Improved methods for the generation of dendritic cells from nonproliferating progenitors in human blood

doi:10.1016/0022-1759(96)00079-8

Journal of Immunological Methods

Volume 196, Issue 2, 27 September 1996, Pages 121-135

https://doi.org/10.1016/0022-1759(96)00079-8 Get rights and content

Abstract

We have investigated an improved method for generating sizable numbers of mature dendritic cells from nonproliferating progenitors in human blood. The procedure uses 1% human plasma in the place of 10% fetal calf serum and involves two steps. The first step or ‘priming’ phase is a 6–7 day culture of T cell depleted mononuclear cells in medium supplemented with GM-CSF and IL-4. The second step or ‘differentiation’ phase requires the exposure to macrophage conditioned medium. This medium cannot be replaced by several known cytokines such as TNF-α, IL-1, IL-6, IL-12 and IL-15, and cannot be inhibited with neutralizing antibodies to IL-1, TNF-α, IL-6 or IL-12 alone, or in combination. Using this two-step approach, we obtain substantial yields. About 1–3 × 10⁶ mature dendritic cells are generated from 40 ml of blood vs. < 0.1 × 10⁶ from noncytokine treated blood. The dendritic cells derive from progenitors found primarily in a radioresistant population of CD14⁺ and adherent blood mononuclear cells and have all the features of mature cells. They include a stellate cell shape, nonadherence to plastic, and very strong T cell stimulatory activity. Strong APC function was evident for both the proliferation of allogeneic T cells in the MLR, and the generation by syngeneic T cells of class I restricted, CTL responses to influenza virus. A panel of dendritic cell restricted markers is also expressed, including CD83, p55, and perinuclear CD68. All of these dendritic cell properties are retained for at least 3 days when the cytokines are removed, suggesting that these populations are stable and terminally differentiated. We suggest that these cells will be effective in vivo as adjuvants for active immunotherapy.

References (37)

N. Bhardwaj et al.
Interleukin-1 production by mononuclear cells from rheumatoid synovial effusions
Cell. Immunol.
(1988)
C. Caux et al.
Recent advances in the study of dendritic cells and follicular dendritic cells
Immunol. Today
(1995)
N. Romani et al.
Generation of mature dendritic cells from human blood: an improved method with special regard to clinical applicability
J. Immunol. Methods
(1996)
J.M. Austyn
Lymphoid dendritic cells
Immunol.
(1987)
A. Bender et al.
Inactivated influenza virus, when presented on dendritic cells, elicits human CD8⁺ cytolytic T cell responses
J. Exp. Med.
(1995)
N. Bhardwaj et al.
Influenza virus-infected dendritic cells stimulate strong proliferative and cytolytic responses from human CD8⁺ T cells
J. Clin. Invest.
(1994)
C. Caux et al.
GM-CSF and TNF-alpha cooperate in the generation of dendritic Langerhans cells
Nature
(1992)
C. Caux et al.
Activation of human dendritic cells through CD40 cross-linking
J. Exp. Med.
(1994)
C. Caux et al.
Human dendritic Langerhans cells generated in vitro from CD34⁺ progenitors can prime naive CD4⁺ T cells and process soluble antigen
J. Immunol.
(1995)
M. Crowley et al.
Dendritic cells are the principal cells in mouse spleen bearing immunogenic fragments of foreign proteins
J. Exp. Med.
(1990)

S. Fossum

The life history of dendritic leukocytes (DL)

S. Fossum

Lymph-borne dendritic leucocytes do not recirculate, but enter the lymph node paracortex to become interdigitating cells

Scand. J. Immunol.

(1989)

D.N.J. Hart et al.

Interstitial dendritic cells

Int. Rev. Immunol.

(1990)

D.C. Helfgott et al.

Interferon-beta 2/intereleukin-6 in plasma and body fluids during acute bacterial infection

J. Immunol.

(1988)

K. Inaba et al.

Dendritic cells pulsed with protein antigens in vitro can prime antigen-specific, MHC-restricted T cells in situ

J. Exp. Med.

(1990)

K. Inaba et al.

Dendritic cells as antigen presenting cells in vivo

Int. Rev. Immunol.

(1990)

L. Klareskog et al.

Immunopathogenesis and immunotherapy in rheumatoid arthritis: an area in transition

J. Intern. Med.

(1995)

D.W. Mason et al.

The rat mixed lymphocyte reaction: roles of a dendritic cell in intestinal lymph and T cell subsets defined by monoclonal antibodies

Immunology

(1981)

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¹: Present address: Institute of Immunology, Marburg, Germany.

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Research reportImproved methods for the generation of dendritic cells from nonproliferating progenitors in human blood

Abstract

Cell. Immunol.

Immunol. Today

J. Immunol. Methods

Lymphoid dendritic cells

Immunol.

Inactivated influenza virus, when presented on dendritic cells, elicits human CD8+ cytolytic T cell responses

J. Exp. Med.

Influenza virus-infected dendritic cells stimulate strong proliferative and cytolytic responses from human CD8+ T cells

J. Clin. Invest.

GM-CSF and TNF-alpha cooperate in the generation of dendritic Langerhans cells

Nature

Activation of human dendritic cells through CD40 cross-linking

J. Exp. Med.

Human dendritic Langerhans cells generated in vitro from CD34+ progenitors can prime naive CD4+ T cells and process soluble antigen

J. Immunol.

Dendritic cells are the principal cells in mouse spleen bearing immunogenic fragments of foreign proteins

J. Exp. Med.

The life history of dendritic leukocytes (DL)

Lymph-borne dendritic leucocytes do not recirculate, but enter the lymph node paracortex to become interdigitating cells

Scand. J. Immunol.

Interstitial dendritic cells

Int. Rev. Immunol.

Interferon-beta 2/intereleukin-6 in plasma and body fluids during acute bacterial infection

J. Immunol.

Dendritic cells pulsed with protein antigens in vitro can prime antigen-specific, MHC-restricted T cells in situ

J. Exp. Med.

Dendritic cells as antigen presenting cells in vivo

Int. Rev. Immunol.

Immunopathogenesis and immunotherapy in rheumatoid arthritis: an area in transition

J. Intern. Med.

The rat mixed lymphocyte reaction: roles of a dendritic cell in intestinal lymph and T cell subsets defined by monoclonal antibodies

Immunology

Research report
Improved methods for the generation of dendritic cells from nonproliferating progenitors in human blood

Inactivated influenza virus, when presented on dendritic cells, elicits human CD8⁺ cytolytic T cell responses

Influenza virus-infected dendritic cells stimulate strong proliferative and cytolytic responses from human CD8⁺ T cells

Human dendritic Langerhans cells generated in vitro from CD34⁺ progenitors can prime naive CD4⁺ T cells and process soluble antigen