CD4 (Leu3, T4)

CD4 (cluster of differentiation 4), also known as Leu-3 and T4, is a glycoprotein primarily expressed on the surface of thymocytes, as well as, specific subsets of mature T-cells, monocytes, marcrophages, dendritic cells and NKT cells. As a T cell co-receptor, CD4 and the TCR complex bind to antigen presenting MHC class II molecules. It is this interaction that triggers T cell activation and influences the immune response.

In biomedical and molecular biology research, the CD4 molecule is of great interest because of its role in the human immunodeficiency virus (HIV). CD4, unfortunately serves as a high affinity receptor for HIV, which the virus utilizes for entry into host T cells. During HIV infection, the production and number of CD4⁺ T cells progressively reduces leaving the host vulnerable to many pathogens. In clinical diagnostics, CD4 counts are frequently used to assess the health of a patient's immune system infected with HIV.

CD4 Structure and Function

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CD4 is a 58 kDa, type 1 transmembrane glycoprotein and a member of the immunoglobulin (IgG) superfamily. It consists of four IgG extracellular domains that are connected to a short transmembrane domain and a short intracellular cytoplasmic tail. This tail contains a unique amino acid sequence that facilitates its interaction with the tyrosine kinase Lck, a key player in ITAM phosphorylation and T cell activation.

During TCR and CD4 recognition of antigen presenting MHC class II molecules, CD4 binds to the β2 region of the MHC class II molecules. As a result, the tyrosine kinase Lck that is bound to the cytoplasmic tail of CD4 is brought into close proximity of the immunoreceptor tyrosine activation motifs (ITAMs) on the cytoplasmic tail of CD3. CD3-ITAM phosphorylation by CD4-Lck, amplifies the T cell signal which leads to the activation of transcription factors that promote T cell activation.

CD4⁺ T cell Subsets

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Interactions between antigen presenting MHC class II molecules, significantly influences the type of T cells that are activated. The following table highlights key details of each major CD4⁺ T cell subset and the characteristics that define each subset.

1. Mark. Exp. = Marker Expression; TF = Transcription Factor; Stim. Cytokines = Stimulatory Cytokines; Inh. Cytokines = Inhibitory Cytokines; SE Cytokines = Secreted Effector Cytokines.

CD4 Antibodies

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Monoclonal CD4 antibodies, designed to recognize the CD4 surface molecules, are frequently used in flow cytometry and immunofluorescence applications to identify immune cell subsets expressing CD4. Cells known to express CD4 surface markers include thymocytes, specific subsets of mature T-cells, monocytes, marcrophages, dendritic cells and NKT cells.

AAT Bioquest offers a comprehensive catalog of CD4 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 CD4 Antibodies

The following table outlines the fluorescence properties of available iFluor® dye labeled anti-human CD4 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 CD4 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 CD4 Antibodies

The following table outlines the fluorescence properties of available mFluor™ dye labeled anti-human CD4 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 CD4 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 CD4 Antibodies

The following table outlines the fluorescence properties of available Alexa Fluor^® dye labeled anti-human CD4 antibodies for use in flow cytometry (FACS) and fluorescence imaging applications. For additional information on Alexa Fluor^® dye-labeled CD4 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 CD4 Antibodies

The following table outlines the fluorescence properties of available classic dye labeled anti-human CD4 antibodies for use in flow cytometry (FACS) and fluorescence imaging applications. For additional information on classic dye-labeled CD4 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 CD4 Antibodies

The following table outlines the fluorescence properties of available phycoerythrin (PE), allophycocyanin (APC), PerCP and tandem dye labeled anti-human CD4 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 CD4 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. Geginat, J., Paroni, M., Maglie, S., Alfen, J. S., Kastirr, I., Gruarin, P., De Simone, M., Pagani, M., & Abrignani, S. (2014). Plasticity of human CD4 T cell subsets. Frontiers in immunology, 5, 630. https://doi.org/10.3389/fimmu.2014.00630
2. Golubovskaya, V., & Wu, L. (2016). Different Subsets of T Cells, Memory, Effector Functions, and CAR-T Immunotherapy. Cancers, 8(3), 36. https://doi.org/10.3390/cancers8030036
3. Li, Y., Yin, Y., & Mariuzza, R. A. (2013). Structural and biophysical insights into the role of CD4 and CD8 in T cell activation. Frontiers in immunology, 4, 206. https://doi.org/10.3389/fimmu.2013.00206
4. Luckheeram, R. V., Zhou, R., Verma, A. D., & Xia, B. (2012). CD4⁺T cells: differentiation and functions. Clinical & developmental immunology, 2012, 925135. https://doi.org/10.1155/2012/925135