CD3 (Leu4, T3)

CD3 (cluster of differentiation 3) is a protein complex commonly expressed on the surface of all mature T cells. It functions primarily as a T cell co-receptor promoting the activation of cytotoxic T cells (CD8⁺ T cells) and T helper cells (CD4⁺ T cells), and ultimately helping to shape the adaptive immune response. As an integral component of T cell activation, CD3 has emerged as a promising target for the development of drugs, namely therapeutic monoclonal antibodies (mAbs), as a form of immunotherapy to treat autoimmune disorders and transplant rejection. A prime example being otelixizumab, which was developed for the treatment of type 1 diabetes and autoimmune disorder.

In flow cytometric analysis, human CD3 is routinely used as a lineage-specific marker differentiating T cells from other lymphocytes. The specificity of CD3 for T cells combined with its appearance at all stages of T cell development makes it an ideal marker for the detection of healthy T cells and T cell carcinomas (e.g. leukemia, lymphoma). In immunohistochemical studies, CD3 is a suitable immunohistochemical marker for T cells in tissue sections.

CD3 Protein Complex

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As a consequence of its short cytoplasmic tail, T cell receptors (TCR) lack the ability to signal upon antigen recognition of peptide major histocompatibility complexes (pMHCs) class I or II molecules. In order to activate the necessary downstream pathways initiating immune cell activation, TCR requires intracellular signaling to be initiated by a complex of signaling molecules known as CD3 proteins.

CD3 Structure

The CD3 protein complex is comprised of four invariant chains - CD3ε, CD3δ, CD3γ and CD3ζ - which are organized into an N-terminal extracellular region and a transmembrane domain, and are identical in all T cells. The N-terminal extracellular region consists of two heterodimer domains, CD3εγ and CD3εδ, and the transmembrane domain consists of a single homodimer CDζζ. Situated at the cytoplasmic tail of each domain are intracellular immunoreceptor tyrosine activation motifs (ITAMs), which are integral components for signal transduction in immune cells. Following TCR recognition of antigen presenting pMHCs, tyrosine residues within these CD3 chain ITAMs are phosphorylated by lck tyrosine kinases associated with the cytoplasmic tails of CD4 proteins. These phosphorylated ITAMs serve as docking sites for SH2 domain containing signaling molecules, such as ZAP-70. Once bound, ZAP-70 phosphorylates the transmembrane adapter protein linker of activated T cells (LAT), which facilitates the binding of various signaling proteins (e.g. SLP-76, phospholipase C, protein kinase C, PI3K), ultimately promoting the transcription of gene products for T cells differentiation, proliferation and cytokine secretion.

Once fully assembled, CD3-TCR complexes contain a total of ten ITAMs: one for each CD3ε, CD3δ and CD3γ and three for each CD3ζ. While the significance of all ten motifs isn't clearly outlined, it is thought to facilitate scalable signaling to induce the appropriate immune response from any magnitude of responses resulting from TCR activity.

CD3 and Its Role In Disease

Mutations in the genes encoding CD3 subunits can lead to severe combined immunodeficiency (SCID), a rare autosomal recessive disorder characterized by severe defects in T- and B-lymphocyte differentiation and function. Deficiencies in CD3 subunits have been associated with the following:

• CD3δ and CD3ε deficiency: associated with severe αβ T cell lymphopenia, results in a T^-B⁺NK⁺ SCID phenotype.
• CD3ε deficiency: leads to a complete block of T cell development.
• CD3γ deficiency: causes partial T cell immunodeficiency.
• CD3ζ deficiency: associated with immunodeficiency 25 and systemic lupus erythematosus (SLE).

CD3 Antibodies

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Monoclonal CD3 antibodies, designed to recognize the ε-chain of the CD3/T-cell antigen receptor complex, are frequently used in flow cytometry and immunofluorescence applications to identify immune cell subsets expressing CD3. Cells known to express CD3 surface markers include ~60-85% of normal peripheral blood lymphocytes, ~65-85% of thymocytes and in Purkinje cells of the cerebellum and gastric parietal cells.

AAT Bioquest offers a comprehensive catalog of CD3 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; FN = Functional Assay; IF = Immunofluorescence; IHC = Immunohistochemistry; IHC-F = Immunohistochemistry (Frozen); ICC = Immunocytochemistry; WB = Western Blot; IP = Immunoprecipitation.

iFluor® Dyes Labeled to CD3 Antibodies

The following table outlines the fluorescence properties of available iFluor® dye labeled anti-human CD3 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 CD3 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 CD3 Antibodies

The following table outlines the fluorescence properties of available mFluor™ dye labeled anti-human CD3 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 CD3 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 CD3 Antibodies

The following table outlines the fluorescence properties of available Alexa Fluor^® dye labeled anti-human CD3 antibodies for use in flow cytometry (FACS) and fluorescence imaging applications. For additional information on Alexa Fluor^® dye-labeled CD3 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 CD3 Antibodies

The following table outlines the fluorescence properties of available classic dye labeled anti-human CD3 antibodies for use in flow cytometry (FACS) and fluorescence imaging applications. For additional information on classic dye-labeled CD3 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 CD3 Antibodies

The following table outlines the fluorescence properties of available phycoerythrin (PE), allophycocyanin (APC), PerCP and tandem dye labeled anti-human CD3 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 CD3 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. Alroy J, Ucci AA, Azabdoaftari G, Banner BF, Cheville JC. Expression of CD3 antigens in renal tubule epithelium and renal oncocytomas. Pathol Res Pract. 2005;201:803-808. doi: 10.1016/j.prp.2005.09.003.
2. Cantrell D. Signaling in lymphocyte activation. Cold Spring Harb Perspect Biol. 2015;7(6):a018788. Published 2015 Jun 1. doi:10.1101/cshperspect.a018788
3. Koretzky GA. The Role of SH2 Domain-containing Leukocyte Phosphoprotein of 76 kDa in the Regulation of Immune Cell Development and Function. Immune Netw. 2009;9(3):75?83. doi:10.4110/in.2009.9.3.75
4. Malissen, B. CD3 ITAMs count!. Nat Immunol 9, 583-584 (2008). https://doi.org/10.1038/ni0608-583
5. Natarajan A, Nadarajah V, Felsovalyi K, et al. Structural Model of the Extracellular Assembly of the TCR-CD3 Complex. Cell Rep. 2016;14(12):2833?2845. doi:10.1016/j.celrep.2016.02.081
6. Smith-Garvin JE, Koretzky GA, Jordan MS. T cell activation. Annu Rev Immunol. 2009;27:591?619. doi:10.1146/annurev.immunol.021908.132706
7. Chen S, Zha X, Yang L, Li B, Liye Z, Li Y (2011) Deficiency of CD3gamma, delta, epsilon, and zeta expression in T cells from AML patients, Hematology, 16:1, 31-36, DOI: 10.1179/102453311X12902908411832