Murine Model of CD40-activation of B cells

Summary

In this video, we demonstrate the procedure of CD40-activation and expansion of murine B cells from splenocytes of C57BL/6 mice, which can be used as a model antigen-presenting cell (APC) to study induction of immunity.

Abstract

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand (CD40L), human B cells can be expanded without difficulties from small amounts of peripheral blood within 14 days to very large amounts of highly-pure CD40-B cells (>10⁹ cells per patient) from healthy donors as well as cancer patients^1-4. CD40-B cells express important lymph node homing molecules and can attract T cells in vitro⁵. Furthermore they efficiently take up, process and present antigens to T cells^6,7. CD40-B cells were shown to not only prime naíve, but also expand memory T cells^8,9. Therefore CD40-activated B cells (CD40-B cells) have been studied as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cell-based immunotherapy1,5,10. In order to further study whether CD40-B cells induce effective T cell responses in vivo and to study the underlying mechanism we established a cell culture system for the generation of murine CD40-activated B cells. Using splenocytes or purified B cells from C57BL/6 mice for CD40-activation, optimal conditions were identified as follows: Starting from splenocytes of C57BL/6 mice (haplotype H-2b) lymphocytes are purified by density gradient centrifugation and co-cultured with HeLa cells expressing recombinant murine CD40 ligand (tmuCD40L HeLa)¹¹. Cells are recultured every 3-4 days and key components such as CD40L, interleukin-4, -Mercaptoethanol and cyclosporin A are replenished. In this protocol we demonstrate how to obtain fully activated murine CD40-B cells (mCD40B) with similar APC-phenotype to human CD40-B cells (Fig 1a,b). CD40-stimulation leads to a rapid outgrowth and expansion of highly pure (>90%) CD19+ B cells within 14 days of cell culture (Fig 1c,d). To avoid contamination with non-transfected cells, expression of the murine CD40 ligand on the transfectants has to be controlled regularly (Fig 2). Murine CD40-activated B cells can be used to study B-cell activation and differentiation as well as to investigate their potential to function as APC in vitro and in vivo. Moreover, they represent a promising tool for establishing therapeutic or preventive vaccination against tumors and will help to answer questions regarding safety and immunogenicity of this approach¹².

Protocol

The protocol for the generation of murine CD40-activated B cells (mCD40B) from splenocytes is divided into two parts: Part A demonstrates the preparation of murine CD40 ligand (CD40L) expressing HeLa cells (tmuCD40L HeLa), which will be used as plate-bound feeder cells. Part B describes the actual murine CD40-B culture. A. Preparation of feeder cells (tmuCD40L HeLa) The tmuCD40L HeLa is an adherent human epithelial cell line, which should never become completely con…

Discussion

Modifications of this protocol are possible, especially regarding the origin of CD40-stimulus and the type of mouse strain used, also yielding in efficient generation of murine CD40-activated B cells. Ahmadi et al. have shown similar results for mouse fibroblasts transfected with murine CD40-Ligand. In this context presentation of xeno-antigen can be excluded with certainty, but intensive studies regarding security and toxicity in vivo gave no signs for inflammation or auto-immunity reaction in this setting usin…

Materials

A. Preparation of Media:

Feeder cell wild type medium	Feeder cell selection medium
Reagent	Concentration	Reagent	Concentration
RPMI 1640		RPMI 1640
L-Glutamine	300 μg/mL	L-Glutamine	300 μg/mL
FBS	10 %	FBS	10 %
HEPES	10 mM	HEPES	10 mM
Gentamycin	15 μg/mL	Gentamycin	15 μg/mL
		Hygromycin B	0.2 mg/mL

mCD40-B washing medium	mCD40-B culture medium
Reagent	Concentration	Reagent	Concentration
D-MEM		D-MEM
L-Glutamine	580 μg/mL	L-Glutamine	580 μg/mL
Glucose	4.5 mg/mL	Glucose	4.5 mg/mL
HEPES	10 mM	HEPES	10 mM
Gentamycin	15 μg/mL	Gentamycin	15 μg/mL
		MEM	0.1mM (1x)
		FBS	10%

B. Miscellaneous Reagents:

Reagent	Company	Order No
RPMI 1640	Invitrogen Gibco®	REF 21875
D-MEM	Invitrogen Gibco®	REF 41965
Dulbecco´s PBS (10x)	PAA	Cat No H15-011
Gencin®	Delta Select	Art No 7395800
FBS	LONZA	Cat No DE14-802C
HEPES Buffer	PAA	Cat No S11-001
MEM NEAA	Invitrogen Gibco®	REF 11140
Hygromycin B	PAA	Cat No P02-015
Recombinant Murine Interleukin-4	Immunotools	Cat No 12340045
Cyclosporin A	Novartis	Cat No NDC 0078-0109-01
Trypsin/EDTA (10x)	Invitrogen Gibco®	REF 15400-054

C. Miscellaneous Supplies:

Reagent	Company	Order No
Sterile pipette tips	Sarstedt
6-well plate	NUNC	Cat No 140675
50mL conical tube	BD Falcon™	Cat No 352070
Tissue culture flask	Sarstedt	Cat No 83.1813.002