Infection and Immunity Weilin Chen, Ph.D Institute of immunology, ZJU NK cells APC and antigen presentation

Contents NK cells –Development ， receptors and function Antigen presenting cells –Dendritic cells Antigen presentation –Four pathways

Natural killer cells Development Receptors Function

NK cells development Bone marrow Blood - In the bone marrow, a common lymphoid progenitor gives rise to the antigen-specific lymphocytes of the adaptive immune system (B and T cells), as well as to a type of lymphocytes that is antigen non- specific; the Natural Killer (NK) cell lineage (express the NK lineage marker, NK1.1). - As NK cells are antigen independent, they belong to the innate immune system. - Mature NK cells that circulate in the blood stream are larger than B and T cells. - They contain a distinctive granular cytoplasm. The release of the lytic granules contributes to the killing of cells infected with distinctive viruses, such as herpes virus. - NK cells also contribute to the killing of abnormal tumor cells, without prior immunization or activation.

NK cells development

In vitro

NK cells development

Activation of NK cells is the net effect of inhibitory and activating signals NK receptors

The “Missing Self” Hypothesis States that altered expression/down-regulation of MHC Class I on target cells leads to spontaneous NK-mediated destruction of the target cell Down-regulation of MHC Class I OR over- expression of NK cell activating molecules leads to NK cell-mediated killing of target cell Hypothesis ： Non-self ； induced self

12 NK receptors: ‘Defense is the best offense” While both KIRs and KLRs sense the presence (absence) of MHC class I molecules, activating as well as inhibitory receptors are found in both families of receptors. - The KIRs are subdivided according to the number of immunoglobulin-like domains (2 or 3 domains) and the length of their cytoplasmic tail: Short tail = activating receptors Long tail = inhibitory receptors - The KLR are heterodimers of CD94 associated with a NKG2 molecule. Six distinct NKG2 isoforms exist in humans. NKG2C/CD94 = activating receptor NKG2A/CD94 = inhibitory receptor NKG2B/CD94 = inhibitory receptor NKG2D homodimer = activating receptor.

Functions

15 Release of cytotoxic granules at the site of contact with infected cells - First contact between a CTL or NK cell with infected cells is via non-specific binding of adhesion molecules (LFA-1 (blue) on T and NK cells with ICAM-1 or ICAM-2 (brown) on target cells). This makes a channel between the target and the cytotoxic cell. - Specific antigen/MHC class I recognition by TCR on CTL, or engagement of the NK’s natural cytotoxic receptors (NCR) (green) by non-MHC ligands (orange) on the surface of the target cell. This results in a polarization of the cell: the actin cytoskeleton (green staining in the immunofluorescence microscopy) at the site of contact is reorganized as to aligning the microtubule-organizing center (MTOC), as well as the secretory apparatus, including the Golgi (GA). The GA-derived lytic granules (stained in red in the photomicrograph) are specifically directed onto the target cell. - The content of the granules is directly released onto the target cell. Target cell NK cell

16 Why are NK cells prominent tumor killers? - The regression of transplanted tumors in a normal mouse model (blue line) is largely due to the action of CTLs recognizing tumor antigens presented on MHC class I (right panel). Albeit the presence of NK cells, this regression is absent in nude mice (red line) in which CTLs do not develop. -Tumor variants that express low levels of MHC class I become susceptible to NK cells, especially in nude mice (have higher levels of NK cells than wild type mice). Thus tumors that are sensitive to NK killing grow less well in nude than normal mice (central panel). - Transfection of MHC class I genes resulting in high expression of this protein restores NK cell resistance but susceptibility to CTL in normal mice (left panel; blue line). Similar to many pathogens, tumor cells can escape the adaptive immune system, by downregulating the expression of MHC class I. This makes them more susceptible to NK cells.

Antigen presenting cells and antigen presentation

A variety of cell types which carry antigen in a form that can stimulate lymphocytes. professional APC:MΦ,DC,B non-professional APC Target cells Antigen presenting cell, APC

The 3 types of professional APCs Constitutively express a high level of MHC II and the co-stimulatory protein,B7. the most effective APC Constitutively express class II MHC but must be activated to produce B7. must be activated by the process of phagocytosis before expressing class II MHC and B7.

Dendritic cells (DC) Dendritic cells were first described by Paul Langerhans (Langerhans cells) in the late nineteenth century. It wasn't until 1973, however, that the term "dendritic cells" was coined by Ralph M. Steinman.

GM-CSF TNF-  IL-4 HSC Myeloid progenitor Lymphoid progenitor DC Mo macrophage DC PMN DC Sources of DCs

Subset of DCs Conventional DCs ， cDC Plasmacytoid DCs ， pDC ： anti viral infection

LPS IL-1,TNFα Maturation of DCs

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antigen presentation, immune activation, immune tolerance Function of DCs

mononuclear phagocyte system, MPS 1.Differentiation and distribution 2. Surface markers 3. function Antigen presenting cells

Bone marrow Blood tissues HSC Myeloid progenitor Pre-monocyte monocyte macrophage Differentiation and distribution of MPS

1. Surface markers: MHC-I/ Ⅱ molecules CKR: M-CSFR CAM: LFA-1,ICAM-1,B7,CD40 FcR, CR1/3/4 2. secretion ： -enzymes: lysosome, myeloperoxidase -cytokines (IL-1,TNF,IL-12) -complement:C1 ～ C9, Bf -coagulation factor, PG, LTs, ACTH, etc. Mononuclear phagocyte system, MPS

Biological functions of MPS lPhagocytosis lantitumor ： indirect or direct killing, ADCC; lparticipating in immune response ： -Ag presenting, providing the second signal ； -CMI lparticipating in immune regulation: -positive regulation ： secrete IL-1/12 ， TNF-α -negative regulation ： PG, TGF-β lMediating inflammation: phagocytosis, secrete inflammation medium

phagocytosis

Antigen presenting cells B cells

The properties of various APCs

Definition Ag presentation -MHC class I molecule pathway -MHC class II molecule pathway - Cross-presentation pathway - CD1 molecule pathway Antigen presentation

Antigen capturing Phagocytosis Pinocytosis Receptor-mediated endocytosis

Antigen processing The conversion of an antigen into a form in which it can be recognized by lymphocytes.

Antigen presentation The process by which certain cells in the body (APC) express antigen peptide- MHC molecule complex on their cell surface in a form recognize by lymphocytes.

sources of antigens * exogenous antigen * endogenous antigen

Y The site of pathogen replication or mechanism of antigen uptake determines the antigen processing pathway used Y Cytosolic compartment Endogenous processing (Viral antigens) Vesicular Compartment Contiguous with extracellular fluid Exogenous processing (Streptococcal, Mycobacterial antigens) Distinct mechanisms of antigen generation are used to raise T cells suited to the elimination of endogenous or exogenous pathogens Intracellular replication Extracellular or Endosomal replication

Pathogens in Cytosol/Vesicles

The pathway of MHC I -associated endogenous Ag presentation endogenous antigen （ such as virus Ag, tumor Ag) cytoplasm degraded by proteasome antigen peptide （ 8-13 AA ） transported to endoplasmic reticulum by TAP Peptide/MHC-I molecule complex to surface of APC submit to CD8 + T

Degradation in the proteasome Cytoplasmic cellular proteins, including non-self proteins are degraded continuously by a multicatalytic protease of 28 subunits

ER membrane Lumen of ER Cytosol Transporters associated with antigen processing (TAP1 & 2) Transporter has preference for >8 amino acid peptides with hydrophobic C termini. TAP-1 TAP-2 Peptide TAP-1 TAP-2 Peptide TAP-1 TAP-2 Peptide TAP-1 TAP-2 Peptide TAP-1 TAP-2 Peptide TAP-1 TAP-2 Peptide TAP-1 TAP-2 Peptide TAP-1 TAP-2 Peptide TAP-1 TAP-2 Peptide TAP-1 TAP-2 Peptide ER membrane Lumen of ER Cytosol TAP-1 TAP-2 Peptide ATP-binding cassette (ABC) domain Hydrophobic transmembrane domain Peptide antigens from proteasome

Endoplasmic reticulum Calnexin binds to nascent class I  chain until  2-M binds TAP-1 TAP-2 Peptide TAP-1 TAP-2 Peptide TAP-1 TAP-2 Peptide TAP-1 TAP-2 Peptide TAP-1 TAP-2 Peptide TAP-1 TAP-2 Peptide TAP-1 TAP-2 Peptide TAP-1 TAP-2 Peptide TAP-1 TAP-2 Peptide TAP-1 TAP-2 Peptide TAP-1 TAP-2 Peptide B2-M binds and stabilises floppy MHC Tapasin, calreticulin, TAP 1 & 2 form a complex with the floppy MHC Cytoplasmic peptides are loaded onto the MHC molecule and the structure becomes compact Maturation and loading of MHC class I

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The pathway of MHC II -associated exogenous Ag presentation Exogenous antigen newly synthesised MHC class II molecule （ in the endoplasmic reticulum ） early endosome li binds in the groove of MHC class II molecule lysosome protease MIIC late endosome li degrade protease Degrade into 13  18AA peptide + MHC class II molecule Ag peptide/MHC class II molecule complex transport to the surface of APC, recognized by CD4 + T Phagocytosis, pinocytosis, FcR-phagocytosis

Y Y Pinocytosis Phagocytosis Membrane Ig receptor mediated uptake Y Uptake of exogenous antigens Complement receptor mediated phagocytosis Y Fc receptor mediated phagocytosis opsonization

Proteases produce ~24 amino acid long peptides from antigens Drugs that raise the pH of endosomes inhibit antigen processing Endosomes Exogenous pathway Increase in acidity To lysosomes Uptake Protein antigens In endosome Cathepsin B, D and L proteases are activated by the decrease in pH

 involve in the assembling and folding of MHC class II molecule;  Block the groove of MHC class II molecule;  Lead the assembled class II molecule to M Ⅱ C. The functions of Ii: CLIP ： class II-associated invariant chain peptide

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Cross-presentation Class I MHC molecules present exogenous Ags to CD8 + T cells Class II MHC molecules present endogenous Ags to CD4 + T cells Cross-presentation of Ags by DC plays an important role in anti-viral infection and anti- tumor immunity.

NKT cells.

CD1 molecular pathway

Antigen presentation

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