T Cell Receptor & T Cell Development

T Cell Receptor & T Cell Development

Questions for the next 2 lectures: How do you generate a diverse T cell population with functional TCR rearrangements? How do you generate a T cell population that is self-mhc restricted? How do you ensure that those diverse T cell receptors are not-self reactive? How do you coordinate lineage specification with MHC specificity and coreceptor expression? - vs. T cell - CD4 vs. CD8 2

T lymphocyte: a key regulator of the immune system

T Lymphocytes Arise from stem cells resident in the bone marrow and migrate to the thymus which serves as an indoctrination center where thymocytes must learn to distinguish self from nonself Thymocytes that cannot make this induction are eliminated, those that can may further differentiate, mature, and graduate as T lymphocytes and enter the circulation

Origin, generation and differentiation of T cells

T Cell Development T cell progenitors migrate from bone marrow and seed thymus. T cell progenitors undergo differentiation to CD4, CD8 and NKT cells in thymus. Mature CD4 and CD8 T cells circulate between blood and lymphoid tissues until they meet antigens presented on dendritic cells in lymphoid tissues. T cells further undergo maturation to become functional memory or effector T cells in LT

Figure 5-2 Thymic involution: Human thymus is fully developed before birth and increases in size until puberty. It then progressively shrinks during adult life. Most thymectomized adults have no problem in T cell immunity because they have enough memory T cells in the periphery, and these T cells are longlived.

T Cell Functions Collectively, T cells display a number of diverse functions: - They often function to initiate, regulate, and fine-tune humoral immune response - They are effector cells responsible for various types of cell mediated immune responses like; DTH, contact sensitivity, transplantation immunity, and cytotoxicity

T Cell Surface Molecules TCR: A very diverse heterodimer that lacks a cytoplasmic tail that would allow direct cytoplasmic signaling once TCR binds an epitope CD3 Complex: It is composed of a group of six invariant accessory molecules; one CD3, one CD3, two CD3ε, and an intracytoplasmic homodimer of ξ or CD247 chains Cytoplasmic signaling occurs through CD3 that noncovalently associate with TCR

TCR Complex Figure 3-6

The CD3 complex is essential for both cell surface expression of the TCR and for signal transduction once the TCR recognizes an antigen Unlike antibodies that can readily bind free antigen, a TCR cannot bind soluble antigens, but only enzymatically cleaved fragments of larger peptides presented as peptide MHC (pmhc) complexes

CD4 or CD8: Most mature T cells express CD4 or CD8 molecules, they function as important co-receptors in association with the TCR By binding to invariant portions of the MHC I (CD8) or MHC II (CD4), they serve to increase the interaction of the MHC-bound antigenic fragment and the TCR

The structures of CD4 and CD8 Figure 3-10

CD8 binds MHC class I CD4 binds MHC class II Most mature T cells are either CD4+ or CD8+. CD8 T cells kill cells infected with intracellular pathogens or tumor cells while CD4 T cells regulate (activate or suppress) other immune cells function.

Both: TCR Vs Immunoglobulin Bind antigen Have Variable region and Constant region Have a binding site that is a heterodimer (composed of 2 different chains) TCRs act only as receptors Igs act as receptors and effector molecules (soluble antigen-binding molecules)

Similarity between TCR and Ig

TCR Structure The TCR is a member of the immunoglobulin supergene family and is composed of two polypeptide chains; a light α or chain and a heavy β or chain Each polypeptide chain of the heterodimer pair contains a variable and a constant region domain The Vα and V regions are encoded by V and J gene clusters The Vβ and V regions are encoded by V, D, and J gene clusters The D gene cluster provides an additional source of variation

Figure 3-7

and TCR gene loci (germline configuration)

The gene clusters undergo DNA rearrangement, similar to that already described for immunoglobulin genes, to synthesize αβ dimers or dimers As with immunoglobulins, the constant domain of the α and β or and chains are encode by constant region genes (Cα and Cβ or C or C ) T cell receptors do not undergo any subsequent changes equivalent to isotype switch, and somatic hypermutation, important to generating diversity of immunoglobulins.

As might be imagined, in the random process of generating diversity, a variety of TCR specificities would be generated for peptides that one may never encounter during his lifetime Three distinct categories of TCR specificities can be identified: - Those that recognize peptides that will never be encountered - Those that recognize peptides produced by potential pathogens or peptides of foreign origin - Those that recognize peptides that are produced by cells of self

TcR gene rearrangement by SOMATIC RECOMBINATION Germline TcR V n V 2 V 1 J C Rearranged TcR 1 transcript Spliced TcR mrna Rearrangement very similar to the IgL chains

TcR gene rearrangement RESCUE PATHWAY There is only a 1:3 chance of the join between the V and J region being in frame V n+1 V n V 2 V 1 J C chain tries for a second time to make a productive join using new V and J elements Productively rearranged TcR 1 transcript

TcR gene rearrangement SOMATIC RECOMBINATION L & V x52 D 1 J C 1 D 2 J C 2 Germline TcR D-J Joining V-DJ joining Rearranged TcR 1 transcript C-VDJ joining Spliced TcR mrna

TcR gene rearrangement RESCUE PATHWAY There is a 1:3 chance of productive D-J rearrangement and a 1:3 chance of productive V D-J rearrangement (i.e only a 1:9 chance of a productive chain rearrangement) V D 1 J C 1 D 2 J C 2 D-J Joining Germline TcR V-DJ joining 2 nd chance at V-DJ joining Need to remove non productive rearrangement Use (DJC) 2 elements

TCR gene rearrangements occur in the thymus n=70-80 n=52 The same RSS and the same enzymes are used to rearrange both the TCR genes and the Ig genes. P and N nucleotides are added at the junctions between rearranged segments

V TCR Chain D region can be read in all frames D GTACTGCAGATT J J starts with ATT No additions: J is in frame V V V D J GTACCTGCAGATT D J GTACCTGCAGGATT D J GTACCTGCAGGCATT One addition and J is out of frame; the ATT start for J is lost (as are all appropriate downstream codons) Two addition and J is out of frame; the ATT start for J is lost (as are all appropriate downstream codons) Three addition and J is in frame; the ATT start for J is is present (as are all appropriate downstream codons)

-chain locus is first to be rearranged

Two chances for productive (=correct reading frame) rearrangement: chain

-chain rearrangement

Multiple rounds of -chain rearrangement can rescue nonproductive TCR

TCR gene rearrangement generates the TCR repertoire Pre-TCR complex stops further gene rearrangement at locus, and induces thymocyte proliferation Finally TCR+ DP cells are made

Germline configuration of and loci TCR D, J and C exons are encoded in the intron between the the Vs and the Js of the TCR locus. The V segments for TCR (4 known) are mixed in with the V segments of the TCR

Figure 3-8 part 2 of 2 Most T cells do not express CD4 or CD8. They are thought to be: First line of defense? Bridge between innate and adaptive responses?

Signals through the TCR and the pre-tcr compete to determine thymocyte lineage

Generation of diversity in the TcR COMBINATORIAL DIVERSITY Multiple germline segments In the human TcR Variable (V) segments: ~70, 52 Diversity (D) segments: 0, 2 Joining (J) segments: 61, 13 The need to pair and chains to form a binding site doubles the potential for diversity JUNCTIONAL DIVERSITY Addition of non-template encoded (N) and palindromic (P) nucleotides at imprecise joints made between V-D-J elements SOMATIC MUTATION IS NOT USED TO GENERATE DIVERSITY IN TcR

The Generation of Diversity (GOD)

T Cell Development T cell precursors migrate from the bone marrow to enter the thymus as thymocytes, they express neither αβtcr nor CD4 or CD8 and are called double negative (DN) cells DN cells proliferate in the subcapsular region of the thymus and differentiate to express low levels of newly generated αβtcr, both CD4 and CD8, and are called double positive (DP) cells

DP cells move inward to the deeper portion of the thymus, where they are fated to die within 3-4 days, unless their TCRs recognize an MHC class I or class II molecules on thymic dendritic cells. This process is called positive selection Although the mechanism of positive selection is yet unclear, partial recognition of class II by CD4 or class I by CD8 molecules must occur T cells that recognize self MHC molecules survive

A DP thymocyte with a TCR that engage MHC class I may become a CD8 + T cell and a DP thymocyte that recognizes MHC class II may become a CD4 + T cell Class I and class II molecules are not displayed on cell surface unless they are loaded with a peptide Only molecules of self origin are available on thymic APCs, and these are presented to the DP thymocyte in the deep or medullary area of the thymus

CD4+CD8+ DP cells: To be CD4 or CD8?

Thymocytes that show strong interaction with MHC molecules or pmhc complexes undergo apoptosis, a process known as negative selection Thymocytes that survive both positive and negative selection migrate from the thymus to populate lymphoid tissues and organs as T cells

Figure 5-3 part 2 of 2

Each thymocyte maturation stage occurs at a distinct location of the thymus Young adult:~5x10 7 thymocytes produced/day ~1.5x10 6 mature cells leave/day

Differentiation Figure 5-3 part 1 of 2 DN (CD4-CD8-) and DP (CD4+CD8+) Immature thymocytes are here More mature SP (CD4+CD8-or CD8+CD4-) thymocytes are here

Positive Selection Positive selection selects T cells that recognize peptides on self MHC This is to assure that mature T cells can respond to antigen-presented on self MHC. Self MHC I and II harboring self peptides on thymic epithelial cells recognize and activate TCRs on some DP thymocytes. DP thymocytes should receive this signal within 3-4 days to survive, otherwise they undergo apoptosis.

Negative Selection Negative selection eliminates T cells with TCRs that bind too strongly to self antigen/mhc complex (autoreactive cells are removed by this process) Dendritic cells and macrophages in corticomedullary junction mediate it. Negative selection cannot eliminate T cells whose receptors are specific for self peptides that are present outside of the thymus These cells enter circulation, but soon to be rendered anergic or unresponsive by other mechanims.

Does receptor occupancy explain positive and negative selection? Low occupancy Survival High occupancy Negative selection

Does the TIME of receptor occupancy explain positive and negative selection? Short occupancy Survival Long occupancy Negative selection Short signaling Long signaling

Stage of maturation can be distinguished by the expression of specific surface molecules DN DP SP

Types of T cells Conventional: Uses TCR Helper (CD4+) and cytotoxic (CD8+) T cells More abundant and highly specific Restricted by classical MHC (I and II) molecules Non-conventional: Uses TCR Primitive with broad specificity Restricted by non-classical molecules

CD4+ T cells T cells with CD4 marker (glycoprotein) represent 70% of T cells in the periphery Named T helper cells Play central role in modulating cellular immunity via secretion of cytokines that mediate: B cell activation Immunoglobulin secretion (quality) Macrophage and dendritic cell activation Cellular chemotaxis and inflammation Two subsets; Th1 and Th2 cells

Th1 and Th2 cells CD4+ T helper cells can be classified into two types based on their cytokine profiles: T helper cell type 1 (Th1) and T helper cell type 2 (Th2). Cytokine profile is influenced by several factors: Nature and dose of antigen Route of administration Type of antigen presenting cell/ costimulation Genetic background The cytokine profile determines the effector function of the helper cell

Differentiation of naive CD4 T cells into different subclasses

The nature and amount of ligand determine CD4 T cell functional phenotype

T Helper (CD4 + ) Subsets Antigen APC FasL Suicide Fas Th1 DR4 Pro-Inflammatory Cytokines Th1 response Cellular Immunity DTH IL-12 IFN- IL-2 LT Th0 IL-4 Th2 IL-4 IL-10 IL-13 IL-5 IL-6 Anti- Inflammatory Cytokines Th2 response, Humoral Immunity and Acute Hypersensitivity

Differences between Th1 and Th2 cells Th1 cell Produces type 1 cytokines IL-2, IFN-, TNF-α, TNF-β Activates macrophages and DCs for intracellular killing of pathogens Mediates CMI Th2 cell Produces type 2 cytokines IL-4, IL-5, IL-10, IL-13 Provides help to B cells in antibody response Mediates allergy and immunity to extracellular pathogens, including parasites

Cytotoxic T cells T cells that express CD8 molecule on their surface and they represent 30% of T cells in the periphery Destroy cells infected by intracellular pathogens and cancer cells Class I MHC molecules (nucleated body cells) expose foreign proteins TC cell releases perforin and granzymes, proteins that form pores in the target cell membrane; causing cell lysis and/or apoptosis

Effector molecules of T cell subsets

Regulation of the Immune Response How does the immune system prevent self reactivity while maintaining reactivity to invaders/non-self? Clonal deletion/inactivation of autoreactive cells Regulatory T cells keep potentially pathogenic self reactive T cells in check through suppressive mechanisms

New T cell phenotypes Regulatory T cells Naturally occurring (CD25 positive) Induced (IL-10 and TGF-β) Some NK T cells Suppressor T cells Th17 cells: produce IL-17, role in acute inflammation, suppress Th2.

Bettelli et al., Nature 2008

Th17 and Treg

Regulatory T Cells (Treg) CD3 +, TcR +, CD4 +, CD25 + Function Suppress the activity of effector Th and Tc cells Inhibition is antigen specific and MHC restricted Inhibition depends on cell:cell contact Appear to function in autoimmunity Development Develop in the bone marrow from DP cells Arise as a result of strong binding to self MHC and self Ag during negative selection. Alternative to cell death or anergy Development and maintenance are dependent upon B7

What are the requirements for Treg development? TCR engagement in the thymus (high affinity but not so high as negative selection) Higher percentage of thymocytes with high affinity TCR in context of auto antigen develop into regs T cells of appropriate affinity are instructed to become Tregs Selective sparing of pre-committed cells from negative selection or promotion of Treg lineage development? higher percentage, but not higher absolute number Preferential elimination of non-regulatory T cells rather than increased production of Tregs TCR engagement serves as survival or expansion signal of Tregs pre-committed to that lineage.

GITR CD4 Foxp3 T reg CD25 The T reg cell phenotype CTLA-4 CD4 Co-receptor for TCR recognition of MHC II/Ag CD25 IL-2R IL-2R component, confers high affinity binding to IL-2R Key T R growth factor CTLA-4 cytotoxic T lymphocyte Ag-4 Binds to B7s (CD80/86) on APC, acts as co-stimulatory molecule for T R (blocking CTLA-4 inhibits T R ) GITR glucocorticoid induced TNF related protein Ligation inhibits TR function (agonist inhibit T R, blocking augments T R ) FoxP3 Forkhead /winged-helix TF critical for T R activity and development Unlike surface markers / receptors, T E do not express FoxP3

Modulation of immune responses by T reg cells T reg cells are crucial for the induction and maintenance of peripheral tolerance to self-antigens T reg cells can also suppress immune responses to 1. Tumor antigens 2. Alloantigen 3. Allergens 4. Microbial antigens Tumor Clearance T reg Microbial Immunity Self-tolerance Transplantation Autoimmunity tolerance Transplant rejection Graft-versus Tumor Self-tolerance Progression host disease Microbial Immunocompetence Allergy Persistence T eff Sheng Cai