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.
6.
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.
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.
13.
14.
15.
16.
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.
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
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.
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.
45.
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