Cluster of differentiation

Importance of cluster of differentiation and the relevance in blood transfusion

1. CD Dr. G.D.A. Samaranayaka

2. CD 34+ CD 8+ CD 4+

3. History Definition Nomenclature Importance Brief description on some CDs

4. Monoclonal Antibodies Monoclonal antibodies ( mAb or moAb ) antibodies made by identical immune cells - clones of a unique parent cell. monovalent affinity - bind to the same epitope. Polyclonal antibodies bind to multiple epitopes usually made by several different plasma cell lineages.

5. History First monoclonal antibody produced in 1975 With the advent of hybridoma technology to produce mAbs – generate very large numbers of mAbs directed against leukocyte cell surface molecules generally using whole cells as immunogen. Initially, surface antigens were named after the monoclonal antibodies that bound to them.

6. The problem was that several mAbs produced by different laboratories (under different names) were actually directed against the same molecule (same antigen different epitopes). Lab A Lab B Lab C

7. This resulted in the chaotic naming of molecules Adaptation of a consistent nomenclature was necessary. The cluster of differentiation (CD) nomenclature system was conceived to classify antigens found on the surface of leukocytes.

8. Cluster of Differentiation The cluster of differentiation (CD) is a group of cell surface molecules providing targets for immunophenotyping of cells.

9. Physiologically, CD antigens do not belong in any particular class of molecules Immunoglobulin (Ig) superfamily Scavenger receptor cysteine-rich (SRCR) superfamily TNF superfamily Regulators of complement/CÆ activation (RCA) family Integral membrane proteins CD act as receptors or ligands to play the functions such as cell signaling and cell adhesion etc. CD system normally plays a role as a cell marker in immune purpose to recognize the molecules in the cells’ surface.

10. Although initially used for just human leukocytes, the CD molecule naming convention has now been expanded to cover both other species (e.g. mouse) as well as other cell types. At present, CD markers range from CD1 to CD371 Some CDs covering a group of closely related proteins or carbohydrates (e.g.,CD1a,CD1b,CD1c,andCD1d). The total number of assigned CDs is 401.

11. HDLA Workshops HLDA (Human Leukocyte Differentiation Antigens) Workshops were created to establish the nomenclature of leukocyte cell surface molecules by using mAbs from different laboratories. The current nomenclature system was adopted in 1982, during the 1st International HLDA Workshop in Paris. The HLDA Workshops have since provided a forum for the characterization and study of leucocyte surface molecules and antibodies against them - compare the ability of monoclonal antibodies to react with human cells and/or human cell proteins. HLDA devised the CD nomenclature, which is sanctioned by the IUIS (International Union of Immunological Societies)/WHO Nomenclature Committee. Latest work shop held in 2014 – HDLA10

12. Current Nomenclature system Monoclonal antibodies that have similar patterns of reactivity with various tissues or cell types are assigned to a cluster group. An antigen well recognized by a cluster or a group of of antibodies can be assigned a cluster of differentiation number, or CD number (e.g. CD1, CD2 etc ) The CD nomenclature is also used to name antigen and the antibodies. Ex:-CD4 designates both the group of mAbs recognizing the CD4 cell surface molecule, as well as the CD4 molecule itself.

13. A lowercase “w” preceding the number designation stands for “workshop” e.g. CDw12 Indicates CD designation is tentative. Denotes an insufficiently characterized Ab or molecule. In some cases, corresponds to a molecule defined by only one Ab submitted to the HLDA Workshops. Most of the provisional CDw -designated Ags of the early workshops turned out to correspond to clusters of mAbs recognizing carbohydrate epitopes, which after proper biochemical identification received their own CD number e.g., CD176 = Thomsen-Friedenreich, carbohydrate Ag

14. Uppercase letters following a CD number designate a spliced variant of the extracellular domain of a cell surface molecule. For example, CD45RA or CD45RO corresponds to splice variants of CD45. A lowercase letter following the CD number - share a common chain e.g., CD1a, CD1b, CD1c, CD1d, or CD1e - β2-microglobulin. Other examples are the integrin chains CD11a, CD11b, and CD11c, - share CD18 as a common chain to form different dimers. In other cases, lowercase letters have been used to name different members of the same gene family , as is the case with CD66 (CD66a, CD66b, CD66c, CD66d, CD66e, and CD66f).

15. The CD nomenclature is also frequently used to describe lymphocyte and leukocyte subsets. The presence or absence of a specific antigen from the surface of particular cell or cell population is denoted with “+” or “-“ respectively. (Ex CD4+, CD34+, CD62-) If a particular CD molecule is expressed at different levels by a cell subset, the superscript “high” or “low” can be added central memory T cells are CD62L high whereas effector memory T cells are CD62L low. CD4 + CD45RA low CD45RO high

16. Importance of CD By monitoring the expression profiles of different CD antigens Identification Isolation Phenotyping of cell types according to their function in various immune processes. The antibodies - useful as markers for cell populations Counting Separation functional study of numerous subsets of cells of the immune system.

17. General structure of membrane antigens

18. Type I Transmembrane proteins COOH-termini in the cytoplasm and their NH2-termini outside the cell. Generally has a signal sequence at the NH2-terminus - cleaved off after the molecule passes into the endoplasmic reticulum. These proteins commonly serve as cell surface receptors and/or ligands. Many belong to the immunoglobulin superfamily. Ex – CD3

19. Type II Transmembrane proteins Opposite orientation to that of type I transmembrane proteins. The NH2-terminus is located inside the cell, and the COOH-terminus is extracellular. Ex – CD70

20. Type III Transmembrane proteins Cross the plasma membrane more than once. Some pass through the bilayer as many as 12 times. Because they cross the membrane multiple times - form channels that often are used to transport ions or small molecules through the lipid bilayer. Ex - CD20 - form a calcium channel for B lymphocytes that is required for B-cell activation.

21. Type IV Transmembrane proteins Type IV proteins are also transmembrane channels but is formed by bringing together a number of independent helical segments rather than connected as a single polypeptide None of the current CD antigens have type IV membrane organization.

22. Type V Transmembrane proteins These proteins use lipid to attach themselves to the plasma membrane. The most common attachment for extracellular proteins - glycosyl-phosphatidylinositol (GPI) anchor.

23. Type of cell CD markers stem cells CD34+, CD31-, CD117 all leukocyte groups CD45+ Granulocyte CD45+, CD11b, CD15+, CD24+, CD114+, CD182+ Monocyte CD4, CD45+, CD14+, CD114+, CD11a, CD11b, CD91+, CD16+ T lymphocyte CD45+, CD3+ T helper cell CD45+, CD3+, CD4+ T regulatory cell CD4, CD25, FOXP3 Cytotoxic T cell CD45+, CD3+, CD8+ B lymphocyte CD45+, CD19+, CD20+, CD24+, CD38, CD22 Thrombocyte CD45+, CD61+ Natural killer cell CD16+, CD56+, CD3-, CD31, CD30, CD38

24. CD3 Belongs to immunoglobulin superfamily. Found on T helper and cytotoxic cells 3 chains - CD3γ, CD3δ and CD3ε These chains associate with a T-cell receptor (TCR) and the ζ-chain to form TCR – complex TCR complex – bind with MHC molecules -> Generation of Activation signal

25. CD3 Useful immunohistochemical marker for T-cells in tissue sections. highly specific presence of CD3 at all stages of T-cell development The antigen remains present in almost all T-cell lymphomas and leukaemias used to distinguish them from superficially similar B-cell and myeloid neoplasms. Human Tonsil stained with anti-CD3 antibody

26. CD4 Surface glycoprotein seen in – T helper cells (T h cells) Monocytes, macrophages and dendritic cells. CD4 amplifies the signal generated by the TCR -> assists the TCR in communicating with an antigen-presenting cell. CD4 also interacts directly with MHC class II molecules on APCs -> antigen recognition.

27. CD4 HIV-1 uses CD4 to entry into host T cells through viral envelope protein gp120. HIV infection leads to a progressive reduction in the number of T cells expressing CD4. CD4 used as a cell marker - CD4+ cell count is used as a prognostic indicator and measure the efficacy of the treatment. CD4 continues to be expressed in most neoplasms derived from T helper cells. CD4 immunohistochemistry - identify most forms of peripheral T cell lymphoma and related malignant conditions.

28. CD8 Transmembrane glycoprotein -co-receptor for the TCR Binds with MHC class I molecules. Predominantly expressed on the surface of cytotoxic T cells also be found on NK cells, cortical thymocytes, and dendritic cells. Plays a main role in antigen recognition

29. CD14 Part of innate immune system CD14 is expressed mainly by macrophages. Also in neutrophils & dendritic cells. Exists in 2 forms membrane glycoprotein (mCD14) - expressed in macrophages and dendritic cells soluble form (sCD14) – liver, breast milk – regulate microbial growth in infant gut CD14 acts as a co-receptor for the detection of bacterial lipopolysaccharide (LPS)

30. CD16 Molecule of the Ig superfamily It is a low affinity Fc receptor FcγRIIIa (CD16a) and FcγRIIIb (CD16b) Found on NK cells, neutrophils, PMNs, monocytes and macrophages. Bind to the Fc portion of IgG antibodies -> activates the NK cell for antibody dependent cell-mediated cytotoxicity (ADCC)

31. CD25 Alpha chain of the IL-2 receptor. Present on activated T cells, activated B cells, some thymocytes & myeloid precursor. Expressed in most B-cell neoplasms, some acute nonlymphocytic leukemias, neuroblastomas, and tumor infiltrating lymphocytes. Used as a marker for hairy cell leukemia and diagnosis of systemic mastocytosis .

32. CD34 AKA - Hematopoietic progenitor cell antigen The CD34 (CD34+ cell) are normally expressed in hematopoietic cells of the umbilical cord and bone marrow, mesenchymal stem cells, endothelial progenitor cells, endothelial cells of blood vessels & mast cells. Cell surface glycoprotein - cell-cell adhesion factor. Mediates the attachment of stem cells to bone marrow extracellular matrix CD34 - adhesion molecule - required for T cells to enter lymph nodes. It is expressed on lymph node endothelia.

33. CD34+ cells can be isolated from blood samples using immunomagnetic or immunofluorescent methods. Antibodies can be used - to quantify and purify hematopoietic progenitor stem cells Injection of CD34+ hematopoietic stem cells - treat various diseases spinal cord injury, liver cirrhosis, peripheral vascular disease, etc

34. CD36 CD36 is a broadly-expressed integral membrane glycoprotein with multiple physiological functions. Found on platelets, erythrocytes, monocytes, differentiated adipocytes, skeletal muscle, epithelial cells, spleen cells AKA - Platelet glycoprotein 4, fatty acid translocase (FAT), scavenger receptor class B member 3 (SCARB3), and glycoproteins 88 (GP88)

35. CD45 Protein tyrosine phosphatase- receptor type C ( PTPRC) Regulate a variety of cellular processes - cell growth, differentiation, mitotic cycle, and oncogenic transformation. Multiple isoforms - CD45RA, CD45RB, CD45RC, CD45RAB, CD45RAC, CD45RBC, CD45RO, CD45R – Present on all differentiated hematopoietic cells, except erythrocytes and plasma cells This gene is specifically expressed in hematopoietic cells. CD45 is a pan-leukocyte protein - routinely used in scientific research to allow identification of cells.

36. Binds many ligands - collagen, thrombospondin, erythrocytes parasitized with Plasmodium falciparum Mutations in the human CD36 gene – lack of platelet glycoprotein IV (GPIV) -> Nak antibody -> Platelet refractoriness CD36 has also been implicated in hemostasis, thrombosis, malaria, inflammation, lipid metabolism and atherogenesis.

37. CD56 AKA - Neural cell adhesion molecule (NCAM). Glycoprotein of immunoglobulin superfamily. expressed on - neurons, glia, skeletal muscle and natural killer cells. Role in cell–cell adhesion, neurite outgrowth, synaptic plasticity, and learning and memory.

38. CD109 Glycosylphosphatidylinositol (GPI)–linked glycoprotein. Macroglobulin/complement family. Found on - subset of hematopoietic stem cells, activated platelets and T cells. Human platelet antigen HPA 15-a ( Gov b ) and 15-b ( Gov a ) associated with CD109. HPA 15 antigens – liable in storage – detection using serological methods is difficult

39. CD114 Granulocyte colony-stimulating factor receptor (G-CSF-R) Cytokine receptors - haematopoietin receptor family. G-CSF-R present on precursor cells in the bone marrow Stimulation by GCSF - initiates cell proliferation and differentiation into mature neutrophilic granulocytes and macrophages.

40. CD117 Stem cell growth factor receptor (SCFR) - AKA proto-oncogene c-Kit encoded by KIT gene. High levels of CD117 - HSCs, multipotent progenitors (MPP), and common myeloid progenitors (CMP). Binds with stem cell factor (c-kit ligand) – stem-cell survival, proliferation, differentiation and mobilization . PBSC collection - G-CSF indirectly activates CD117. Direct CD117 agonists are currently being developed as mobilization agents. Activating mutations gene are associated with gastrointestinal stromal tumors, testicular seminoma, melanoma, acute myeloid leukemia.

41. References Cluster of Differentiation (CD); Ma Hongbao , Margaret Young, Yang Yan; New York Science Journal 2015;8(7) CD Nomenclature 2015: Human Leukocyte Differentiation Antigen Workshops as a Driving Force in Immunology; Pablo Engel, Laurence Boumsell , Valter Gattei , Vaclav Horejsi , Robert Balderas, Bo-Quan Jin , Fabio Malavasi , Frank Mortari , Menno C. van Zelm,Reinhard Schwartz- Albiez , Heddy Zola, Armand Bensussan , Hannes Stockinger , and Georgina Clark; Journal of Immunology 2015; 195:4555-4563; Cell surface antigen CD109 is a novel member of the α 2 macroglobulin/C3, C4, C5 family of thioester-containing proteins; Martin Lin, D. Robert Sutherland, Wendy Horsfall , Nicholas Totty, Erik Yeo, Rakash Nayar , Xiang-Fu Wu and Andre C. Schuh; Blood 2002 99:1683-1691 Guide to human CD antigens; Abcam William’s Haematology – 8 th edition Rossis’s Transfusion Medicine – 5 th edition Wikipedia

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