projections (Fig. 2 b). These clusters grew progressively in size releasing individual cells (Fig. 2, c and d ) that, by day 14, exhibited long delicate and highly motile cytoplasmic processes (Fig. 2, c and d, 15-s interval). Cytocentrifugation and Giemsa staining of CD40L-cultured CD34HPC revealed that a proportion of cells displayed numerous and long dendrites (Fig. 2 e). A detailed immunophenotypic analysis of CD40-generated DC at day 14 showed high levels of MHC class II DR antigen (Fig. 2 i), and the costimulatory molecules B7-1 (CD80) and B7-2 (CD86) (Table 1). At variance with GM–CSF/TNF-derived DC, CD40-derived DC were negative for CD1a and CD40 (Table 1). Interestingly, CD40generated DC expressed high levels of the transactivating factor rel-B (Fig. 2 h), associated with mature DC but absent from dendritic Langerhans cells (26). A small proportion of CD40-derived DC could bind recombinant HIVgp120 (Fig. 2 f  ) and expressed CD26 (Fig. 2 g). Occasional DC exhibited beaded dendrites reminiscent of those observed on follicular dendritic cells associated with iccosome release (Fig. 2 g) (27). The nondendritic population from CD40L cultures were nongranulocytic monocyte-like cells as determined by May–Grünwald–Giemsa staining (data not shown). Independent analysis of five different CD34HPC samples cultured 14 d over CD40L cells demonstrated 29.9% ± 1.4% motile cells by phase contrast microscopy, 37.4% ± 3.7% cells with dendrites by Giemsa staining, and 39.6% ± 3.6% DC with strong HLA DR expression (data not shown). Taken together, these results demonstrate that a population of CD34HPC can differentiate into DC after CD40 triggering.

Bottom Line: 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.

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