1. Leukocyte Differentiation Antigens Cell Adhesion Molecules NK cells
Linrong Lu (鲁林荣)
Institute of Immunology Zhejiang University School of Medicine Medical Research Building A801
Tel:

2. Contents Concept of leukocyte differentiation antigens （白细胞分化抗原）
Concept of cluster of differentiation
（CD分子，分化群）
Cell adhesion molecules
NK cells

3. Leukocyte differentiation antigens
Leukocyte differentiation antigens: Cell surface molecules which may appear or disappear from the cell membrane of leukocytes in the different stages of differentiation and activation.
（第6版p58）
Leukocyte differentiation antigens are also found on cells other than leukocytes, such as erythrocytes, endothelial cells, epithelial cells.

4. Classification of LDA Immunoglobulin superfamily
Cytokine receptor family
C-Type lectin superfamily
Integrin family
TNF superfamily
TNF receptor superfamily

5. Cluster of differentiation (CD): Cell surface molecules can be recognized by particular monoclonal antibodies. All of the monoclonal antibodies that react with a particular membrane molecule are grouped together as a cluster of differentiation (CD).
Each new mAb that recognizes a cell surface molecule is analyzed for whether it falls into a recognized CD designation; if not, it is given a new CD designation that reflects a new membrane molecule.
CD1~CD363, up to 363 most recently (as of 2010) in human

6. Human CD Groups CD（example）
T cell CD2、CD3、CD4、CD5、CD8、CD28、CD152(CTLA4)、
CD154(CD40L)、CD272(BTLA)、CD278(ICOS)、CD294(CRTH2)
B cell CD19、CD20、CD21、 CD40、CD79a(Ig)、CD79b (Ig)、
CD80 (B7-1)、CD86 (B7-2) 、CD267(TACI)、CD268(BAFFR)、
CD269(BCMA)、CD307(IRTA2)
Myeloid cell CD14、CD35(CR1)、CD64 (FcRI)、CD256(APRIL)、
CD257(BAFF)、CD312(EMR2)
Platelet CD36、CD41、CD42aCD42d、CD51、
CD61 、CD62P
NK cell CD16 (FcRIII) 、CD56(NCAM-1)、CD94、CD158(KIR)、
CD161(NKR-P1A)、CD314(NKG2D)、CD335(NKp46)、
CD336(NKp44)、CD337(NKp30)
Non-lineage CD30、CD32(FcRII)、CD45RA、CD45RO、CD46(MCP)、
CD55(DAF)、CD59、CD252(OX40L)、 CD279(PD1)、
CD281CD284(TLR1-TLR4)、CD289(TLR9)、 CD305(LAIR-1)、
CD306(LAIR-2)、CD319(CRACC)

7. Human CD Groups CD（example）
Adhesion molecules CD11a-CD11c、CD15、CD15s(sLx)、CD18 、
CD29 、CD49aCD49f、CD54(ICAM-1)、
CD62E、CD62L 、CD324(E-cadherin)、
CD325(N-cadherin)、CD326(EpCAM)
Cytokine/ CD25(IL-2R)、CD95(Fas)、CD116-CDw137、CD178(FasL)、
Chemokine receptors CD183(CXCR3)、CD184(CXCR4)、CD195(CCR5)、
CD261CD264(TRAIL-R1TRAIL-R4)
Endothelia CD105(TGF-RIII)、CD106(VCAM-1)、CD140(PDGFR)、
CD144、CD299(DCSIGN-related)、
CD309(VEGFR2)、JAM1(CD321)、JAM2(CD322)
Carbohydrate structures CD15u、CD60aCD60c、CD75、CDw327CDw329
Dendritic cells CD80,CD86,CD85
Stem cells CD133、CD243
Erythrocytes CD233CD242
Stromal cells CD （FGFR1-FGFR4）

8. The CD system is commonly used as cell markers
cytotoxic

9. Cell types and subtypes defined by CDs
Type of cell
CD markers
stem cells
CD34+,CD31-
all leukocyte groups
CD45+
Granulocyte
CD45+,CD15+
Monocyte
CD45+,CD14+
T lymphocyte
CD45+,CD3+
T helper cell
CD45+,CD3+,CD4+
Cytotoxic T cell
CD45+,CD3+,CD8+
B lymphocyte
CD45+,CD19+ or CD45+,CD20+
Thrombocyte
CD45+,CD61+
Natural killer cell
CD16+,CD56+,CD3-
B cell
T cell
CD4+ T cell
Tregs
(CD4+CD25+)

10. Rules for assigning a CD number
at least one Workshop—characterized antibody and good molecular data.
the antigen is expressed on the surface of cells involved in immune reactions.
the antibodies react with primary cells (not just transfectant or recombinant protein).
the antibody is available.
CD acts as Receptors or Ligands. Initiates a signal cascade, altering the behavior of the cell, or has other functions, such as cell adhesion.

11. Functions of CDs
Mediate cell-cell interaction: Homing, activation, effecter functions

12. Functions of CDs
2. Receptors recognize exogenous or soluble substance: pathogens, antibodies, cytokines

13. CDs related to T cell activation

14. TCR signaling

15. CD4 and CD8 •transmembrane glycoprotein •co-receptor of T cell
*CD4: first and second domains bind to nonpolymorphic region of MHC Ⅱmolecules
* CD8: IgV-like domain of  chain binds to nonpolymorphic 3 region of MHCⅠmolecules
APC
•function
*for cell adhesion
*Involved in TCR-CD3 signal transduction

16. Structure of CD4

17. Structure of CD8

18. CCR5
1.CD4 is a primary receptor used by HIV-1 to gain entry into host T cells.
2.HIV-1 attaches to CD4 with a protein in its viral envelope known as gp120. The binding to CD4 creates a shift in the conformation of gp120 allowing HIV-1 to bind to other surface receptors on the host cell, the chemokine receptors CCR5 .Following a structural change in another viral protein (gp41), HIV inserts a into the host cell that allows the outer membrane of the virus to fuse with the cell membrane.
3.HIV infection leads to a progressive reduction in the number of T cells possessing CD4 receptors. Therefore, medical professionals refer to the CD4 count to decide when to begin treatment for HIV-infected patients. Normal blood values are x106/L.

19. 又称CDl52，为同源二聚体，表达于活化T细胞，而静止T细胞则不表达。CTLA-4与CD28分子有一定的同源性，也能与CD80和CD86结合。 CTLA-4分子胞浆内含免疫受体酪氨酸抑制基序（immunoreceptor tyrosine-based inhibitor motif，ITIM ) CTLA-4分子胞浆区可与磷酸酶SHP-1及SHIP结合，使CD3分子内的ITAM中已磷酸化的酪氨酸去磷酸化，对T细胞活化产生抑制作用，对T细胞活化起负调节作用

20. CD40 and CD40L (CD154)
CD40L主要表达在活化CD4+T细胞、部分CD8+T细胞和 T细胞,CD40L结合到B细胞表面CD40产生的信号，是B细胞进行免疫应答和淋巴结生发中心形成的重要条件。

21. CDs related to B cell activation
BCR complex
co-receptor
CDs related to B cell activation

22. CD79 /CD79  Heterodimer cytoplasmic domains contain ITAM. Function:
1. transfers the signals that lead to B cell activation.
2. transports the Ig

23. BCR signaling

24. CD19/CD21/CD81
CD19/CD21/CD81 interactions with complement associated with antigen play a role in antigen-induced B-cell activation.
CD21=CR2, C3dR, EBV R

25. CDs constituting FcR family

26. Different types of human Fc receptors

27. Role of FcRI (CD64) in the Ab-mediated opsonization of microbes

28. Role of FcRIII (CD16) and FcRI in ADCC(antibody-dependent cell-mediated cytotoxicity)

29. Cell adhesion molecules, CAM
A group of proteins involved in adhesion between cells or between cells and extracellular matrix (ECM) 29

30. CAMs are grouped depending on their adhesion functions.
CAMs and CD molecules are respectively designated according to the different aspects of these molecules.
CAMs are grouped depending on their adhesion functions.
CD molecules contain the majority of CAMs that have obtained CD designation. A minority of CAMs have not been given CD designation. 30

31. Categories of CAM 1. IgSF (immunoglobulin superfamily)
2. Integrin family
3. Selectin family
4. Cadherin family
5. Others: CD44 31

32. Integrin
Integrin family is so named because the molecules of this family primarily mediate the cellular adherence to extracellular matrix, enabling cells and extracellular matrix to form the integration. 32

33. Structure:
Heterodimeric proteins consisting of two non-covalently bound polypeptides
( chain 1 out of 18, and  chain 1 out of 8).

34. Components of integrin family
1: VLA (very late activation antigen, 12 members), ligand: VCAM-1(vascular cell adhsion molecule-1)
T cell activation (co-stimulation and adhesion)
2: LFA-1 (CD11a,lymphocyte function associated antigen-1, 4 members);Mac-1(CD11b) ligand: ICAM-1(intercellular adhesion molecule-1)
T cell activation, circulation, inflammation
3: gpIIb ligand:Fg (fibrinogen) Platelet aggregation
Distribution
Broad distribution on the surface of cells.

36. Selectin family
Selectin family includes L-selectin, P-selectin and E-selectin, which play important roles in leukocyte adhesion to endothelium, inflammation and lymphocyte homing.

37. 1. Structure
Transmembrane glycoproteins, with a number of extracellular domains homologous to those seen in complement control proteins (CCP). The extracellular region also contains a domain related to the EGF receptor and a distal C type lectin-like domain.
2. Members of selectin family L-selectin: leukocytes (Cd62L) P-selectin: platelets and megakaryocytes (Cd62P) E-selectin: activated endothelia (Cd62E)
3. Ligand: carbohydrates, such as CD15s

39. Functions of CAM
1. Serving as co-receptors and providing co-stimulatory signals in immune cells recognition and activation
CD4/CD MHC II/I (coreceptor)
CD B7 (costimulation)
2. Adhesion between leukocytes and vascular endothelial cells in inflammation
3. Lymphocyte homing and recirculation

40. The sequence of events in the migration of blood leukocytes to sites of infection. Selectins mediate weak tethering and rolling of blood neutrophils on the endothelium; integrins mediate firm adhesion of neutrophils to the endothelium, and chemokines activate the neutrophils and stimulate their migration through the endothelium to the sites of infection.

41. Role of CAMs in migration of naïve and effector T cells
Role of CAMs in migration of naïve and effector T cells. Naïve T cells home to lymph node as a result of L-selectin binding to its ligand on HEVs(high-endothelial venules), which are present only in lymph nodes. Activated T cells home to sites of infection in peripheral tissues, and this migration is mediated by E-selectin, P-selectin and integrins.

42. Leucocytes adhesion to blood vessel

43. Application of CD, CAM and Their mAb
1. Elucidation of pathogenesis
CD4 and AIDS, gp120 binds to CD4
2. Application in diagnosis
leukemia, AIDS (ratio of CD4+ to CD8+ T cells)
3. Prophylactic and Therapeutic application
Graft rejection: Anti-CD3, Anti-CD25
non-Hodgkin’s lymphoma: Anti-CD20

44. Natural Killer cells

46. NK cells - What are they? 3rd lineage of lymphocytes
Human CD3-,CD56+
Mouse CD3-,NKp46+ (in B6 mice NK1.1+)
3rd lineage of lymphocytes
Function in innate immunity to protect against viruses, bacteria, & tumors
Produce cytokines & kill abnormal cells

47. NK Cells - Where do they come from?
Progenitor in bone marrow
Thymus not required
nude mice have normal NK cells
Do not rearrange a,b,g or d-TcR or Ig
normal NK cells in scid and Rag-/- mice

49. NK Cells - Where do they live?
~5-20% peripheral blood lymphocytes
~5% lymphocytes in spleen
Abundant in liver
Low frequency in thymus, bone marrow, uninfected lymph nodes and lymphatics
>90% of lymphocytes in decidual tissue

50. NK Cells - What do they do?
Cell mediated-cytotoxicity
Antibody-dependent cellular cytotoxicity (ADCC)
Early g-interferon production
Secretion of TNFa, LTa, GM-CSF, IL-5, M-CSF, IL-3, IL-10, IL-13, MIP-1a, MIP-1b, RANTES, etc.

51. NK cells- How do they kill?
Predominantly perforin / granzymes
Kagi et al Nature 369:31, 1994
Secreted or membrane TNFa
Degliantoni et al J Exp Med 162:
Fas ligand
Arase et al J Exp Med 181:1235, 1995
TRAIL
Zamai et al J Exp Med 188:2375, 1998

52. NK cells - Cytokine Activation
**Interferon-a/b
augments cytolytic activity
**IL-15
required for development, induces proliferation, increases cytotoxicity
IL-12 & IL-18
augments INFg production
IL-2
induces proliferation, increases cytotoxicity…..physiological relevance?

53. NK cells - what are they good for?
Protection from viruses & tumors

54. What are NK cells are good for?
Humans lacking NK cells susceptible to Varicella zoster & CMV
Biron et al. N Engl J Med 320:
Mouse NK cells protect against CMV - requires perforin and IFNg
Bukowski et al. J Immunol 131: , J Exp Med 161: ; Welsh et al. J Exp Med 173:1053, 1991; Scalzo et al. J Exp Med 171:1469, 1990; Orange et al. J Exp Med 182:1045, 1995
Mouse NK cells protect against mousepox - requires IFNg
Jacoby et al. Arch. Virol. 108:49, 1989; Chaudhri et al. PNAS 101:9057, 2004; Fan et al. PLoS Pathogen 8:e30, 2008
Mouse NK cells protect against Ebola virus - requires perforin, not IFNg
Warfield et al J Exp 200:169, 2004

56. Immune surveillance for ‘ Missing Self’
NK cells preferentially kill cells that have lost MHC class I
Provides protection against cells escaping T cell recognition （tumor and viral-infected cells tend to reduce the expression of MHC I to avoid the attack from T cells）
Predicts existence of inhibitory receptors for MHC class I that spare normal cells from NK cell attack
Karre et al. Nature 319:675, 1986

57. RBC, neural cells
Normal healthy cells
Virus-infected cells, tumor
Stressed cells
Virus-infected cells, tumor

58. Human Chr
19q13
Killer cell Ig-like Receptors (KIR)
Activating & inhibitory
NK receptors

59. Killer Cell Ig-like Receptors (KIR)
Ig superfamily
7-12 functional genes on human chromosome 19q13
Extensive allelic polymorphism (no rearrangement)
Mono-allelic expression possible
Inhibitory KIR recognize polymorphic HLA-A, -B, and –C
Activating receptors have no intrinsic signaling capacity..associate with DAP12 ITAM-adapter protein
Expressed by subsets of NK cells and memory T cells (usually CD8+ T cells)

60. Human NK Receptors for HLA class I

61. Human KIR Haplotypes Differ in Gene Content and Organization
Uhrberg et al. 1997; Wilson et al. 2000; Vilches et al. 2000; Hsu et al. 2002
Human KIR Haplotypes Differ in
Gene Content and Organization
3DL1
3DL3
2DL3
2DS2
2DL2
2DL5B
2DS3
2DP1
2DL1
3DP1
2DL4
2DL5A
2DS4
2DS5
2DS1
3DL2
3DS1
A haplotype
3DL3
2DL3
2DL1
2DL4
3DL1
2DS4
3DL2
B haplotype
3DL3
2DS2
2DL2
2DL1
2DL4
3DS1
2DL5A
2DS5
2DS1
3DL2
In addition to this variability within an individual, individuals within a population also differ in the KIR genes they contain in their genomes. The KIR gene family is located in a region of human Chromosome 19 and the gene organization of the family is depicted here. The so-called “frame-work” genes (3DL3, 2DL4 and 3DL2, depicted here in orange) are present on all chromosomes in all individuals. The genes between these framework genes, (depicted in blue for genes that encode functional proteins, and pink for the pseudogenes) can vary on different chromosomes. Population studies have defined 2 broad KIR haplotype groups, or the group of KIR genes found together on a single chromosome. The group A haplotypes contain the minimum number of KIR genes possible, and have 2DS4 as their only activating receptor. The group B haplotypes typically contain more activating KIR and are more variable in terms of their gene content. The B haplotype shown here is but 1 example of the many B haplotypes that have been described.
Generated by gene conversion - exon swapping
Difficult to distinguish loci versus alleles
Group A haplotype
3DL3
2DL3
2DL1
2DL4
3DL1
2DS4
3DL2 55 7 15 26 49 11 38
# alleles:
3 x 106 possible combinations in the group A haplotypes
(Courtesy P. Parham)

62. Mice don’t have KIR genes Mice have Ly49 genes that do the same job

63. Humans don’t have functional Ly49 genes

64. Ly49 Receptors C-type lectin-like superfamily Polygenic & polymorphic
Extensive allelic polymorphism (no rearrangement)
Inhibitory Ly49 recognize polymorphic H-2D and H-2K
Activating receptors have no intrinsic signaling capacity..associate with DAP12 ITAM-adapter protein
Expressed by subsets of NK cells and memory T cells (usually CD8+ T cells)

65. Inhibitory and activating Ly49 receptors
Ligand = MHC class I
Ly49D,H
Ly49A,C,G,I...
DAP12 - - - + +
ITIM
Recruit phosphatases -
inhibit responses
ITAM
Recruit Syk & Zap70
kinases - activate

66. NKG2D
C-type lectin-like superfamily
1 gene, non-polymorphic, conserved mice - humans
Homodimer expressed on all NK cells, gd T cells, and CD8+ T cells
R in transmembrane associates with D in DAP10 transmembrane
DAP10
10 kd homodimer
Cytoplasmic YINM recruits Grb2 & p85 PI3-kinase

67. NKG2D ligands in mice and humans
Many genes
Many alleles

68. NKG2D ligands MHC class I-like Bind with nM affinity to NKG2D
don’t require peptide or b2-microglobulin
Bind with nM affinity to NKG2D
Low levels expressed on healthy tissues
Induced on virus-infected cells and tumor cells
Induced by DNA damage
Elevated in autoimmune diseases

69. What is the biological role of the NKG2D ligands?
“Danger signals” to alert the immune system to infection or genomic damage

70. NK cells - Influence on Adaptive Immunity
Early INFg production by NK cells may ‘skew’ CD4+ T cells towards Th1
INFg production by NK cells may cause IgG class switching in B cells
Cross-talk with dendritic cells

71. Thank you !