ImmunoregulationLu.Linrong@gmail.com

The ability of the immune system to sense and regulate its own responses:

1. To determine the type and scale of a response;

2. To ensure Fast Response upon exogenous antigen stimulation;

3. To switch off the response properly and restore the immune system to a resting state when the response to a given antigen is no long required.

Immunoregulation

Immunoregulation Immune Response and Regulation: Including Positive & Negative regulation. Positive regulation ensures fast response. While negative regulation after the clearance of infection is very essential for restore the immune homeostasis. Immunoregulation and intervention: Upon the understanding of immunoregulation mechanisms, we can deliberately enhance or take away one of the control to enhance or block an immune response.

Immunoregulation and Disease: Since immune regulation is achieved at many different levels by different mechanisms, any defect in this system will cause an immune disorder.

Immune regulation happens at different stages of an immune responseDuring Innate Immune Response TLR mediated inflammatory response Cytokine signaling

During adaptive immune response By Inhibitory receptorsBy TregsOther Immune regulation mechanismsRegulation by antibodyby the idiotype and anti-idiotype network

Immune Response

Toll-like receptors and recognition of pathogensK. Takeda & S. Akira, Cell. Microbiol. 5: 143-53, 2003diacyl- triacyl-lipopeptide flagellin:; LPS: (Lipopolysaccharides)

Toll-like receptor signaling pathwaysMyD88 pathway andTRIF pathway;

Transcription factors

Cytokine expression

Innate response is critical for an effective immune response.

Excessive response lead to endotoxic shock (sepsis) Sepsis is a serious medical condition that is characterized by a whole-body inflammatory state (called a systemic inflammatory response syndrome or SIRS) and the presence of a known or suspected infection. The body may develop this inflammatory response to microbes in the blood, urine, lungs, skin, or other tissues. A lay term for sepsis is blood poisoning.

The Key event for innate immune response: Recognition of microbe patterns by pattern recognition receptors including TLR.

Regulation of TLR4 signaling

Inflammatory cytokines Innate TLR signaling :

IRAK-M: interleukin-1-receptor associated kinase M

SOCS1: suppressor of cytokine signaling 1.

MyD88s: myeloid differentiation primary-response protein 88 short SIGIRR: single immunoglobulin IL-1R-related molecule

Rab7b promote endocytosis and degradation Cao X 2007Rab10TLR4 signaling is regulated by membrane traffickingBiosynthesis

Replenishment

Rab10 promote trafficking onto cell surface. Wang D et al in Lu lab 2010Endocytosis

DegradationRecycle

Regulation by signaling pathways (cytokine receptors signaling)

Regulation by signaling pathways (SOCS family)

Virus-infected cell produces interferon to act on neighboring cellsInfected cell makes interferon, uninfected cells respond to interferon and become refractory to viral growth (Antiviral State)

Production of interferon by infected cells

Summary of the first part:

Regulation of TLR signaling

The role of SOCS in cytokine signaling(inducible, ubiqutin ligases)

Amplifciation of IFN signaling in antiviral response.

Regulation by Inhibitory receptors1. Immune cells are activated by Ligand binding to their activating receptors

2. Activation of Immune cells is mediated by Protein Phosphorylation

3. Phosphorylation is mediated by protein kinases; Dephosporylation is catalyzed by protein phosphotases.

1). Activating receptor: ITAM (immunoreceptor tyrosine-based activation motif) MotifsYxxL/V Recruit : kinases, adaptor proteinsInduce activation signal2). Inhibitory receptor:ITIM (immunoreceptor tyrosine-based inhibitory motif) MotifsI/Vx YxxLRecruit Protein phosphatasesTransduce inhibitory singnalRegulation by Inhibitory receptorsTwo type of receptors

Regulation by Inhibitory receptorsActivating receptors mediate cellular activation

Cell typeActivation Triggeractivation motifKinase recruitedB cellBCR ITAM (Ig Ig)LynT cell TCR ITAM (CD3)LckNK cellNKG2D ITAM DAP12Syk..

Regulation by Inhibitory receptors

B cell, mast cell: FcRIIBPIRBCross-linking immune complex or anti-idiotype antibody with BCR

T cell: CTLA-4, PD-1CTLA-4 and PD-1 transmit signals that inhibit lymphocyte activation, providing a pathway of classical feedback inhibition.

NK, CTL: KIRLy49 (type I), CD94/NKG2A (type II)

Inhibitory receptors

Once the ITIM of FcRIIB is phosphorylated, SH2-containing SHIP are recruited, which influences PI3K and ERKpathways.

PIRBpaired immunoglobulin-like receptor B ITIMs recruites SHP1which dephosphorylates various protein-tyrosine kinases, including SYK and Bruton's tyrosine kinase (BTK). Inhibitory receptors on B cell

Inhibitory receptors

Inhibitory receptors for T cell. CTLA-4

Y201 YVKMY208

Inhibitory receptors

Dynamic regulations of T cell activation by CD28 and CTLA4:

CD28 and CTLA-4 has the same ligand B7.

Resting T cell only express CD28, which is an activating coreceptor provides 2nd signaling to ensure T cell activation.

Activated T cells start to express CTLA-4 on their surface, because CTLA-4 has 100 folder higher affinity to B7, CTLA-4 activation become dominant which inhibit the TCR signaling. Before After activation activation

Other inhibitory receptors on T cells

Inhibitory receptors on NK cells

Immune surveillance for Missing Self by NK cells Existence of inhibitory receptors for MHC class I spare normal cells from NK cell attack which ensures NK cells preferentially kill cells that have lost MHC class I. 1. Viral-infected cells or tumor cells tends to reduce their surface expression of MHC I to avoid CD8 cell attacks. 2. NK cells thus Provides protection against cells escaping T cell recognition. 3. NK cells will also kill foreign cells with mismatched MHC class I molecule. Karre et al. Nature 319:675, 1986Inhibitory receptors on NK cells

Summary of Part II:

Definition of Inhibitory receptors ITIM /ITAM (how do they work)

Typical inhibitory receptors

Story of CTLA4

NK cell function regulated by inhibitory receptors(KIR in human, Ly49 in mouse NKG family in both)

Immunoregulation by Tregs

CD4+FoxP3+ TregsCD8+ TregsOther regulatory cells

Summary of CD4+ FoxP3+ Treg cells

1) Natural Treg: constitute approximately 10% of CD4+ T cells in spleen and lymph nodes of normal mice, 10~30% of CD4+T cells in peripheral blood of human. Naturally anergic and suppressive, appear to be produced by the normal thymus as a functionally distinct subpopulation of T cells. 2) Surface markers CD25 (IL-2R), IL-2R, CTLA-4, GITR, CD44high, CD45RB, CD5high, ICAM-1high, LFA-1high, partly CD62L. The expression pattern of some accessory molecules is in part similar to primed , activated, effector, or memory T cells.3) Inducible TregiTreg-- that differ in terms of their development, specificity, mechanism of action and dependence on T-cell receptor and co-stimulatory signaling.

CD4+FoxP3+ Tregs

CD25+ Activated T cells can suppress autoimmune diseasesCD25-PECD4+FoxP3+ TregsSakaguchi et al. 155 (3): 1151. (1995)

Another classic experiment

FoxP3+ is the transcription factor that program the development of TregsAlexander Y. RudenskyUniversity of WashingtonShimon SakaguchiKyoto UniversityRIKENCD4+FoxP3+ TregsNature Immunol 4:330-336.

I P E XImmune deficiency/dysregulationPolyendocrinopathyEnteropathy (Often have Ab against gut epithelium)X-linked inheritancePatients have mutations in FOXP3 gene. They suffer from inflammatory diseases similar to that seen in mice deficient in CD4+CD25+ Treg cells.

IPEX Outside (Clinical Findings) First described in 1982 by Powell et al. as a syndrome of diarrhea, polyendocrinopathy, and fatal infection in infancy. Neonatal onset diabetes mellitus() Hypothyroidism () Enteritis (diarrhea/villous atrophy)() Hemolytic anemia & thrombocytopenia. Dermatitis Dermatitis (eczema) Death by 1-2 years of age

IPEX

*PROLINE-RICH DOMAINZINC-FINGER LEUCINEZIPPERFORKHEAD DOMAINNCFOXP-3 is the master control gene for Treg development, encodes a novel forkhead/winged helix transcription factor

FOXP3 Mutations

IPEXScurfyFoxp3TregFoxp3

Probability of selection

Avidity of TCR-MHC-peptidePositive selectionNegative selection

CD25+CD4+ nTregsHigh affinity interaction with MHC/self peptide is required

IL-2 is required for development of CD4+CD25+ nTregs, anti-IL-2 treatment early after birth leads to autoimmune diseases in adults Expansion of nTresgs in thymus is likely autoantigen-drivenIL2 or IL2R deficiency lead to autoimmune diseases

TregTGF-Nakamura et al., JEM, 2001CD4+CD25+ TregsTGF-TGF-

TregTGF-TGF- CD4+CD25+ TregChen et al., JEM, 2003TGF- and TCRcostimulation induces Foxp3 expression in CD4+CD25-naive responder T cells.

TregTGF-Zhou et al., Immunity, 2009The Cytokine Milieu Determines CD4+ T Cell Differentiation and Conversion

Mechanisms used by TregsCD4+FoxP3+ Tregs

Combined effects of Tregs

Tregs in Clinic Tregs Part IIPart III Treg

TregSLETreg TregDNASLE

SLETregFas Construction and characterization of a novel DNA vaccine that is potent antigen-specific tolerizing therapy for experimental arthritis by increasing CD4+CD25+Treg cells and inducing Th1 to Th2 shift in both cells and cytokines.Vaccine2009

Clinical Applications of Human Treg Cells in Allo-HSCTRiley et al., Immunity, 2009

CD4+FoxP3+ TregsAs a immune research modelSuppressor cell activityHow to identify? What drives its development?How dose it work?Where can it be used?How to use it to explain immune phenomenon? Apply into clinic?

Summary of Part III

What is CD4+FoxP3+ Treg (Properties)

The central role of FOXP3

IPEX

Working hypothesis

CD8 regulatory cellsCD8 cells were first discovered as suppressors: Cantor H, Boyse EA . J Exp Med 1975; 141: 1376 1389.

Jandinski J, Cantor H, Tadakuma T, Peavy DL, Pierce CW. J Exp Med 1976; 143: 1382 1390.Role of CD8 in control autoimmune disease:

Role of CD8+ T Cells in Murine Experimental Allergic Encephalomyelitis Hong Jiang, Sheng-le Zhang, and Benvenuto Pernis Science 22 May 1992 256: 1213-1215

Less Mortality but More Relapses in Experimental Allergic Encephalomyelitis in CD8-/- Mice Dow-Rhoon Koh, Wai-Ping Fung-Leung, Alexandra Ho, Dawn Gray, Hans Acha-Orbea, and Tak-Wah Mak Science 22 May 1992: 1210-1213.

Several cell surface molecules have been associated with CD8+ Treg in different experimental systems. But the definition of CD8+ Treg is not clear because of the lacking of reliable surface marker.

For example:CD11c+CD8+ T Cell: Have a essential role in immunotherapy of Rheumatoid Arthritisand

CD122+CD8+ T Cell: Have a essential role in the recovery from Experimental Autoimmune Encephalomyelitis (EAE)

CD28-CD8+ T Cell: Behave as regulatory T cells, since transfer into CD8-deficient mice significantly suppressed EAE

Qa-1-restricted CD8+ Treg CD11c+NKG2A+CD8+

CD8 regulatory cells

CD8 regulatory cellsQa-1-restricted CD8 suppressor cells as an example

Lu L. et.al. PNAS 2008;105:19420-19425Increased susceptibility to autoimmune disease in Qa1 mutated miceCD8 regulatory cells

Qa1/NKG2A interactionShi and Van Kaer Nature Reviews Immunology 6, 751760 Other regulatory cells-NK cells

Other regulatory cells-NKT cells(4) NKT cells are cells positive for both CD4 T cell and NK cell surface markers. NKT cells recognize CD1d-restricted lipid ligands. Upon TCR activation, NKT cells can exert regulatory function by secreting various cytokines including IL-4IL-10TGF-

Other regulatory cells -DCs

Nature Immunology 5, 1124 - 1133 (2004)Other regulatory cells DCs & microenvironment

1.6. Regulation by apoptosisNegative immunological regulation by cell apoptosis

Two forms of apoptosis

FAS mediated apoptosisCytokine starvation

Regulation by cytokinesTGF- blocks T cell proliferation by inhibiting IL-2 production via Smad3 and by downregulating the expression of cyclinD2 and E, CDK4, and c-myc. TGF- inhibits differentiation of Th1 and Th2 cells through suppressing the expression or function of T-bet/Stat4 and GATA-3/NFAT. Mechanisms involved in TGF- inhibition of CTL differentiation are not well understood, although inhibition of c-myc and T-bet expression is suggested. TGF- induces FoxP3 expression and the generation of Tregs. TGF- also prevents T cell activation-induced cell death (AICD) through inhibiting c-myc-mediated FasL expression and other unknown mechanismsTGF- regulation of T cell responses

Th differentiation governed by signal III (cytokines)Signal ISignal IISignal IIITh differentiation

Th subsets can regulate each other.

Th1IFN- to inhibit Th2

Th2IL-10to inhibit Th1

IL4 and IFN produced by Th1 and Th2 cells inhibit Th17 TregTGF- inhibit Th1, Th2

Th17 cells can produce IL21 to inhibit Treg Th differentiation

Regulation by neuroendocrine system 1. Lymphocytes express receptors for many hormones, neurotransmitters and neuropeptides.

2. The nervous system directly or indirectly controls the output of various hormones especially under stress, in particular corticosteroids, growth hormone, prolactin, thyroxine and adrenalin.

3. A particularly important control is mediated by corticosteroids, endorphins and enkephalins (), all of which may be released during stress, and all of which are immunosuppressive in vivo.

Regulation by endocrine system

Corticosteroids inhibit Th1 cytokine production, expression of IL-2R and MHC, but induce Th2 cytokine production and TGF- production.

Estrogen influences the more active immune responses in female

e.g. higher serum Ig and is susceptible to autoimmune disease

--

nerve-endocrineMicroenvironmentRegulatory cellsInhibitory receptorsCytokinesCell signalingApoptosisInside CellWhole BodySummary of Immunoregulation

CD4+FoxP3+ Tregs

Inhibitory receptors

()

EAE)Lee YK et al PNAS 108:4615-4622 Wu H-J et al, Immunity 32: 815-827.T

1. Lee YK et alProinflammatory T-cell responses to gut microbiota promote experimental autoimmune encephalomyelitis. PNAS 108:4615-4622 2. Wu H-J et al, Gut-residing segmented filamentous bacteria drive autoimmune arthritis via T helper 17 cells. Immunity 32: 815-827.3. Hooper LV, Littman DR, and Macpherson AJ. Interactions Between the Microbiota and the Immune System Science 336:1268-1273, 2012

* NKT; *

******Toll.TIRToll/IL-1R domain)SIGIRRST2TIRAPMyD88MyD88s myd88IRAK4IRAK-MIRAK1IRAK4SOCS1IRAK1TOLL

Toll-like receptor (TLR)-signalling pathways are negatively regulated by several molecules that are induced by the stimulation of TLRs. IRAK-M (interleukin-1-receptor (IL-1R)-associated kinase M) inhibits the dissociation of the IRAK1IRAK4 complex from the receptor. SOCS1 (suppressor of cytokine signalling 1) probably associates with IRAK1 and inhibits its activity. MyD88s (myeloid differentiation primary-response protein 88 short) blocks the association of IRAK4 with MyD88. The TIR (Toll/IL-1R)-domain-containing receptors SIGIRR (single immunoglobulin IL-1R-related molecule) and ST2 have also been shown to negatively modulate TLR signalling. IB, inhibitor of NF-B; IKK, IB kinase; NF-B, nuclear factor-B; TIRAP, TIR-domain-containing adaptor protein; TRAF6, tumour-necrosis-factor-receptor-associated factor 6.**TproteasomeJAK STATJAKSTATSTATSOCS.JAKSTATInhibition of the Janus kinase (JAK)signal transducer and activator of transcription (STAT)-signalling pathway by SOCS proteins through distinct mechanisms. SOCS1 binds directly to tyrosine-phosphorylated JAKs through the SH2 domain, resulting in the inhibition of kinase activity. SOCS3 inhibits JAKs through binding to the receptor. By contrast, CIS does not affect the activity of JAKs. Instead, CIS inhibits STATs by competing with STATs for docking sites on the receptor.

*SOCS1995ubiquitin ligase ()JAK***** SerThrTyr*ITAMmotif ITIMmotif*ITAMBTNKG2DNKITAM123.* TCTLA4,PD1BFcFcrRIIBNKTNKKIRLyCD94/NKG

*BBFCITIMSHIPPI3KEARPIRBBTKSykPLC*TCTLA-4 cytotoxic T-lymphocyte antigen 4 TCtla-4,Y201Y208.Y201YVKMITIM motifctla-4AP-2AP50Tctla-4SrcPP2ASHP-2CD3TCRMAP kinases NF-B AP-1NFAT*TCD28ctla-4123.**NKNKMHC INKMHC INK**NKmissing selfMHCNKMHC I+NKNKMHC IMHC Icd8 TNKMHC INK*NK**CD4+FoxP3+CD4 T*CD4+FoxP3+TT12TGF-betaTreg*Treg1995sakaguchi**FoxP3************TregTGFb,DC*********TCD8TCD8*Qa1CD4TCD8T

*NK*NKT*DC.DCTGF-betaTregDCIL6Th17.*2004DCDCdifferentiated DCLNstoma*

FASTFasFasLFasFasLcaspase*TGF-beta*TTMHCTCRCD28TTT*TTh1Th2Th1INFTh2IL4Th17Treg*****