Actively helping customers, employees and the global community during the coronavirus SARS-CoV-2 outbreak.  Learn more >>

CD4 (Leu3, T4)

CD4, 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.



Table 1. CD4 At A Glance

Specification
Value
SynonymsLeu-3, T4, OKT4
Cellular localizationCell membrane
Cellular ExpressionT cells, double positive thymocytes (e.g. CD4+CD8+), γ/δ T cells, innate lymphoid cells, macrophages, monocytes
Receptor TypeType 1 transmembrane glycoprotein, member of IgG superfamily
LigandAffinity for peptide-MHC Class II molecules, Interleukin-16 (IL-16), HIVgp120
FunctionMHC Class II molecule co-receptor, HIV receptor
Associated Signaling PathwaysT cell differentiation and T cell signal transduction pathways



CD4 Structure and Function


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 ?2 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


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.

 

Table 2. Overview of CD4+ T cell subsets.

Subset
Phenotype
Mark. Exp.
TF
Stim. Cytokines
Inh. Cytokines
SE Cytokines
Target cells
Function
Th1CXCR3+CD178, CD183, CD195, CD212, CD218αEomes, Hlx, Runx3, STAT1, STAT4, T-betIFN-γ, IL-2, IL-12, IL-18, IL-27IL-4, IL-10, IL-21IFN-δ, IL-2, LTα, TNF-βCytotoxic T cells, Dendritic cells, MacrophagesTh1 cells are mainly involved in controlling intracellular pathogens. They induce cell-mediated immunity through the secretion of cytokines, which increases macrophage recruitment and phagocytosis. Th1 cells also stimulate cytotoxic T cell responses. Autoimmune diseases such as rheumatoid arthritis, multiple sclerosis and diabetes are classified as Th1-dominant diseases.
Th2CRTH2+CD183, CD193, CD194, CD198, CD294GATA-3, GFI-1, IRF4, MAF, STAT5, STAT6IL-2, IL-4, IL-25, IL-31, IL-33, TSLPIFN-γ, TGF-βIL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-13, IL-25, IL-31B cells, Basophils, EosinophilsTh2 cells are mainly involved in the humoral response. They are required for IgE antibody production, as well as some subclasses of IgG. They contribute to host defense against parasites and in allergic responses.
Th9Not DeterminedCD196GATA-3, IRF4, PU.1, STAT6IL-4, TGF-βIFN-γ, IL-27CCL17, CCL22, IL-9, TGF-βBasophils, Eosinophils, Mast CellsTh9 cells are important for host defense against parasitic infection. They have also been shown to contribute to the development of autoimmune disease.
Th17CCR6+CD161+CD45RO, CD126, CD161, CD194, CD196IRF4, RORC2, Runx1, STAT3IL-1, IL-6, IL-21, IL-23, TGF-βIFN-γ, IL-2, IL-12, IL-27IL-17A, IL-17F, IL-21, IL-22, IL-23, IL-24, IL-26, TNF-αNeutrophilsTh17 cells play a role in host defense against bacteria and fungi. They are also involved in the induction of autoimmune tissue injury, which is linked to insufficient regulation by T regulatory (Treg) cells.
Th22Not DeterminedCD140a, CD140b, CD194, CD196AhRIL-6, IL-23, TNF-αTGF-βIL-13, IL-22, TNF-αEpithelial cells, Stromal cellsTh22 cells may be involved in host defense against microbial pathogens, as well as to promote tissue repair and remodeling.
TFHCXCR5+ICOS+CD185, CD279Bcl-6, MAF, STAT3CXCL13, IL-6, IL-21, IL-27Not DeterminedIFN-γ, IL-4, IL-6, IL-10, IL-17A, IL-17F, IL-21B cellsTFH cells stimulate B cells to mature into antibody-secreting plasma cells. These cells are primarily located in the periphery within B cell follicles of secondary lymphoid organs.
TregCD25+CD127-CD25, CD39, CD73, CD103FOXP3, Helios, Smad3, STAT5Not DeterminedNot DeterminedIL-10, IL-35, TGF-βAll other T cell subsetsTregs develop in the thymus. They are primarily involved in the regulation of immune responses and maintenance of immune tolerance to prevent autoimmunity.

Note

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



CD4 Antibodies


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.

Table 3. Overview of monoclonal anti-human CD4 antibodies.

Immunogen
Class
Species Reactivity
Host, Isotype
Clone
Applications¹
Recognizes CD4MonoclonalHumanMouse, IgG1, kappaRPA-T4FC, IHC-F, IF, ELISA, WB
Recognizes CD4MonoclonalHumanMouse, IgG1, kappaSK3FC, IHC-F, IF, ELISA, WB
Recognizes CD4MonoclonalHumanMouse, IgG2b, kappaOKT-4FC, IHC-F, IF, ELISA, WB
Recognizes CD4MonoclonalHumanMouse, IgG2b, kappaHIT4aFC, IHC-F, IF, ELISA, WB

Applications

  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..


Table 4. Available iFluor™ dye-labeled CD4 antibody conjugates.

Label
Ex (nm)
Em (nm)
ε¹
Φ²
CF 260 nm³
CF 280 nm?
iFluor™ 35034444820,0000.950.830.23
iFluor™ 40540242537,0000.910.480.77
iFluor™ 43043349540,0000.780.680.3
iFluor™ 45045150240,0000.820.450.27
iFluor™ 48849151675,0000.90.210.11
iFluor™ 51452755480,0000.830.250.11
iFluor™ 53254356390,0000.680.260.16
iFluor™ 546541557100,0000.670.250.15
iFluor™ 555556569100,0000.640.230.14
iFluor™ 560559571130,0000.570.0480.069

Note

  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.


Table 5. Available mFluor™ dye-labeled CD14 antibody conjugates.

Label
Ex (nm)
Em (nm)
ε¹
Φ²
CF 260 nm³
CF 280 nm?
mFluor™ UV37535438835,0000.940.0990.138
mFluor™ Violet 45040644525,0000.920.3380.078
mFluor™ UV46036245615,0000.860.350.134
mFluor™ Violet 50042649735,0000.810.7690.365
mFluor™ Violet 51040950430,0000.860.4640.366
mFluor™ Violet 54040053215,0000.641.3920.529
mFluor™ Blue 570552564120,0000.080.2280.179
mFluor™ Green 62052161750,0000.060.8950.569
mFluor™ Yellow 630546625110,0000.010.2830.413
mFluor™ Red 700657694250,0000.0290.1350.127

Note

  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.



Table 6. Available Alexa Fluor® dye-labeled CD4 antibody conjugates.

Label
Ex (nm)
Em (nm)
ε¹
Φ²
CF 280 nm³
Alexa Fluor® 35034344119,000N/D0.19
Alexa Fluor® 48849952073,0000.920.11
Alexa Fluor® 555553568155,0000.10.08
Alexa Fluor® 59459061892,0000.660.56
Alexa Fluor® 647650671270,0000.330.03
Alexa Fluor® 680681704183,0000.360.05
Alexa Fluor® 700696719205,0000.250.07
Alexa Fluor® 750752776290,0000.120.04

Note

  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.


Table 7. Available FITC, TRITC and other classic dye-labeled CD4 antibody conjugates.

Label
Ex (nm)
Em (nm)
ε¹
Φ²
CF 280 nm³
Pacific Blue™40445546,0000.780.2
FITC49151673,0000.790.254
Pacific Orange™40055125,000N/D0.6
TRITC54457085,0000.10.34

Note

  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 phcoerythrin (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.


Table 8. Available PE, APC, PerCP and tandem dye-labeled CD4 antibody conjugates.

Label
Ex (nm)
Em (nm)
ε¹
Laser Line (nm)
PE5665741,960,000488 nm, 532 nm, 561-568 nm
PE-Texas Red®5676151,960,000488 nm, 532 nm, 561-568 nm
PE-Alexa Fluor® 6105676271,960,000488 nm, 532 nm, 561-568 nm
PE-Cy5®5656661,960,000488 nm, 532 nm, 561-568 nm
PE-iFluor™ 6475696661,960,000488 nm, 532 nm, 561-568 nm
PE-Alexa Fluor® 7005667211,960,000488 nm, 532 nm, 561-568 nm
PE-Cy7®5667781,960,000488 nm, 532 nm, 561-568 nm
PE-iFluor™ 7505667781,960,000488 nm, 532 nm, 561-568 nm
APC651660700,000633-647 nm
APC-iFluor™ 700651710700,000633-647 nm

Note

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