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人CD40激活的B细胞的生成

Biology

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Liebig, T. M., Fiedler, A., Zoghi, S., Shimabukuro-Vornhagen, A., von Bergwelt-Baildon, M. S. Generation of Human CD40-activated B cells. J. Vis. Exp. (32), e1373, doi:10.3791/1373 (2009).

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Abstract

CD40激活的B细胞(CD40 - B细胞)已被确定为肿瘤免疫治疗的免疫刺激抗原提呈细胞(APC)1-3的替代来源。树突状细胞(DCs),最好的APC,CD40 - B细胞有几个不同的生物和技术性能相比。与区议会,B细胞的MHC和共刺激分子(图1b)的表达增加,表现出强烈的的迁徙能力和目前的T细胞抗原提呈效率,刺激白细胞介素4和CD40配体(CD40L)。然而,在不成熟或成熟的DCS相比,CD40 - B细胞表达完整的淋巴结归巢CD62L,CCR7/CXCR4组成的黑社会,和白细胞功能抗原-1(LFA1,CD11a/CD18),归巢到次级淋巴器官所必需的(图1a)3。 CD40 - B细胞可以产生而没有从非常小,它可以进一步扩大在体外大量高纯度CD40的B细胞(> 10 9细胞每名患者)从健康的捐赠者以及癌症金额外周血困难患者(图1c,d)项1,4。

在这个协议中,我们演示了如何获取充分激活的人PBMC CD40 - B从细胞。细胞培养的关键分子CD40配体,白细胞介素4(IL - 4)和环孢素A(CsA),这是在3-4天养殖周期的补充。 CD40的刺激对于实验室的目的是提供NIH/3T3细胞表达重组人CD40配体(tCD40L NIH/3T3)5。为了避免污染与非转染的细胞,表达人CD40配体的转染,定期进行检查(图)。

CD40 - B细胞培养,经过14天,包括95%以上的纯B细胞和CD40 - B细胞超过65天的扩张是没有任何损失的功能:1,4经常可能。 CD40 - B细胞的有效占用,过程和目前T细胞抗原6。他们不只有总理naϊve,而且还扩大记忆性T细胞7,8。 CD40激活的B细胞可用于研究B细胞的活化,分化和功能。此外,他们代表一个有前途的治疗或预防接种抗肿瘤9工具。

Protocol

PBMC的人CD40激活的B细胞产生的协议分为两部分:A部分演示NIH/3T3细胞,将用于板约束的饲养层细胞表达CD40配体的准备。 B部分介绍了各种实际的CD40 - B的文化。

A.制备饲养细胞(N​​IH/3T3 tCD40L)

tCD40L NIH/3T3壁小鼠成纤维细胞系,这不应该成为完全融合。因此,这些细胞被劈裂每周两次。超过6周以上的培养,不推荐。

  1. 从小学文化的老媒体取出,用无菌吸管和10 mL 1X PBS中洗细胞。吸的PBS洗涤后。
  2. 添加4 mL的胰蛋白酶/ EDTA在75厘米 2瓶5-10分钟,在37 ° C。使用温柔攻分离的细胞。
  3. 野生型中添加10毫升,并轻轻旋转。
  4. 转移到50 mL试管细胞悬液,用无菌吸管和旋转5分钟在225 XG细胞。
  5. 去除上清,重悬沉淀在野生型中的10毫升。计数细胞悬液等分的细胞数,并准备适当数量的细胞50毫升管:
    1. 1.5 × 10 6细胞传代
    2. 0.2 × 10 6细胞/孔的CD40 - B细胞培养用于照射
    3. 其余冻结(如需要)。
  6. 旋转细胞在225 5分钟XG。
  7. 去除上清。
    1. 传代培养:重悬于1.5 × 10 6细胞10毫升,75厘米2细胞培养瓶中的野生型中(细胞密度为1.5 × 10 5细胞/ mL),加入的G - 418 [0.7毫克/毫升]和孵化细胞37℃,5%的CO 2 。每周两次分裂的细胞。
    2. 对于CD40 - B细胞培养:您需要1.2 × 10 6 6孔板细胞。在野生型中重悬的细胞密度0.1 × 10 6细胞/ mL,他们在78 Gy的照射。板2毫升细胞悬液,每口井,并在37℃,5%的CO 2。使用B -细胞的刺激,这准备板块tCD40L NIH/3T3细胞是贴壁时(至少4个小时:坚持用显微镜检查;不要等到超过24 h开始的B细胞刺激)。 (继续与B)

B. CD 40 - B细胞培养

一,制备外周血CD40的刺激(0天):

请注意:在继续之前确定,饲养层细胞是贴壁。随时添加新的解决办法白细胞介素4和环孢素A培养基中,使用前。

  1. 取外周血,新鲜或适当解冻。悬浮外周血1X PBS毫升,两次把它们洗干净,和自旋向下第一时间为7分钟,第二次在7分钟190 XG删除其他细胞在265 XG。弃上清,并在20毫升的PBS重悬细胞。确定细胞的细胞悬液分装数。
  2. 离心5分钟在225 XG所需的细胞量。对于6孔板4 × 10 6细胞/孔的需要,因此24 × 10 6细胞每盘。
  3. 去除上清,重悬CD40 - B的文化与50 U /白细胞介素4毫升新鲜培养基中的外周血单个核细胞在1 × 10 6细胞/ mL,作为一种生长因子和0.63微克/毫升环孢素A到防止T细胞的产物(给定的浓度是指一毫升培养液)。
  4. 去除上清从6孔板前培养与tCD40L NIH/3T3细胞。
  5. 每孔2 mL的PBS第一和第二步在2毫升的CD40 - B的清洗介质,清洗附着tCD40L NIH/3T3细胞。
  6. 轻轻添加4毫升的外周血单个核细胞悬液(1 × 10 6细胞/ mL),每孔6孔板。
  7. 细胞在37 ° C的5%的CO 2。
  8. 第7天,reculture细胞(3.2继续。)

二。复垦的CD40的B细胞(7天,然后每隔3-4天):

  1. 从6孔板CD40 - B细胞10毫升到50毫升管的移液器和池他们resuspending收获集群。
  2. 离心7分钟在225 XG,并完全取代CD40 - B的清洗介质的上清。虽然计数细胞数量的等分,5分钟旋转225 XG细胞。悬浮CD40的B细胞CD40 - B的培养基中,在浓度为1 × 10 6细胞/ mL 。
  3. 在50 U / mL的浓度和0.63μg/ mL的环孢素A的中期添加新鲜的白细胞介素4的解决方案。
  4. 从6孔板与tCD40L NIH/3T3细胞预孵育取出上清。
  5. 每孔2 mL的PBS第一和第二步在2毫升的CD40 - B的清洗介质清洗贴壁细胞。
  6. 6细胞/ ml)CD40 - B的悬挂。
  7. 孵育板在37℃,5%的CO 2。
  8. 再次传代培养细胞每隔3-4天,结束与高纯度的人CD40 B细胞激活后,共14天。

C.故障排除 - CD40 - B细胞,如果不增长?

  1. 你有检查饲养层细胞的CD40的配体表达?
  2. 板势必给料机,用于刺激细胞不应超过24h以上吗?
  3. 支原体可能是一个污染?
  4. 白细胞介素4的解决方案,为补充新鲜解冻,并有适当的生物活性?
  5. 环孢素A中添加正确的浓度?

图1
图1,点击这里为图1的放大版本。

图2
图2,点击这里为图2的较大版本。

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Materials

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

DOWNLOAD MATERIALS LIST

References

  1. Schultze, J. L. CD40-activated human B cells: an alternative source of highly efficient antigen presenting cells to generate autologous antigen-specific T cells for adoptive immunotherapy. J Clin Invest. 100, 2757-2765 (1997).
  2. Schultze, J. L., Grabbe, S., vonBergwelt-Baildon, M. S. DCs and CD40-activated B cells: current and future avenues to cellular cancer immunotherapy. Trends Immunol. 25, 659-664 (2004).
  3. Bergwelt-Baildon, M. von CD40-activated B cells express full lymph node homing triad and induce T-cell chemotaxis: potential as cellular adjuvants. Blood. 107, 2786-2789 (2006).
  4. Wiesner, M. Conditional immortalization of human B cells by CD40 ligation. PLoS ONE. 3, 1464-14 (2008).
  5. Urashima, M., Chauhan, D., Uchiyama, H., Freeman, G. J., Anderson, K. C. CD40 ligand triggered interleukin-6 secretion in multiple myeloma. Blood. 85, 1903-1912 (1995).
  6. Lapointe, R., Bellemare-Pelletier, A., Housseau, F., Thibodeau, J., Hwu, P. CD40-stimulated B lymphocytes pulsed with tumor antigens are effective antigen-presenting cells that can generate specific T cells. Cancer Res. 63, 2836-2843 (2003).
  7. von Bergwelt-Baildon, M. S. Human primary and memory cytotoxic T lymphocyte responses are efficiently induced by means of CD40-activated B cells as antigen-presenting cells: potential for clinical application. Blood. 99, 3319-3325 (2002).
  8. Kondo, E. CD40-activated B cells can be generated in high number and purity in cancer patients: analysis of immunogenicity and homing potential. Clin Exp Immunol. 155, 249-256 (2009).
  9. Mason, N. J. RNA-loaded CD40-activated B cells stimulate antigen-specific T-cell responses in dogs with spontaneous lymphoma. Gene Ther. 15, 955-965 (2008).

Comments

8 Comments

  1. I would like to know if the 3T3 cells seeded are not confluent in the 6-well plate after ²4 hours incubation, can the plate be used for co-culture with PBMC?

    Reply
    Posted by: Ping Lung C.
    October 21, 2009 - 5:48 AM
  2. The 3T3 cells are adherent after 1² to ²4h incubation and yes they can be used for co-culture.

    Reply
    Posted by: Alexander S.
    October 21, 2009 - 8:49 AM
  3. This is quite interesting, but I wonder what is the practical advantage over using an activating anti-CD40 like www.epitomics.com/pdf/440²-1.pdf or simply a soluble CD40L like www.biomol.de/datenblaetter/biomol_de/94894.pdf or ?

    Reply
    Posted by: Oliver T.
    October 21, 2009 - 5:40 PM
  4. You are absolutely right. It would be much simpler to use a soluble ligand instead of the CD40L-expressing cell line. Unfortunately, none of the ligands we tested worked for our purpose. All were able to induce activation of B cells but none of them induced the robust proliferation that we observe with the CD40L-expressing NIH3T3 cells.
    The only ligand that was able to induce proliferation was a trimeric ligand produced by Immunex. Howerver, to our regret Immunex stopped its development and it is not available anymore.

    Reply
    Posted by: Alexander S.
    October 22, 2009 - 10:07 AM
  5. Apart from research developments, what do you think about the use of CD40L-stimulated B cells in clinical settings (such as cellular adiuvants), and how, in these cases, can we avoid the use of reagents and cells not adequate?

    Reply
    Posted by: Anonymous
    November 14, 2009 - 12:11 PM
  6. where did you obtain the cd40ligand expressing 3t3 cells???

    Reply
    Posted by: Anonymous
    May 6, 2010 - 6:56 PM
  7. Hi,
    I am encountering problem with the feeder cell monolayer, after putting the B-Cells on the top of monolayer, cells from monolayer it starts coming up and ultimately leading to loss of B-cells too.
    Have you any time encountered any such issue with monolayer (Cd40)?

    Reply
    Posted by: Anonymous
    April 4, 2011 - 4:56 PM
  8. i wanna know in daetail about culturing cells and about protein purification

    Reply
    Posted by: Mustafa A.
    September 10, 2012 - 7:34 AM

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