This document provides information on cluster of differentiation (CD) markers, which are cell surface molecules used to identify and study immune cells. It discusses the history of CD markers and the CD nomenclature system. It also describes different types of CD markers (types I-V), common CD markers like CD3, CD4, CD8, CD16, CD25, CD34, CD36, CD45, CD109, CD114, and CD117, and their roles in immune cell identification, function, and disease.

1. CD
▪Dr. G.D.A. Samaranayaka

4. History
Definition
Nomenclature
Importance
Brief description on some CDs

5. 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.

7. 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.

8. ▪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

9. ▪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.

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

11. ▪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.

12. ▪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.

17. 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

18. 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.

19. ▪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

20. ▪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).

21. ▪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+CD45RAlowCD45ROhigh

22. 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.

23. General structure of
membrane antigens

24. 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

25. 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

26. 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.

27. 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.

28. 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.

29. 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

30. 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

31. 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

32. CD4
▪Surface glycoprotein seen in – T helper cells (Th
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.

33. 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.

34. 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

35. 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)

36. 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.

37. 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.

38. ▪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

39. 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)

40. 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.

41. ▪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.

42. 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 (Govb) and 15-b (Gova) associated
with CD109.
▪HPA 15 antigens – liable in storage – detection using serological
methods is difficult

43. 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.

44. 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.

46. References
1. Cluster of Differentiation (CD); Ma Hongbao, Margaret Young, Yang Yan; New York
Science Journal 2015;8(7)
2. 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;
3. Cell surface antigen CD109 is a novel member of the α2 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
4. Guide to human CD antigens; Abcam
5. William’s Haematology – 8th edition
6. Rossis’s Transfusion Medicine – 5th edition
7. Wikipedia

47. Thank You