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.
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.
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.
B. CD40-B cell culture
I. Preparation of PBMCs for CD40-stimulation (day 0):
Please note: before you continue ascertain that feeder cells are adherent. Always add fresh solutions of interleukin-4 and cyclosporin A to the growth medium immediately before use.
II. Recultivation of CD40-B cells (day 7 and then every 3-4 days):
C. Trouble-shooting – What if CD40-B cells do not grow?
Figure 1. Please click here for a larger version of figure 1.
Figure 2. Please click here for a larger version of figure 2.
A. Preparation of Media:
Feeder cell wild type medium | Feeder cell selection medium | ||
Reagent | Concentration | Reagent | Concentration |
DMEM-Ham’s / F12 | DMEM-Ham’s / F12 | ||
L-Glutamine | 365 μg/mL | L-Glutamine | 365 μg/mL |
FBS | 10 % | FBS | 10 % |
HEPES | 10 mM | HEPES | 10 mM |
Gentamycin | 15 μg/mL | Gentamycin | 15 μg/mL |
G-418 | 0.7 mg/mL |
CD40-B washing medium | CD40-B culture medium | ||
Reagent | Concentration | Reagent | Concentration |
IMDM | |
IMDM | |
L-Glutamine | 584 μg/mL | L-Glutamine | 584 μg/mL |
HEPES | 25 mM | HEPES | 25 mM |
Gentamycin | 15 μg/mL | Gentamycin | 15 μg/mL |
rh Transferrin | 50 μg/mL | ||
rh Insulin | 5 μg/mL | ||
AB-Humanserum | 10 % |
B. Miscellaneous Reagents:
Reagent | Company | Order No |
DMEM / Ham’s F12 | PAA | Cat No: E15-813 |
IMDM | Invitrogen Gibco® | REF 21980-032 |
Dulbecco’s PBS (10x) | PAA | Cat No H15-011 |
AB-Human Serum | Invitrogen | Cat No 34005100 |
holo-Transferrin human | Sigma | Cat No T0665 |
Insulin human | Sigma | Cat No I2643 |
Gencin® | Delta Select | Art No 7395800 |
Recombinant Human Interleukin-4 | Immunotools | Cat No 11130045 |
Cyclosporin A | Novartis | Cat No NDC 0078-0109-01 |
FBS | LONZA | Cat No DE14-802C |
HEPES Buffer | PAA | Cat No S11-001 |
G-418 Sulphate | PAA | Cat No P02-012 |
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 |