CD40 (TNFRSF5)

CD40 (cluster of differentiation 40), also known as TNFRSF5, is a transmembrane protein primarily expressed on the surface of B cells, antigen presenting cells (APC), dendritic cells, macrophages and monocytes, as well as on non-hematopoietic cells, non-immune cells under inflammatory conditions and tumors. CD40 functions mainly as a co-stimulatory protein that binds to CD154, also known as CD40 ligand or CD40L, which is present on activated T helper cells. It is this interaction, the binding of CD40 and CD40L that plays a pivotal role in adaptive immunity. For instance, interactions between CD40L expressing T cells and CD40 expressing B cells initiate B cell proliferation and differentiation into antibody-secreting plasma cells. As an integral component of various immune and inflammatory responses, including the aforementioned B cell activation and immunoglobulin class switching, CD40 has emerged as a promising target for the development of drugs, namely therapeutic monoclonal antibodies (mAbs), as a form of cancer immunotherapy. One such example is Dacetuzumab, a humanized agonistic anti-CD40 mAb which was designed to activate CD40-mediated signaling in various cell types.

CD40 Mediated-Signaling

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Human CD40 is a 45-50 kDa, type I transmembrane glycoprotein and member of the tumor necrosis factor receptor (TNFR) superfamily. It consists of four components, a 193 amino acid long extracellular domain, a 21 amino acid long leader sequence, a 22 amino acid long transmembrane domain and a 62 amino acid long intracellular domain. Upon interaction with its ligand CD40L on activated T helper cells, CD40 is internalized into the B cell initiating several signaling events that result in key cellular and humoral immune responses. Since the cytoplasmic domain of CD40 does not contain any enzymatic activity, it recruits and binds with TNF-receptor associated factors - TRAF1, TRAF2, TRAF3, TRAF5 and TRAF6 - which act as mediators trigger downstream signaling events. One of the most important downstream events activated by this interaction is the NF-κB signaling pathway which is essential for maintaining homeostasis and in pathogenic processes such as cell proliferation, death and inflammatory responses.

CD40 Antibodies

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Monoclonal CD40 antibodies, designed to recognize human CD40 surface molecules, are frequently used in flow cytometry and immunofluorescence applications as a pan-B marker identifying B cells from terminally differentiated plasma cells. Cells known to express CD40 surface markers include B cells, pre-B cells, macrophages, monocytes, endothelial cells and follicular dendritic cells. In clinical research, diseases associated with CD40 dysfunction include CD40L deficiency and Type 3 Hyper-IgM immunodeficiency.

AAT Bioquest offers a comprehensive catalog of CD40 antibodies purified by affinity chromatography and conjugated to a variety of fluorophores under optimal conditions that minimize unconjugated fluorophore and antibody. Available fluorophores include:

• iFluor® dyes - bright, photostable dyes with optimized flow cytometry (FACS), fluorescence imaging and in vivo imaging applications.
• mFluor™ dyes - bright, photostable dyes with optimized for flow cytometry (FACS) applications.
• Alexa Fluor^® dyes - suitable for flow cytometry (FACS) and fluorescence imaging applications.
• Classic dyes - suitable for flow cytometry (FACS) and fluorescence imaging applications.
• Phycobiliproteins and Tandem dyes - intensely bright dyes for flow cytometry (FACS) and multiparametric analysis.

1. FC = Flow Cytometry; ELISA = Enzyme-linked immunosorbent assay; IF = Immunofluorescence; IHC-F = Immunohistochemistry (Frozen); WB = Western Blot.

iFluor® Dyes Labeled to CD40 Antibodies

The following table outlines the fluorescence properties of available iFluor® dye labeled anti-human CD40 antibodies for use in flow cytometry (FACS) and fluorescence imaging applications. Conjugates made with iFluor® dyes exhibit superior brightness and photostability, outperforming Alexa Fluor^® conjugates and other spectrally similar conjugates. For additional information on iFluor® dye-labeled CD40 antibodies and availability of other clones click on any label in the table below.

1. ε = molar extinction coefficient at their maximum absorption wavelength (Units = cm^-1M^-1).
2. Φ = fluorescence quantum yield in aqueous buffer (pH 7.2).
3. CF at 260 nm is the correction factor used for eliminating the dye contribution to the absorbance at 260 nm (for oligo and nucleic acid labeling).
4. CF at 280 nm is the correction factor used for eliminating the dye contribution to the absorbance at 280 nm (for peptides and protein labeling).

mFluor™ Dyes Labeled to CD40 Antibodies

The following table outlines the fluorescence properties of available mFluor™ dye labeled anti-human CD40 antibodies for use in flow cytometry (FACS). Each mFluor™ dyes is optimally excited by one of the major laser lines commonly equipped in flow cytometers, such as the 405 nm, 488 nm, 532 nm, 561 nm or 633 nm laser lines. For additional information on mFluor™ dye-labeled CD40 antibodies and availability of other clones click on any label in the table below.

1. ε = molar extinction coefficient at their maximum absorption wavelength (Units = cm^-1M^-1).
2. Φ = fluorescence quantum yield in aqueous buffer (pH 7.2).
3. CF at 260 nm is the correction factor used for eliminating the dye contribution to the absorbance at 260 nm (for oligo and nucleic acid labeling).
4. CF at 280 nm is the correction factor used for eliminating the dye contribution to the absorbance at 280 nm (for peptides and protein labeling).

Alexa Fluor^® Dyes Labeled to CD40 Antibodies

The following table outlines the fluorescence properties of available Alexa Fluor^® dye labeled anti-human CD40 antibodies for use in flow cytometry (FACS) and fluorescence imaging applications. For additional information on Alexa Fluor^® dye-labeled CD40 antibodies and availability of other clones click on any label in the table below.

1. ε = molar extinction coefficient at their maximum absorption wavelength (Units = cm^-1M^-1).
2. Φ = fluorescence quantum yield in aqueous buffer (pH 7.2).
3. CF at 280 nm is the correction factor used for eliminating the dye contribution to the absorbance at 280 nm (for peptides and protein labeling).

Classic Dyes Labeled to CD40 Antibodies

The following table outlines the fluorescence properties of available classic dye labeled anti-human CD40 antibodies for use in flow cytometry (FACS) and fluorescence imaging applications. For additional information on classic dye-labeled CD40 antibodies and availability of other clones click on any label in the table below.

1. ε = molar extinction coefficient at their maximum absorption wavelength (Units = cm^-1M^-1).
2. Φ = fluorescence quantum yield in aqueous buffer (pH 7.2).
3. CF at 280 nm is the correction factor used for eliminating the dye contribution to the absorbance at 280 nm (for peptides and protein labeling).

PE, APC, PerCP and Tandem Dyes Labeled to CD40 Antibodies

The following table outlines the fluorescence properties of available phycoerythrin (PE), allophycocyanin (APC), PerCP and tandem dye labeled anti-human CD40 antibodies for use in flow cytometry (FACS). Phycobiliproteins are uncharacteristically bright due to their high molar extinction coefficients and quantum yields, an enviable quality when imaging low-abundance targets. However, since phycobiliprotiens photobleach rapidly, they are not recommended for microscopy. For additional information on phycobiliprotein-labeled CD40 antibodies and availability of other clones click on any label in the table below.

1. ε = molar extinction coefficient at their maximum absorption wavelength (Units = cm^-1M^-1).

References

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1. Chung, H.W., Lim, J. Clinical significance of elevated serum soluble CD40 ligand levels as a diagnostic and prognostic tumor marker for pancreatic ductal adenocarcinoma. J Transl Med 12, 102 (2014). https://doi.org/10.1186/1479-5876-12-102
2. Elgueta, R., Benson, M. J., de Vries, V. C., Wasiuk, A., Guo, Y., & Noelle, R. J. (2009). Molecular mechanism and function of CD40/CD40L engagement in the immune system. Immunological reviews, 229(1), 152-172. https://doi.org/10.1111/j.1600-065X.2009.00782.x
3. Tan, J., Town, T., Mori, T., Obregon, D., Wu, Y., DelleDonne, A., Rojiani, A., Crawford, F., Flavell, R. A., & Mullan, M. (2002). CD40 is expressed and functional on neuronal cells. The EMBO journal, 21(4), 643-652. https://doi.org/10.1093/emboj/21.4.643
4. Twohig, J. P., Cuff, S. M., Yong, A. A., & Wang, E. C. (2011). The role of tumor necrosis factor receptor superfamily members in mammalian brain development, function and homeostasis. Reviews in the neurosciences, 22(5), 509-533. https://doi.org/10.1515/RNS.2011.041
5. Vonderheide, R. H., & Glennie, M. J. (2013). Agonistic CD40 antibodies and cancer therapy. Clinical cancer research : an official journal of the American Association for Cancer Research, 19(5), 1035-1043. https://doi.org/10.1158/1078-0432.CCR-12-2064