1. Dendritic Cell Subsets June 3, 2005 MIMG 261 Stephan Krutzik, Ph.D. skrutzik@mednet.ucla.edu

2. Dendritic cell (den-'dri-tik 'sel) A cell found in the T-cell area of lymphoid tissue with a branched or dendritic morphology that is the most potent stimulator of T-cell responses. Non-lymphoid tissue also contain dendritic cells, but these are not able to stimulate T-cell responses until they are activated and migrate to lymphoid tissue.

3. Myeloid dendritic cells Tissue DC- sites of host pathogen encounter respond to bacterial and viral infections Trigger Ag-specific T cells First identified 1973 as spleen cells with tree-like processes Plasmacytoid dendritic cell circulating DC, lymphoid tissue respond to viral infection and produce large amounts of IFN-  Trigger Ag-specific T cells First appreciated as DC in 1997

4. Human dendritic cell subsets Myeloid markers lymphoid markers function Human dc can be distinguished based on key features…

5. Mouse dendritic cell subsets

6. Iwasaki et al Nat Imm Mouse Human TLR1 TLR2 TLR3 TLR4 TLR5 TLR6 TLR8 TLR1 TLR2 TLR4 TLR5 TLR6 TLR8 TLR7 TLR9 TLR1 TLR2 TLR3 TLR4 +/- TLR6 TLR9 TLR1 TLR2 TLR3 TLR5 TLR6 TLR7 TLR expression in dendritic cell subsets GM-CSF + IL-4

7. LANGERHANS CELL Discovered in 1868 by Paul Langerhans Specialized myeloid dendritic cell in both mouse and human Represent 2-3% of all epidermal cells Located in suprabasal layers of epidermis Member of the dendritic cell family of professional APC Are critical for the generation of T cell responses to haptens, proteins, and alloantigens

8. 1.Antigen capture (maturation) 2.Migration to lymph node 3.T cell activation The life of a myeloid dendritic cell

9. Modes of maturation “Danger Signals” Non-self: TLR ligands (PAMPs) Self: CD40L, TNF- , IL-1, IL-6, IFN- 

10. DC maturation upon activation with TLR ligand Maturation leads to increased expression of co-stimulatory molecules, CD80, CD86 and higher levels of MHCII TLR Mature Dendritic cell immature Dendritic cell TLR2/1 TLR4 media

11. CCR7 is induced upon DC maturation A= + control B= PBS-injected epidermis C= TNF-injected epidermis D= TNF-injected epidermis with depletion of MHCII+ cells before RNA isolation Mature Dendritic cell CCR7 immature Dendritic cell CCR2, CCR4, CCR6, CXCR3 CCR7 ligands CCL19 and CCL21, are expressed by stromal cells in the T cell zone of the lymph nodes

12. CCR7 deficiency affects DC migration Anti-DEC205 (LC marker) staining Lymph node single cell suspension FITC Forster et al Cell 1999

13. Adoptively transfer CFSE- labeled OT-II cells OVA +/- CpG 72hr Proliferation CCR7 deficiency affects T cell activation OVA +/- CpG 72hr Proliferation WTCCR7-/- + CpG ----- CFSE: Carboxy-fluorescein diacetate, succinimidyl ester Ohl et al Immunity 2004

14. Role of dendritic cells in T cell activation IL-12 Antigen presentation molecules Human MHCI MHCII CD1a, b, c, d Mouse MHCI MHCII CD1d

15. Mouse myeloid dendritic cell subsets

16. Mouse myeloid dendritic cell subsets trigger distinct T cell responses Th2Th1 CD8-CD8+ CD8+ and CD8- DC were isolated from spleen, loaded with KLH peptide and injected into footpad of syngeneic mice LN harvested and reactivity toward KLH was measured Maldonado-Lopez et al J Exp Med 1999)

17. Production of IL-12 by DCs IL-12 p70 (pg/ml) UntreatedPansorbin + IFN- + GM-CSF CD8 - CD8 + CD8 - CD8 + Exp 111.5 <8.013.8190.1 Exp 212.711.133.3812.2 Exp 317.719.893.3854.8 Lack of Th1 skewing in IL-12 p40 -/- CD8+ DC drive Th1 T cells via IL-12

18. PhagocytosisAntigen presentation MaturationMigration

20. Plasmacytoid Dendritic Cells Innate immunity Secrete high levels of IFN-  Adaptive immunity Trigger T cell activation

21. Plasmacytoid dendritic cells “Interestingly, pathologists have long observed the presence of plasma cells that express the T cell marker CD4 and lack intracellular immunoglobulins within the T cell zones of human lymph nodes. These plasma cells were suggested to represent terminally differentiated T plasma cells of unknown secretory functions. Paradoxically, these so called plasmacytoid T cells were found not to express other T cell markers such as CD3 and TCRs. They expressed MHC class II antigens, HLADR and HLADQ, and the invariant chain CD74.” Early on (1970’s) an IFN-a producing MHCII leukocyte was observed but could not be identified--‘interferon producing cell’ (IPC) Found in circulation and in lymphoid tissue Lymphoid origin based on expression of pre-T cell receptor and IgH D-J recombination Flt3 ligand part of their development

22. Depletion using lineage specific antibodies CD4+CD11c-CD3- sorted Discovery of plasmacytoid dendritic cell Characterized peculiar “plasmacytoid T cell” in tonsil area that was CD4+ but lacked lineage markers for T cell, B cell, monocytes, Langerhans cells, granulocytes Freshly isolated pDC undergo apoptosis

23. Grouard, j ex med 1997 pDC survival is IL-3-dependent pDC undergo apoptosis unless cultured with IL-3 pDC cultured with IL-3 have an immature DC phenotype

24. Plasmacytoid dendritic cells have capacity to trigger T cell proliferation

25. Unique features of plasmacytoid dendritic cells Express high levels of CD123 (IL-3R  ) CD11c- (CD11c lo in mouse) Express TLR7, TLR9 Secrete high levels of IFN-  CD45RB, CD45RA, B220, Ly6 (mouse) Can be sorted using CD123+ (IL-3R  ), DR+, Lin- (CD3, CD14, CD16, CD19, CD56) CD11c-, CD4+, Lin- (human) BDCA-2 or BDCA-4 (human) CD11c+, B220+ (mouse)

26. Migration dependent on CD62L and CCR7 which interact with HEV and CCL19, CCL21 (expressed by stromal cells and HEV in T cell rich area of lymph node) lymphatics Migration of plasmacytoid dendritic cells mDC first travel to nonlymphoid tissue and then to lymphoid tissue pDC travel directly to lymphoid tissue

27. activated pDC Colonna et al Nat Imm Unactivated pDC Antigen presentation IFN- 

28. pDC function T cell activation and polarization pDC IL-3 CD40L Th2 OX40L TLRL Th1 IFN- 

29. pDC-derived DC induce Th2 responses mDC or pDC CD40L 7 daysCD3/CD28 ELISA Rissoan et al Science 283:19 1999) pDC=CD11c-, CD4+, Lin- mDC=monocytes (GM-CSF + IL-4 5 days)

30. pDC-derived DC can also induce Th1 responses pDC R-848 or IL-3 7 daysPMA/ionomycin Intracellular flow

31. Plasticity of pDC-derived DC

32. Human plasmacytoid dendritic cells have a unique TLR expression pattern Expression of TLR7, TLR9 allow pDC to detect viral invasion and initiate immune response TLR7- ssRNA TLR9- CpG DNA

33. pDC function continued A major role of pDC during viral infection is the secretion of high levels of type I interferons (IFN- , IFN-  ) Type 1 interferons have a very global impact on the immune response IFN (U/ml) PBMCpDC2-deppDC2-enrichpDC2CD11c + DCMoDC1DC2 Exp. 1500ND2,80089,800120ND<12.51,100 Exp. 240<12.518020,000<12.5350<12.5 Exp. 3700402,800638,0007090ND HSV infected cells

34. TLR ligand (TLR7, TLR9) monocyte Mature Dendritic cell immature Dendritic cell IL-12 Th1 NK cells pDC have a broad impact on immune response via IFN-  generic cell MHCI B cell Plasma cell IFN-  cytotoxicity

35. CD40L CpG Th2 Tr cells (CD4+CD25+) pDC have a broad impact on immune response OX40L IFN-  OX40 T cells (CD4+CD25-)

36. Myeloid vs. plasmacytoid dendritic cell