Generation of Human CD40-activated B cells

Summary

In this video we present the ex vivo generation and expansion of human CD40-activated B cells (CD40-B) from peripheral blood mononuclear cells (PBMC) by stimulation with CD40 ligand and interleukin-4.

Abstract

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer immunotherapy ^1-3. Compared to Dendritic cells (DCs), the best characterized APC, CD40-B cells have several distinct biological and technical properties. Similar to DCs, B cells show an increased expression of MHC and co-stimulatory molecules (Fig.1b), exhibit a strong migratory capacity and present antigen presentation efficiently to T cells, after stimulation with interleukin-4 and CD40 ligand (CD40L). However, in contrast to immature or mature DCs, CD40-B cells express the full lymph node homing triad consisting of CD62L, CCR7/CXCR4, and leukocyte function antigen-1 (LFA1, CD11a/CD18), necessary for homing to secondary lymphoid organs (Fig.1a) ³. CD40-B cells can be generated without difficulties from very small amounts of peripheral blood which can be further expanded in vitro to very large amounts of highly-pure CD40-B cells (>10⁹ cells per patient) from healthy donors as well as cancer patients (Fig.1c,d) ^1,4.

In this protocol we demonstrate how to obtain fully activated CD40-B cells from human PBMC. Key molecules for the cell culture are CD40 ligand, interleukin-4 (IL-4) and cyclosporin A (CsA), which are replenished in a 3-4 day culture cycle. For laboratory purposes CD40-stimulation is provided by NIH/3T3 cells expressing recombinant human CD40 ligand (tCD40L NIH/3T3) ⁵. To avoid contamination with non-transfected cells, expression of the human CD40 ligand on the transfectants has to be checked regularly (Fig.2).

After 14 days CD40-B cell cultures consist of more than 95% pure B cells and an expansion of CD40-B cells over 65 days is frequently possible without any loss of function ^{1, 4}. CD40-B cells efficiently take up, process and present antigens to T cells ⁶. They do not only prime naϊve, but also expand memory T cells ^7,8. CD40-activated B cells can be used to study B-cell activation, differentiation and function. Moreover, they represent a promising tool for therapeutic or preventive vaccination against tumors ⁹.

Protocol

The protocol for the generation of human CD40-activated B cells from PBMC is divided into two parts: Part A demonstrates the preparation of CD40 ligand expressing NIH/3T3 cells, which will be used as plate-bound feeder cells. Part B describes the actual CD40-B culture.

A. Preparation of feeder cells (tCD40L NIH/3T3)

The tCD40L NIH/3T3 is an adherent murine fibroblast cell line, which should never become completely confluent. The cells are therefore splitted twice per week. Culturing over more than 6 weeks is not recommended.

1. Remove old medium from the primary culture with a sterile pipette and wash c....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
A. Preparation of Media:
1. Feeder cell wild type medium
DMEM-Ham’s / F12
L-Glutamine (365 μg/mL)
FBS (10%)
HEPES (10 mM)
Gentamycin (15 μg/mL)
2. Feeder cell selection medium
DMEM-Ham’s / F12
L-Glutamine (365 μg/mL)
FBS (10%)
HEPES (10 mM)
Gentamycin (15 μg/mL)
G-418 (0.7 mg/mL)
3. CD40-B washing medium
IMDM
L-Glutamine (584 μg/mL)
HEPES (25 mM)
Gentamycin (15 μg/mL)
4. CD40-B culture medium
IMDM
L-Glutamine (584 μg/mL)
HEPES (25 mM)
Gentamycin (15 μg/mL)
rh Transferrin (50 μg/mL)
rh Insulin (5 μg/mL)
AB-Humanserum (10%)
B. Miscellaneous Reagents:
DMEM / Ham’s F12	PAA Laboratories	Cat No: E15-813
IMDM	Invitrogen	REF 21980-032
Dulbecco’s PBS (10x)	PAA Laboratories	Cat No H15-011
AB-Human Serum	Invitrogen	Cat No 34005100
holo-Transferrin human	Sigma-Aldrich	Cat No T0665
Insulin human	Sigma-Aldrich	Cat No I2643
Gencin®	Delta Select	Art No 7395800
Recombinant Human Interleukin-4	Immunotools	Cat No 11130045
Cyclosporin A	Novartis AG	Cat No NDC 0078-0109-01
FBS	Lonza Inc.	Cat No DE14-802C
HEPES Buffer	PAA Laboratories	Cat No S11-001
G-418 Sulphate	PAA Laboratories	Cat No P02-012
Trypsin/EDTA (10x)	Invitrogen	REF 15400-054
C. Miscellaneous Supplies:
Sterile pipette tips	Sarstedt Ltd
6-well plate	Nalge Nunc international	Cat No 140675
50mL conical tube	BD Biosciences	Cat No 352070
Tissue culture flask	Sarstedt Ltd	Cat No 83.1813.002