Cell Mediated Immunity

W. Robert Fleischmann, Ph.D.

Department of Urologic Surgery

University of Minnesota Medical School

rfleisch@umn.edu

(612) 626-5034

Objectives

• To understand the role of cell-mediated immunity

• To understand the identity, mechanism of activation, and method of killing by cytotoxic T cells

• To understand the identity, mechanism of activation, and method of killing by natural killer (NK) cells

• To understand the method of killing by antibody-mediated cellular cytotoxicity (ADCC)

Arvak Petrosian, age 2 years, is brought from Armenia to a private clinic in the U.S. that specializes in diagnosis and treatment of unusual diseases.

Arvak has a history of failure to gain weight. He appears to be anorexic. In addition, he is noted to have eczema.

He has recently been diagnosed with Type I diabetes. According to his Armenian physicians, he is now showing signs of hypothyroidism.

What are your thoughts?What tests would you wish to request?

A complete blood cell count shows Low levels of neutrophilsLow levels of RBCsLow levels of platelets

T and B cell mitogen stimulation is normal.Antibody isotypes and sub-types are at normal levels.

What are your thoughts?What tests would you wish to request?

Urinalysis shows the presence of protein in the urine.Blood chemistry shows elevated levels of serum

creatinine and urea nitrogen.Liver enzymes are significantly elevated.

What are your thoughts?What tests would you wish to request?

Arvak is diagnosed as havingEczemaEnteropathy (destruction of cells in the intestine)DiabetesNeutropeniaAnemiaThrombocytopeniaThyroiditisGlomerulonephritisHepatitis

What are your thoughts?

Role of Cell-Mediated Immunity• Humoral immunity mediated by antibody is responsible for

the protection of the extra-cellular environment from pathogens and toxins.

• The principal roles of cell-mediated immunity are to recognize and destroy pathogen-infected cells and cells that have undergone genetic alterations (tumor cells).

• The importance of cell-mediated immunity can be seen in patients who lack a thymus (DiGeorge Syndrome).– Although antibodies are not made, these patients deal relatively

well with extracellular bacteria.• DiGeorge patients do suffer repeated infections because no memory.

– Infections with viruses, intracellular bacteria, and fungi are difficult to clear and become life-threatening in DiGeorge patients.

The Mediators of Cell-Mediated Immunity

• Antigen-nonspecific effector cell– Natural Killer cells– Macrophages– Neutrophils– Eosinophils

• Antigen-specific effector cells– CD4+ T cells (aka, helper T cells, Th cells)– CD8+ T cells (aka, cytotoxic T cells, CTLs,

Tc cells)

Different Effector T Cells Can Be Recognized by the Effector Molecules

They Produce

Cytotoxic T Cells

Cytotoxic T Cells• Naïve CTL cells are called CTL precursors (CTL-P) to

indicate their immature state.• A CTL-P cell matures only after being activated by

interaction with a Th1 cell.• Maturation requires three sequential signals.

– Antigen-specific signal is transmitted by the TCR upon recognition of peptide:Class I antigen presented by a professional antigen presenting cell (licensed antigen-presenting cell) or by a tissue cell.

– Costimulatory signal is transmitted by CD28:B7 interaction between the CTL-P cell and the licensed antigen-presenting cell.

– A signal induced by IL-2 secreted by a Th1 cell, results in proliferation and differentiation of the antigen-activated CTL-P cell to a CTL.

Activation of Effector CTLs from

Naïve CTL-Ps

• Before an APC can activate a CTL-P cell by peptide:MHC class I presentation, it must be “licensed” to do so.

– APC must present peptide via MHC Class II to a Th1 cell.

– Somehow, this interaction licenses the APC to present peptide via MHC Class I antigen to the CTL-P cell.

• It is not known whether the Th1 cell and the CTL-P cell are bound to the APC at the same time. Note: Naïve CTL-P = naïve CTL precursor

Importance of Licensing of APC• Licensing of APC requires interaction of the

APC with the Th1 cell.• Thus, presentation of antigen to the Th1 cell via

MHC class II and to the CTL-P cell via MHC class I acts a control to prevent recognition of self antigen (antigen must be a phagocytosed antigen to be expressed on Class II).– This is a function best carried out by a dendritic cell

acting as an APC.– Infection of a dendritic cell by a virus assists in the

presentation of antigen associated with MHC class I.

Activation of Memory CTLs• Th1 cell not required to provide IL-2.• Autocrine IL-2 is sufficient.

Naïve CTL-P Versus Mature CTL• Naïve CTL-P

– Does not express IL-2 or IL-2R and only begins to express them after it begins to be activated.

– Expresses the CD45RA isoform of CD45.– Expresses a low level of cell adhesion molecules CD2 and LFA-1.– Does not exhibit cytotoxic activity.

• Mature CTL– Expresses high affinity IL-2 receptor and requires high levels of IL-

2 to proliferate.– Synthesizes low levels of IL-2.– Expresses the CD45RO isoform of CD45.– Expresses a high level of cell adhesion molecules CD2 and LFA-1– Exhibits cytotoxicity.

• Memory CTL cells may not require Th1 help.– Requires only low levels of IL-2 (produced by activated CTL-Ps)

for memory CTLs to become mature effector CTLs.

Overview of CTL Killing • Primary events– Conjugate formation– Reorientation of cytoplasmic

granules in CTL– Granule exocytosis– Dissociation– Target cell death by apoptosis

Pictures of CTL Killing

Note that the T cell approachesthe target cell, forms a close binding to the target cell, andreorients its granules (small arrow) toward the target cell.

CTL Killing: Binding of Target Cell

• The TCR-CD3 membrane complex on the CTL recognizes peptide bound to MHC class I on a target cell.

• LFA-1 on CTL binds to ICAMs on target cell.• Antigen activation converts LFA-1 from a low-

affinity state to a high-affinity state, resulting in the formation of the conjugate.

• After 5-10 minutes, the LFA-1 returns to a low-affinity state, resulting in dissociation of the CTL from the target cell.

Bound CTLs Kill Targets by Two* Mechanisms

• Perforin/Granzyme are secreted from granules– Perforin molecules form pore– Granzyme molecules activate apoptosis by

cleavage of caspases

• FasLigand protein on cell membrane surface– Membrane-bound FasL binds to Fas on the

membrane of the target cell to initiate cell killing– Activates apoptosis by cleavage of caspases

* CTLs can also kill by TNF production and secretion

CTL Killing: Degranulation Events• The CTLs (but not naïve CTL-Ps) contain intracellular granules.

– Contain monomers of perforin• Perforin monomers polymerize to form pore in the target cell• Perforin has some homology with C9 and forms pores similar to complement pores

– Contain serine proteases called granzymes or fragmentins• Granzymes taken up by receptor mediated endocytosis (granzyme binds to

mannose 6-phosphate receptor) or through perforin pore (perforin-assisted pathway)

• Granzymes induce apoptosis

Apoptosis Inductionby FasL and Granzyme

• FasL on the CTL binds to Fas (TNF family receptor) on the target cell, activating its death domains to bind to FADD (FAS-associated Death Domain) that, in turn, activates caspase-8

• Granzymes initiate apoptosis by activating caspase-8

Natural Killer Cells

Natural Killer Cells (NK Cells)• NK cells make up 5-10% of the population of

circulating lymphocytes.• NK cells play major roles in killing virus-

infected cells, intracellular pathogen-infected cells, and tumor cells.

• NK cells produce a number of important cytokines, including IFN-.– IFN- can tilt the immune response toward Th1

cells by inhibiting Th2 and by inducing IL-12 production by macrophages and dendritic cells.

– IFN- can activate macrophages and NK cells.

• NK cell activity is stimulated by IFN-, IFN-, IFN-, TNF-, IL-12 and IL-15.

NK Cells Are an Early Defense Against Viruses

NK Cells versus CTLs• NK cells express CD16, FcRIII.• NK cells do not need to be educated by the thymus.• NK cells do not undergo rearrangement of receptor

genes and, thus, do not express T cell receptors or CD3.

• NK cell killing is not MHC restricted (identical killing levels are seen for allogeneic and syngenic tumor cells).

• NK cells do not show immunologic memory that can be primed by re-exposure to antigen.

• NK cell killing is similar to CTL killing– FasL expressed on surface and can kill cells expressing Fas– Perforin and granzyme released from granules– TNF expressed on surface and secreted

NK Cell Receptors• NK cells have both activation and inhibition

receptors.• NK cell receptors fall into two categories but

members of each group can be activation or inhibition receptors.– Lectin-like receptors

• Bind proteins rather than polysaccharides• Can be activation or inhibition receptors

– Immunoglobulin-like receptors (aka, KIR = killer cell immunoglobulin-like receptors)

• Bind to most class I MHC molecules• Can be activation or inhibition receptors, but most are

inhibition receptors

Opposing Signals Model ofNK Cell Killing

• If an antigen recognition signal is given to the NK cell and the level of MHC class I is high, no killing occurs.

• If an antigen recognition signal is given to the NK cell and the level of MHC class I is low, killing occurs.

NK Cells and CTL Have Complementary Activities

NK Cells• Kill cells that mask

the presence of foreign antigen on MHC class I

CTLs• Kill cells that

express foreign antigen on MHC class I

NKT Cells

NKT Cells• NK cells express lectin-like receptors and KIR.• CTL express T cell receptor and CD3.• NKT cells express T cell receptor but differs from CTL.

– Maturation is in the thymus.– The T cell receptor is invariant (specific gene segments encode

TCR and TCR chains).– The T cell receptor does not recognize MHC-bound peptides but

does recognize a glycolipid presented by CD1d.– NKT cells do not exhibit memory.– NKT cells express cell surface markers characteristic of NK cells.– NKT cells may play a role in killing bacteria and tumor cells but

precise role is uncertain.• Bacteria killing via expression of glycosyl ceramides (glycolipid)?

• Tumor cell killing via expression of glycolipids that are specific to tumor cells?

Antibody-DependentCell-Mediated Cytotoxicity

(ADCC)

ADCC Killing• Effector cells associated with

ADCC– NK cells– Macrophages– Monocytes– Neutrophils– Eosinophils

• Effector cells bind antigen via antibody bound to Fc receptor

• Killing is mediated by– Cytolytic enzyme release by

macrophages,neutrophils,and eosinophils

– TNF release by NK cells, monocytes, and macrophages

– Perforin release by NK cells and eosinophils

– Granzyme release by NK cells

Arvak Petrosian

Because of the number of organ systems under autoimmune attack, Arvak is diagnosed with a rare condition called IPEX syndrome (immune dysregulation, polyendocrinophathy, enteropathy, X-linked syndrome).

This syndrome is caused by mutations in the FOXP3 gene.

Arvak Petrosian

FOXP3 gene function is required for the development of Treg cells.

In the absence of appropriate Treg cell function, CD8+ cytotoxic T cells are active and destroy normal tissue cells that they recognize. Thus, peripheral tolerance is lost, resulting in autoimmunity.