MICR439 Exam #3 PDF

Lecture 17: Self-MHC Restriction of TCR In 1974, Zinkelnagel and Doherty showed that antigen (ag) recognition by t cells is not specific for the antigen alone - rather, it is specific for the ag associated with the MHC molecule. T cells recognize the antigen only when it is p...

Lecture 17: Self-MHC Restriction of TCR In 1974, Zinkelnagel and Doherty showed that antigen (ag) recognition by t cells is not specific for the antigen alone - rather, it is specific for the ag associated with the MHC molecule. T cells recognize the antigen only when it is presented by the self-MHC molecule. ○ Self-MHC restriction: phenomenon differentiates recognition of the antigen by t and b cells. ○ Doherty and Zinkelnagel were awarded the Nobel Prize for their work on the topic in 1996 Model for MHC Restriction of TCR Two models were proposed for the MHC restriction: ○ The dual receptor model: two separate TCR, one for antigen and one for the MHC molecule. ○ The altered-self model: single TCR recognizes an alteration in self-MHC molecules induced because of association with the foreign antigen. Kappler and Marrack showed that specificity for both antigen and MHC molecules reside in a single receptor. At present, the altered-self model is an accepted model for the MHC restriction of TCR. T Cell Receptor (TCR) Responsible for antigen recognition by T cells. Expressed on most thymocytes and all mature T cells. Resembles the B cell receptor (BCR) in mant ways. ○ Like b cells, t cells possess an antigen-specific and clonally-restricted receptor. ○ Genomic organization of TCR gene families and the mechanism of diversity generation in TCR chains are similar to BCR chains. ○ Similar to IG-alpha and Ig-beta of BCR, TCR is associated with a signal-tranducing complex called CD3. TCR also differs from BCR (Ig) in many ways. ○ The spectrum of antigens recognized by TCR is much more restricted than BCR. ○ TCR is membrane bound and in contrast to BCR (Ig), it does not occur in a soluble form, therefore, T cells do not secrete their TCRs. ○ In contrast to BCR, most TCRs except a few, are not specific to antigen alone - rather, they are specific to antigen combined with MHC molecule. ○ A small percentage of T cells recognize lipid or unprocessed antigens that may or may not be associated with MHC-related molecules. TCR Structure There are 2 types of T cell receptors: alpha-beta TCR and gamma-delta TCR.  These TCRs are mutually exclusive, therefore, there is no expressive combination. They develop independently of one another. The alpha-beta T cells function in adaptive immunity, but the gamma-delta T cells participate in innate immunity. TCRs are transmembrane and insoluble proteins, and are members of the Ig superfamily. ○ There are roughly 10,000 - 30,000 TCR molecules on the surface of a T cell. Unlike mIg, TCR is a heterodimeric glucoprotein with a single antigen binding site and resembles an Ig FAB fragment. Amino acids in antigen binding sites establish contacts with the antigenic peptide and MHC molecules to which it is bound. Unlike Ig molecules which can undergo isotype switching, a TCR’s ‘C’ region is fixed for the life of a given T cell clone. Short connecting sequences located between the TCR ‘C’ domain and transmembrane domain is analogous to the Ig hinge. Crystal Structure V domains in both alpha and beta chains contain 4 complimentary determining (CDR), or hypervariable (HV) regions: CDR1, CDR2, CDR3, and HV4 - all of which are equivalent to present in Ig light and heavy chains. The various CDRs are involved in peptide-MCH (pMHC) recognition. HV4 is a region of amino acid variablity, but it does not contact the peptide within the pMHC complex directly. The V domains of the TCR gamma and delta chains also contain CDR1, CDR2, CDR3, and HV4 regions. The TCR-CD3 Complex Immunoreceptor tyrosine-based activation motifs (ITAMs) interact with tyrosine kindases and play important role in signal transduction. The cytoplasmic tails of TCR chains are too short for signal transduction Like Ig-alpha and Ig-beta chains in B cells, additional proteins (CD3 dimers) are responsible for signaling through TCR after antigen interaction. ○ CD3 dimers do not affect the interaction with the antigen. The CD3 complex contains 3 heterodimeric proteins made up of variable combinations of 5 ITAM-containing invariant polypeptide chains which associate to form 3 dimers. They include the following: ○ Epsilon ○ Delta ○ Zeta ○ Eta ○ Gamma TCR non-covalently associated with CD3, forming a TCR-CD3 complex, or commonly known as the TCR complex.  The CD3 complex has 2 major functions: ○ The CD3 chains are required for intracellular signaling. Upon engagement of TCR by pMHC, tyrosine residues in the CD3 ITAMs are phosphorylated by an intracellular signaling kinase known as Lck. Additional signaling kinases are then recruited to propagate the signaling cascade. ○ CD3 expression is required for the surface expression of alpha-beta or gamma-delta TCR. In the ER, TCR physically associated with the CD3 complex, and finally transports to the T cell surface. CD4 & CD8 Co-Receptors The TCR binds to the antigen-MHC complex. Additional accessory membrane molecules also play an important role in antigen recognition and T cell singal transduction/activation. ○ The most important of these are CD4 and CD8 molecules. These are considered co-receptors. ○ Despite their equivalent functions, both CD4 and CD8 proteins show little similarity in either structure or amino acid sequence. Some intraepithelial T cells express CD8-alpha-alpha homodimers. Mature alpha-beta T cells are either CD4+ or CD8+ cells, and in humans, about ⅔ are CD4+ and ⅓ are CD8+. Most mature gamma-delta T cells express neither CD4 not CD8; however, some gamma-delta T cells in the gut are CD8+. CD4 and CD8 are referred to as co-receptors. This is due to one of the proteins co-localizing with the TCR, binding to the same MHC molecule on the APC/target cell which is engaged by the particular TCR. CD4 and CD8 molecules bind to conserved regions (outside of the peptide binding groove) of MHC class I & class II molecules. Co-receptor binding does not depend on the identity of the antigenic peptide by TCR. CD4 +ve T cells are largely helper T cells (Th). CD8 +ve T cells are largely cytotoxic T cells (CTLs). CD4 and CD8 have 2 main functions: ○ Stabilization of the TCR-pMHC binding by the interaction of CD4 with MHC class II and CD8 with MHC class I. ○ Recruitment of Lck to the TCR-CD3 complex. Although neither CD4 nor CD8 is absolutely required for the initial engagement of TCR-alpha-beta by pMHC, their binding to the MHC molecules greatly enhance TCR-pMHC binding. CD4 or CD8 binding to MHC molecules brings Lck into close proximity of CD3 chains and Lck then phosphorylates ITAMs and leads to T cell activation. Lecture 18 TCR Genes TCR-alpha, -beta, -gamma, & -delta polypeptide chains are encoded by the TCRA, TCRB, TCRG, and TCRD loci. ○ Note that in both mouse and human, TCRA and TCRD loci are present on chromosome 14 and TRCD locus is nested withing the TCRA locus. ○ Reference table 8-1 on slide 3 of lecture 18. Similar to Ig, each TCR chain has a variable (V) and a constant (C) domain, encoded by V and C exons. Similar to the Ig heavy chain, the TCR V exon is made up of small, V, D, and J gene segments in the TCRB and TCRD loci which are assembled at the DNA level by V(D)J recombination. ○ Analogous to the Ig light chain, the TCRA and TCRG loci contain V and J segments; however, there is no D segment. Only 1 C-alpha exon is present in the TCRA in both mice and humans. 2 C-beta exons in TCRB are present in both mice and humans; however, they are not functionally different as in T cells. There is no mechanism analogous to isotype switching in B cells. 2 C-gamma exons in TCRG in humans. 3 functional C-gamma exons in TCRG in mice: C-gamma-1, -2, & -4. C-gamma-3 is a pseudogene. 1 C-delta exon in TCRD is present in both mice and humans. Although TCRD has many similarities to the Ig heavy chain, it also exhibits some differences, including: ○ In both mice and humans, the TCRD is nested within the TCRA locus. This presents the expression of both TCRD and TCRA on the same T cell. ○ In addition to the use of one D-delta segment to create a VDJ exon, multiple D-delta gene segments can be used in tandem to create a VDDJ or VDDDJ exon. These mutations dramatically enhance the junctional diversity found in the TCR-delta chains. Order of Gene Rearrangement TCR genes are in the germline configuration when T cell progenitors leave the bone marrow and enter the thymus. ○ In the thymus, an immature thymocyte rearranges its TCR genes and becomes either an alpha-beta or gamma-delta T cell; depending on the signals it receives both through its TCR and from the local environment. TCRA/TCRB Rearrangement Irrevocable commitment of a thymocyte to TCR-alpha/beta lineage depends upon V(D)J recombination, resulting in a function TCR-beta gene. ○ TCRB locus will rearrange prior to the TCRA locus.  ○ Simultaneously in the TCRB on the maternal and paternal chromosomes, V(D)J recombination first joins a D-beta gene segment to a J-beta segment, and then a V-beta segment to the D-beta-J-beta segments. The functionality of the completed TCR-beta gene is tested via formation of the pre-TCR signaling complex. Successful intracellular signaling through pre-TCR indicated that the candidate TCR-beta protein is functional, and TCR-beta rearrangement is successful. The assembly of a functional TCR-beta gene on one chromosome suppresses its rearrangement on other chromosomes. This is referred to as allelic exclusion. ○ In a thymocyte, if TCR-beta is unsuccessful on both chromosomes, then the cell will die through apoptosis instead of rearrangeing to its TCR-alpha gene or becoming a gamma-delta T cell. If a functional TCR-beta gene is produced, then V and J gene recombination of the TCRA starts on both chromosomes. If a productive TCR-alpha chain gene rearrangement takes place on either chromosome, TCR-alpha chain can combine with TCR-beta chain to produce a functional surface TCR-alpha-beta. ○ If TCRA rearrangement fails on both chromosomes, the cell will die by apoptosis. TCRG and TCRD Rearrangement In thymocytes which finally become gamma-delta T cells, rearrangement of genes starts simultaneously but independently in TCRG and TCRD loci on both chromosomes. In these cells although TCRA lous physically surrounds TCRD, TCRA does not undergo rearrangement. VJ joining of TCR-gamma gene segments occurs in usual manner. TCR-delta D gene segments can be combined with each other to form tandem D-D or D-D-D units. The D, D-D, or D-D-D entities are joined to J-delta and then finally to V-delta to complete the V exon. V(D)J Recombination The same RAG recombinases and DNA repair enzymes that executes V(D)J recombination in the Ig loci act on TCR loci in thymocytes to produce functional TCR genes. Despite the duplication of V(D)J recombination apparatus in B and T cells, the Ig genes are not rearranged in developing T cells and the TCR genes in developing B cells. Similar to BCR genes, the TCR genes are flanked by the same 12-RSS and 23-RSS sequences. In TCRB locus, RAG recombinases follow the same 12/23 rule to juxtapose only rhose RSSs that are not of hte same type. D segments in the TCRB and TCRD loci are flanked by 5’ side by 12-RSS and by a 23-RSS on the 3’ side.  This in TCRD may falilitate the tandem joining of D segments prior to addition of the J-delta segment, which happens less frequently in the TCRB locus. TCR Gene Transcription and Protein Assembly Unlike Igh genes, which contain separate exons for mIg or sIg, the TCR genes have only an exon encoding a transmembrane domain. After translation into the ER, disulfide bonding links TCR alpha and beta chains, or TCR gamma or delta chains, which lateer associate with CD3 molecules and appear on the cell surface as the TCR complex. TCR Diversity The isotype switching and somatic hypermutation mechanisms which create diversity in antigen activated B cells do not operate in T cells. Diversity in T cell repertoire is established completely by mechanisms operating before antigenic stimulation. These mechanisms include: ○ Multiplicity of germline segments. ○ Combinatorial diversity. ○ Junctional diversity. ○ The alpha-beta or gamma-delta chain pairing. Multiplicity and Combinationatorial Joining of Germline Gene Segments In mice and humans, the number of different V and D gene segments in TCR loci are lower than corresponding genes in Ig loci, but number of TCRA J segments is greater than the number of Ig J segments. Overall, the contribution of this source of diversity to the maximum theoretical TCR repertoire is less than for the Ig repertoire. The actual diversity derived from combinatorial sources is more limited than the theoretical diversity. Fortunately, what is lost in combinatorial diversity is compensated for by variable D segment inclusion (D, D-D, or D-D-D) in TCR-beta (less) and TCR-delta loci (more frequent). Although the Ig loci contains higher number of D gene segments, only one D segment can join to an Ig J segment. Reference slide 21 in the lecture for more. Junctional Diversity Similar to B cells, both P nucleotides and N nucleotides can be added to VD and DJ joints in TCR chains and give rise to amino acids that are not encoded in the germline. Because more than one D-delta segment may be included in tandem in a TCR-delta chain, many more opportunities for P and N nucleotides addition occur at each D-D or D-J joint. Junctional diversity contributes to billions of possible TCR-delta chains to the TCR repertoire. Chain Pairing The random pairing of TCR-alpha and -beta chains also contribute to TCR repertoire. The total number of possible alpha-beta heterodimers approaches 10^18 in humans and 10^15 in mice. These numbers compare to the 10^11 specificities estimated for the Ig repertoire. Due to death of T cell clones before they ever meet their antigen, as well as the processes of central and peripheral tolerance; a human and mouse have roughly 2x10^7 and 2x10^6 functional alpha-beta T cell clones, respectively. TCR-Antigen Interaction The interaction between a TCR-alpha/-beta and its pMHC peptope underlies the fundamental aspects of the cell mediated adaptive immune response. ○ The strength of binding between a thymocyte’s TCR and various pMHCs encountered in the thymus determines positive or negative selection of thymocytes. ○ The strength of binding between a mature alpha/beta T cell’s TCR and pMHC presented by APC in the periphery determines whether the T cell will: Be activated to proliferate and differentiate into effector cells. Become anergic or non-responsive. Both the TCR alpha/beta chains are usually involved in binding to oth the MHC molecule and the peptide, and this binding occurs virtually simultaneously. Reference slide 26 of the lecture for more. The area of contact between TCR and pMHC is relatively small, and only a few peptide residues generally make contact with a TCR. The binding affinity of a TCR is significantly lower than that of an antibody for its antigen. ○ Relatively modest affinity of TCR has many implications: The initial contact between T cells and APCs/target cells is established not by TCR-pMHC interaction but rather by binding of complementary pairs of adhesion molecules. The contact between CD4/CD8 and MHC molecules is also important in holding cells together. TCR can bind with varying affinities to a broad range of pMHCs and such promiscuity facilitates thymic selection. Lecture 19 Comparison of B and T Cell Devleopment B and T lymphocytes are derived from the same hematopoietic progenitors, but their development differs in many ways: ○ Thymus is required for the generation of T cells, but not for the B cells. B cells develop in the bone marrow.  ○ Naive B cells are produced throughout the life. In contrast, thymus involutes around puberty, and so, the production of new naive T cells is sharply reduced after puberty. ○ MHC molecules are involved in the establishment of central tolerance of T cells, but not for B cells. TCR must recognize the host’s MHC molecules. ○ TCR, unlike BCR, is fixed for life of the T cell and cannot undergo the somatic hypermutation like BCR in B cells. ○ Most B cells result from a single developmental program, but functional T cells can result from many different pathways, including the following: Alpha/beta & gamma/delta T cells. Th/Tc cells. T regulatory cells. Thymus A flat, bilobed structure above the heart which is a site of T cell development and maturation. ○ T cells which develop in the thymus are called thymocytes. In ‘nude mice’ the thymus fails to develop. In ‘nude mutants’ the mouse strain has a defect in the development of thymic stromal cells, and so lacks all mature T cells. Nude mice show absence of cell mediated immunity and as a result, increase in infectious diseases. Aging is accompanied by a decline in thymic function and this plays a role in decline in immune function during aging. DiGeorge syndrome is a primary immunodeficiency (PID) in humans, in which thymus development is impaired or it does not develop at all. ○ This mat lead to abnormal T cell numbers, but not always. Colonization of Thymus The fetal thymus is ‘colonized’ or ‘seeded’ with hematopoietic progenitors which then proliferate and mature in the thymus into functional naive T cells. T cells are derived from hematopoietic cells are derived from HSCs which are present in fetal liver in a fetus and bine marrow in adolescents/adult individuals. Proliferating HSCs differentiate into MPPs. they further differentiate into CMP, CLP, & MCP; leaving bone marrow or liver, entering into the blood circulation. Circulating CLPs give rise to a slightly more differentiated progenitor called the NK/T precursor, which can generate NK and T cells, but NOT B cells. Upon entering the thymus, a NK/T cell precursor differentiates into alpha/beta or gamma/delta T cells, lymphoid DCs, or NK/NKT cells based on the cytokines and stromal cell ligands it encounters. The fetal thymus is colonized by NK/T precursors in distinct waves that occurs both before or after birth.  Earlier prenatal waves give rise only to gamma-delta thymocytes, but subsequent waves of NK/T precursors just before birth and shortly thereafter give rise to both alpha-beta and gamma-delta thymocytes. After birth, NK/T precursors destined to become T cells are more biased towards alpha-beta T cell lineage such that gamma-delta T cells become a minor population. In addition, bone marrow also becomes the dominant site of NK/T precursors generation. Repertoire of T cell specificities in neonates is significantly less diverse than in older individuals because of less active terminal deoxytransferase (TdT) enzymes responsible for junctional diversity. Thymocyte Maturation in the Thymus Thymocytes at different developmental stages are morphologically very similar so they are distinguished based on patterns of surface marker expression, or by their TCR gene rearrangement status. Using these criteria, thymocytes are divided into 3 phases: ○ Double negative phase (DN, DN1-DN4): expresses neither CD4 nor CD8. ○ Double positive (DP) phase: expresses both CD4 and CD8. ○ Single positive (SP) phase: expresses either CD4 or CD8, but not both. Once SP thymocytes emerge from thymus and enter the circulation and secondary lymphoid organs, they are considered mature naive CD4+ or CD8+ peripheral T cells. Several selection processes occur during these transitions. The Thymic Environment The development of thymovices through the DN, DP, and SP phases is totally dependent on the stromal cells. The most important stromal cells are: ○ Cortical thymic epithelial cells (cTECS). ○ Medullary thymic epithelial cells (mTECS). ○ Thymic DCs. ○ Thymic fibroblasts. Thymic DCs, cTECS, and mTECS are vital for the establishment of T cell central tolerance during the DP phase. Expression of Notch1 on thymocytes directs them to T cell lineage and thus it is a key protein in T-lineage specification. cTECs and mTECs express cell surface ligands for Notch1. Once Notch1 binds to its ligand, cytoplasmic domain of Notch1 interacts with transcription factors (GATA-3) to promote T cell development and suppress B cell development. Continues Notch1 signaling is required to sustain the survival of thymocytes until they pass through the DN stage.  Thymic fibroblasts secrete ECM components such as collagen which aid in concentrating cytokines crucial for thymocyte’s development and in controlling thymocytes adhesion to stromal cells. The DN Phase DN1 subset: ○ TCR genes in germline configuration and resides in the thymic cortex. ○ cTECs supply stem cell factor (SCF) that binds to c-kit on DN1 cells and delivers a survival signal. ○ Transcription factor GATA-3 is vital for the generation of DN1. DN2 subset: ○ Murine DN2 are known as Pro-T cells (progenitor T cells). ○ Present primarily in the outer cortex and TCR genes remain in germline configuration. ○ Commence expression of the CD3 chains. ○ Under the influence of IL-7 and SCF, DN2 thymocytes start to proliferate rapidly. DN3 Subset DN3 cells stop proliferation and remain in the outer cortex. DN3 stage is critical in T cell development as 5 key events occur. ○ DN3 thymocytes become restricted to T cell lineage and generate mature alpha/beta and gamma/delta T cells. ○ TCRG, TCRD, and TCRB loci commence V(D)J recombination with concomitant upregulation of RAG and TdT. ○ DN3 cells destined to become alpha/beta T cells express a functional ‘Pre-TCR complex’ that allows them to determine if a functional TCR-beta chain has been produced. ○ Successful rearrangement at the TCR-beta locus induces the cessation of further rearrangement at TCRG and TCRD loci in these cells. ○ These DN3 thymocytes become early ‘pre-t cells’ that are fully committed to the alpha/beta T cell lineage and express a diverse repertoire of TCR-beta chains. In DN3, thymocytes that generate alpha/beta T cells, the TCR-beta locus is the first to undergo VDJ recombination. DN3 thymocytes that have successfully arranged their TCR-beta chains undergo ‘beta-selection’, and cells that survive beta selection are said to have passed the pre-TCR checkpoint. ○ This results in the proliferation and differentiation of DN3 cells. This process incolces the expression of a glycoprotein known as the ‘pre-t-alpha chain’ on the DN3 cells which act as a surrogate for the real TCR-alpha chain, which has yet to rearrange. Pre-TCR-alpha chain assembles with a successfully arranged and translated beta chain as well as CD3 complex proteins.  This precursor TCR/CD3 complex is known as the ‘pre-TCR’ and acts as a sensor by initiating signal transduction. If the TCR-beta rearrangement on both chromosomes has been unsuccessful, the cell neither attempts to rearrange its TCRA genes nor becomes a gamma-delta cell; instead, it dies by apoptosis. Only 10% of DN3 thymocytes successfully rearrange their TCR-beta genes, are beta-selected, and enter the cell cycle. DN4 Thymocytes Murine DN4 cells are also called ‘late pre-T cells’ are slightly larger in size than DN3 cells. DN4 cells are concentrated in the subcapsular region of the thymic cortex. DN4 cells contain a functionally rearranged TCR-beta gene, where they downregulate their expression of CD25, RAG, and TdT. ○ These cells start expressing very low level of CD4 and CD8. The Double Positive (DP) Phase In both humans and mice, the DP phase of alpha-beta T cells development is dominated by the thymic selection processes that shape the mature alpha-beta T cell repertoire. CD4 and CD8 expression levels are upregulated and they play important roles in directing thymocyte development. DP thymocytes move towards the thymic medulla. TCR-alpha/beta pool expansion and TCRA locus rearrangement: ○ DP thymocytes receive signals through Pre-TCR for proliferation. ○ V(D)J recombination in borth TCRA loci start - the TCRD loci is deleted. ○ Newly synthesized TCR-alpha chains combine with TCR-beta chains to form complete TCR-alpha/beta heterodimers. ○ TCRA rearrangement continues on both chromosomes until positive selection delivers a survival signal. A small population of DP thymocytes can also commit to NKT cells which play an important role in innate immunity and express a TCR that includes an invariant TCR-alpha chain (thus also called iNKT cells). Thymic Selection and Establishment of Central T Cell Tolerance Thymic selection shapes the TCR repertoire of DP thymocytes based on the affinity of TCRs for the MHC/peptides they encounter in the thymus. The establishment of central T cell tolerance requires that thymocytes within TCRs that recognize self antigen be eliminated before they leave the thymus. Selection process which includes both positive and negative selection events allows only self MHC-restrivted and nonself-reactive T cells (tolerant to self) to mature and leave the thymus. Finally, functionally distinct mature CD4+ and CD8+ subpopulations that exhibit class II and class I MHC restriction respectively exits the thymus. The Thymus as a Testing Ground for T Cells Failute to successfully rearrange TCR genes, to pass positive selection, or to pass negative selection results in cell death. Positive selection preserves the 1-2% of developing thymocytes whose TCRs recognize self pMHCs neither too strongly nor too weakly. Estimated 98% of all thymocytes never meet the selection criteria and die by apoptosis in the thymus. Positive Selection Thymic stromal cells, including epithelial cells, macrophages, and dendritic cells play essential roles in positive and negative selection. These cells express MHC class I and high levels of MHC class II. This takes place in the CORTEX of the thymus. ○ T cells which recognize self-MHC with low or intermediate affinity receive a ‘survival/protective’ signal from specialized APCs in the thymus, and are positively selected. ○ Positive selection ensures ‘self-MHC restriction.’ ○ Cells that fail positive selection are eliminated within thymus by apoptosis. ○ During positive selection, gene rearrangement may continue, but MUST stop following selection. Negative Selection Negative selection which is also called ‘central tolerance’ takes place in the medulla of the thymus. AIRE+: autoimmune regulator mTECs. In negative selection, T cells which demonstrate too high an affinity for self-MHC molecules alone or self-antigen presented by self-MHC by mTECs, thymic dendritic cells or macrophages are ‘deleted’ in the medulla. Negative selection ensures ‘self-tolerance.’ Somehow, the thymic APCs signal apoptosis in reactive cells. Selection of Single Positive (SP) CD4+ and CD8+ Cells Depending of specificity, double positive (DP) T cells downregulate either CD4 or CD8 to become single positive (SP) CD4+ or CD8+ T cells. Lineage commitment requires changes in genomic organziation and gene expression that results in: ○ Silencing of a coreceptor gene. ○ Expression of genes associated with a specific lineage function. The mechanism which regulates this is not fully understood, but is likely linked to the specificit of the TCR for either Class I or Class II MHC. Both CD4+ and CD8+ SP thymocytes loiter in the medulla of the thymus for a short time before they receive a final proliferation signal and expand their numbers.  Majority of CD4+ SP thymocytes differentiate into mature naïve T helper (Th) cells and CD8+ SP thymocytes usually differentiate into mature naïve cytotoxic T cells (Tc). These cells then exit the thymus into the blood and travel to secondary lymphoid organs, taking up the residence as fully functional mature CD4+ or CD8+ T cells. Maintenance of Self-Tolerance Negative selection of thymocytes can get rid of cells which express a high affinity for self-antigens. However, negative selection in thymus is not perfect because of 2 reasons: ○ Thymocytes which have low affinity to self-antigens do escape the negative selection. ○ Thymocytes have not browsed the right ‘tissue-specific’ antigen/MHC combination. The body has evolved many other mechanisms to avoid autoimmunity. ○ T regulatory cells negatively regulate immune responses. ○ Peripheral mechanisms of tolerance will be mediated through T cell anergy. Lecture 20 T Cell Activation Similar to B cells, the complete activation of naive T cells generally requires 3 different signals: ○ Engagement of the antigen receptor (TCR) by antigen. ○ Costimulation. ○ Receipt of cytokines. These signals differ slightly between B and T cell activation and between naive Th and Tc cells. Additional differences in the activation of effector and memory T cells also exist. Meeting of Naive T cells and DCs Activation of most naive cells happens in the paracortex of the lymph node (LN) where antigen loaded mature DCs congregate and naive T cells recirculate. Immature migratory and lymphoid resident DCs capture the materials form their microenvironment including foreign substances and pathogens. In case of infection and inflammation, pro-inflammatory cytokines and DAMPs/PAMPs induce that maturation of DCs. Maturing migratory DCs enter LN via an afferent lymphatic and settle in the paracortex surrounding the high endothelial venules (HEVs). Resident DCs are already present there. Mature DCs process their captures antigens and display antigenic peptides on MHC II via exogenous processing and on MHC I via cross presentation. If a DC is infected by a pathogen, intracellular antigens may also be processed via endogenous pathway and presented on MHC I or displayed on MHC II by autophagy.  Naive T helper and cytotoxic T cells circulate in the blood and throughout the secondary lymphoid tissues, such as LN. Mostly a naive T cell enters the nove via its HEVs and inspects the pMHCs displayed by mature DCs in the vicinity of these vessels. T cells ‘crawl’ slowly over the surface of a DC in a process facilitated by several adhesion molecule paits which hold the T cells and DC together for pMHC screening. pMHCs that are boud with sufficient affinity/avidity by TCR have the potential to activate the T cell. Signal 1 for T Cell Activation Signal 1 is delivered to T cells when specific pMHCs present on the DCs bind to multiple copies of a TCR expressed on a naive Th or Tc cell surface. TCR engagement by pMHC leads to a conformational change in CD3 chains which allow the phosphorylation of CD3 ITAMs by Lck kinase associated with CD4 and CD8. The additional intracellular signaling enzymes are then recruited to cytoplasmic tails of CD4 and CD8 and CD3 chains. ○ Together, these enzymes mediate a cascade of chemical reactions that lead to activation of many other enzymes. ○ When this activation cascade occurs for multiple TCRs, the TCR receives signal 1. Because of low affinity, a single pMHC engagement with TCR does not engage a single TCR long enough to achieve complete activation of a naive T cell. Similarly, transient interaction between a few pMHC-TCR pairs is also not sufficient. Sustained interaction between naive T cell and DC for many hours is needed for the proper and sufficient T cell activation. TCRs and pMHCs required for sustained signaling are gathered together by the formation of an immunological synapse between T cell and DC interface. ○ Both cells undergo rearrangement of their actin cytoskeletons and polarization. Immunological synapse results in the formation of 3 concentric rings, each containing various signaling adhesion and cytoskeletal molecules that cluster around TCR-pMHC pairs. ○ Inner ring: central supramolecular activation cluster (cSMAC) and it is composed of aggregated TCRs and costimulatory molecules. ○ Middle ring: peripheral supramolecular activation cluster (pSMAC) contains signaling adaptor talin and a large number of intefrins and other adhesion molecules. ○ Outer ring: distal supramolecular activation cluster (dSMAC) which mainly contains actin-based cytoskeletal structures and large proteins. TCR-Mediated Signaling Formation of an immunological synapse is not required for the initiation of TCR signaling, but sustained T cell activation is more effective if an IS forms. Signal 2 for T Cell Activation In most cases, the engagement of TCRs my pMHCs is not sufficient to fully activate a naive Th or Tc cell, and signal 2 in the form of costimulator signal is required. In Th cells, the receipt of signal 1 leads to upregulation of costimulatory molecule CD28 on T cell surface. CD28 molecule on T cell binds to its ligand on the surface of DC to convey the signal 2 to T cell. Initially, DC does not express optimal levels of B7. However, delivery of signal 1 to T cells and initial CD28 binding to B7 upregulation of the expression of CD40L on T cells. ○ Once CD40L on Th engages CD40 expressed by DC, the DC greatly increases its B7 expression and its binding to CD28 on Th cell, and as a result, vigorous signal 2 is delivered. ○ Delivery of signal 2 enhances the activating intracellular signaling induced by signal 1. Tc cells upregulate CD28, but most Tc cells do not express CD40L after receiving signal 1. Thus, Tc cells cannot induce a DC to initiate CD40 signaling and upregulates B7 expression. Instead, Tc cells rely on CD28 engagement resulting from interaction with a DC that already expressed B7 due to a previous interaction with antigen-activated Th cell. It is relieved that these DCs have been licensed for Tc activation. This licensing of DCs by Th cells is one form of T cell help provided by Th cells for Tc responses. ○ In oth Th and Tc cells, CD28 signaling lowers the T cell activation threshold necessary to activate new gene transcription and pushes it to proliferation and differentiation. In the absence of CD28 costimulation (signal 2), naive T cells are anergized instead of activated, and fial to respond to pMHC. This is a very important concept in T cell biology. Costimulation via CD28/B7 interaction has several molecular effects: ○ IL-2R expression is induced on T cell surface, allowing the cell to receive signal 3. ○ Th cells start to secrete large quantities of IL-2 and other important cytokines and chemokines. ○ The expression or upregulation of additional costimulatory and regulatory molecules is induced in both Th and Tc cells. ○ Intracellular signaling supporting T cell survival, proliferation, and metabolism is promoted. Negative Regulation of Signal 2 The potentially destructive power of T cells must be tightly regulated to ensure it is applied only when appropriate.  The TCR and costimulatory signaling pathways are negatively regulated at multiple steps. The 2 most important negative regulators of T cell activation are PD-1 (programmed death-1) and CTLA-4 (cytotoxic T lymphocyte associated molecule 4). PD-1 is expressed by T cells, B cells, and some DCs while CTLA-4 expression is exclusively restricted to T cells. PD-1 expression on a T cell is induced within hours of its activation, while expression of the PD-1 ligand on DC surface is induced by inflammatory cytokines. PD-1 and TCR engagement together transmits an inhibitory signal that shuts down early steps of the TCR signaling pathway. In contrast to PD-1, CTLA-4 is not expressed on the T cell surface until 1-2 days after T cell activation, giving adaptive response time to eliminate the threat before T cell activation in damped down. Although CD28 and CTLA-4 are structurally similar glycoproteins, they act antagonistically. CTLA-4 competes with CD28 for binding to the B7 costimulatory ligands. As CTLA-4 has a much higher affinity for B7 proteins than does CD28, CTLA-4 displaces CD28 and recruits inhibitory molecules to the TCR complex. ○ This is an example of feedback inhibition, a common regulatory feature of the immune system. CD28 and CTLA-4 CD28 is expressed on both resting/naive & activated T cells. CTLA-4 (CD152) is virtually undetectable on resting T cells & is expressed on activated T cells. Both CD28 & CTLA-4 are members of Ig superfamily. Two forms of B7: B7-1 (CD80) & B7-2 (CD86) (members of Ig superfamily). B7-1 and B7-2 have similar extracellular domains but differ markedly in their cytoplasmic domains. Both B7 molecules are constitutively expressed on dendritic cells. Both B7 molecules are induced on activated macrophages and activated B cells. Signal 3 for T Cell Activation A naïve Th or Tc cell that has received signals 1 and 2 upregulates the receptors (mainly IL-2R) which permit it to receive signal 3 in the form of cytokines (mainly IL-2), chemokines and growth factors. These assisting cytokines are referred to as ‘Signal 3’. Cytokines stimulate a cascade of intracellular signals that enhance both proliferation and/or survival of T cells. IL-2 is one of the best-known cytokines involved in T-cell activation & plays a key role in inducing optimal T-cell proliferation. IL-2 is produced by activated T cells & acts in an autocrine manner.  Signal 3 is also provided by other cytokines (produced by APCs, T cells, NK cells & others) known as ‘polarizing cytokines’. Although a Th cell on its own can make sufficient IL-2 to meet its requirement (autocrine IL-2), a Tc cell usually cannot produce enough IL-2 needed for its proliferation. Thus, another component of T cell help provided to Tc cells by Th cells is the production of IL-2 (and possibly other cytokines) necessary of Tc proliferation. A naïve T cell upon activation proliferate and generates daughter cells that differentiate into effector T cells. Effector T cells differ from naïve T cells in several important ways besides function, including tissue of residence, preferred APC, costimulatory requirements, duration of TCR signaling needed for activation, dominant metabolic pathways, rate of cell division, sensitivity to cell death mechanisms and life span. Lecture 21 T Helper (Th) Cell Differentiation When a naive Th cell is fully activated, it starts producing copious amounts of IL-2 and proliferates vigorously. The progeny generated are called Th0 cells. 48-72 hours after activation, these Th0 cells terminally differentiate into various subsets of resting effector cells. Th1, Th2, and Th17 cells are the most important subsets. ○ Other subsets include Th9, Th22, and follicular Th (fTh) cells. Th0 cells cna also generate ‘induced regulatory T cells’ (iTreg) which play important role in peripheral tolerance. Type of effector Th subset generated from Th0 cells is determined by: ○ Cytokines and other factors present in the immediate microenvironment. ○ The nature of the DC by which the naive T cells was activated. Different pathogens supply PAMPs that bind to different PRRs and cause the DC to mature into different subsets. These DCs secrete different panels of cytokines and deliver signals that direct Th cell differentiation such that Th effectors suited for eliminating the pathogen are produced. Some Th subsets secrete cytokines that facilitate effector functions specialized for killing intracellular pathogens. ○ However, some Th subsets promote effector functions designed to counter extracellular pathogens. Following their generation and differentiation in a secondary lymphoid tissue, most resting Th effectors migrate back to the site of inflammation or tissue containing the antigen. In this site, presentation of same antigen (which originally activated naive T cells) by an APC activates these Th effectors and causes them to secrete subset-specific panels of cytokines and mediates the effector function.  These differentiation paths are not fixed for the life of a T cell clone, and one type of effector can become another type if circumstances change and its transcription program shifts in response. Th1 Cells Intracellular pathogens such as viruses and intracullular bacteria trigger macrophages and DCs to produce IFN-gamma, IL-12, and !L-27. These cytokines cause activation of transcription factor STAT4 in Th0 cells which causes these cells to commit to Th1 subset. At the site of inflammation or in tissues Th1 cells are stimulated by antigen and activates transcription factor T-bet. ○ T-bet drives IFN-gamma production. ○ Th1 cells oppose Th2 cell differentiation. Th2 Cells Most extracellular pathogens do not induce IL-12 production by macrophages and DCs. ○ Instead, they stimulate an unknown cell type (might be a mast cell or NKT cell) to secrete IL-4. In the presence of IL-4, Th0 cells experience activation of STAT6, which drives the differentiation to Th2 effectors. At the site of inflammation or in tissues, Th2 cells are stimulated by antigen and activates transcription factor GATA-3 GATA-3 drives Th2 signature cytokines IL-4, IL-5, and IL-13 production. Th2 cells oppose Th1 cell differentiation. Cross Regulation of T Helper Subsets by Transcriptional Regulators Helper T cell subsets often cross-regulate each other. The cytokines they secrete typically enhance their own differentiation and expansion, inhibiting commitment to other helper T-cell lineage. ○ This effect is known as cross-regulation. This is particularly true of the Th1/Th2 pair, as well as the Th17/iTreg pair. IL-4 and IL-5 produced by Th2 cells suppress the expansion of Th1 population. IFN-gamma produced by Th1 cells inhibits the expansion of Th2 population. Th17 Cells Th17 effector cells counter infections of the skin and mucosae (particulatly in the lungs and intestine) that are initiated by certain species of extracellular bacteria and fungi. Th0 cells exposed to a combination of immunosuppressive cytokine TGC-beta plus pro-inflammatory cytokines IL-6 and/or IL-21 experience activation of transcription factors STAT3 and IRF4 which cause Th17 effector generation. IL-23 is required for the continued survival and terminal differentiation of Th17 cells into functional effectors. In the inflammatory site, antigen-stimulated Th17 effectors activate transcription factor ROR-gamma-t that drives the production of IL-17, IL-21, IL-22 and IL-26.  Both IFN-gamma (produced by Th1) and IL-4 (produced by Th2) suppresses Th17 differentiation. Th17 cells play an important role in autoimmune and/or autoinflammatory diseases in both humans and mice. Induced Regulatory T Cells (iTREG) Cells Th0 cells can differentiate into ‘induced regulatory T cells’ (iTreg) cells if exposed to TGF-beta in addition to IL-2. Exposure to these cytokines activates transcription factor STAT5. Regulatory T cells can shut down the functions of other effector T cell subsets, suppressing the adaptive response. Once differentiated, iTreg cells activate Foxp3 transcription factor which induces secretion of IL-10 and TGF-beta. ○ Both these cytokines then act on other effector T cells to curtail their responses. IL-6 and IL-21 are potent repressors of TGF-beta-driven Foxp3 expression. ○ Thus, Th0 cells exposed to TGF-beta are induced to become Th17 cells rather than iTreg if IL-6 and/or IL-21 is also present. ○ This balance between the generation of cells is fine-tuned by the surrounding environment. Activation of Th Effector Cells Localiztion: ○ After differentiation, Th effector cells may remain in the LN to provide T cell help to naive Tc cells in the paracortex and naive B cells in the primary follicles. ○ Alternatively, Th effector cells may leave LN and go to other tissues (under the skin and the mucosa) or the site of inflammation. ○ All the Th effector cells initially express CCR7. which permit the migration of the effector T cells from paracortex to the primary lymphoid follicles, where naive B cells are present. ○ As the response progresses, Th1, Th2, and Th17 cells express different panels of chemokine receptors, and thus exhibit differential trafficking patterns. Th1 cells move to site of inflammation. Th2 cells move to sites such as mucosae. Th17 cells move to SALT and MALT and to inflamed tissues. Interaction with APCs: ○ Effector Th cells encountering with APCs either in the LN or at the site of attack are activated essentially in the same way as naive T cells, but with some important differences: Effector Th cells express higher levels of adhesion molecules which facilitate rapid and more effective TCR triggering. ○ Thus, effector T cells are activated by significantly lower quantities of antigen/pMHC compared to naive cells.  For Th effector cells activation, far less costimulation by APC is required. Thus, these cells respond efficiently to pMHC presented by DCs, macrophages or B cells or by non-HSCs such as gut or skin epithelial cells. In general, B cells are the principal APCs presenting antigen to Th2 cells, whereas macrophages predominate as APCs in interaction with Th1 and Th17 cells. Differential Costimulatory Requirements: ○ While CD28-B7 is the major costimulatory mechanisms fornaïve T cell activation, effector T cells appear to require only low level of CD28-B7 costimulation for activation. ○ CD28 signaling downregulates the expression of chemokine receptors & this prevents the effector cells from migrating away from the site where Ag has been encountered. ○ Two supplementary costimulatory pairs important for effector T cells activation are OX40-OX40L & ICOS-ICOSL. ○ In Th1 responses, OX40 expressed on a Th1 cell surface binds to OX40 ligand (OX40L) expressed on APCs. ○ Similarly, inducible costimulatory (ICOS) molecule, which is upregulated on Th2 and TH17 cells only after activation, binds to ICOS ligand (ICOSL) expressed on APCs. ○ ICOS is rarely expressed by Th1 effectors. Lecture 22 Superantigens Bypass normal immune recognition requirement by non-specifically ligating TCR and MHC simultaneously. They are viral or bacterial proteins that bind simultaniously to the v-beta domain of the tCR and to the alpha-chain of MHC class II. This produces an activating singal that induces T-cell activation and proliferation; however, this does not bypass the need for costimulation. There are 2 types of superantigens: ○ Endogenous superantigens: cell-membrane proteins encoded by certain viruses that infect mammalian cells, such as minor lymphocyte stimulating (Mls) determinants from mouse mammary tumor virus (MTV). ○ Exogenous superantigens: soluble proteins secreted by bacteria, such as a variety of exotoxins secreted by gram POSITIVE bacteria, such as: Staphylococcal enterotoxins. TSS toxin (TSST1). Exofoliative-dermatitis toxin (ExFT). Mycoplasma-arthritidis toxin (MAS).  Streptoccal pytogenic exotoxins. ○ T cell activation by superantigens in polyclonal and results in overproduction of Th cell cytokines, leading to systemic toxicity. Th1 Effector Functions Supply help to Tc and D cells, providing cell mediated and humoral defense against intracellular pathogens. Plays an important role in delayed type hypersensitivity (DTH). Secrets a panel of cytokines dominated by IL-2, IFN-gamma, and lymphotoxin (LT) - sometimes called Th1 cytokines. IL-2 drives T and B cell proliferation and enhances reactive O2 species intermediates (ROI) production by macrophages. IFN-gamma and LT activate macrophages, increase phagocytosis, and upregulates nitric oxide production. It also increases NK cells and macrophages expression of high affinity Fc-gamma-R molecules that promote ADCC. ○ Further, it promotes the isotype switching of B cells to IgG1 and IG3 in humans, as well as IgG2 and IgG3 Abs in mice, which are effective against intracellular pathogens. ○ IgG1 and IgG3 Abs in humans are best suited for opsonization, phagocutosis, and complement activation. ○ IgG1 and IgG3 Abs bind with high affinity to FcR on NK cells, macrophages, and other phagocytes, all which further increase ADCC. ○ Th1 cytokines increase the antigen presenting potential of macrophages by upregulating MHC Class II and TAP. ○ Th1 cells support the activation of Tc cells by producing IL-2 and by providing CD40/D40L contacts for DC listening. Th2 Effector Functions Th2 cells promote humoral responses as these cells secrete cytokines IL-3, IL-4, IL-5, IL-6, IL-10 & IL-13 (sometimes called Th2 cytokines). Major functions of Th2 cells are to establish CD40-CD40L contacts with B cells & to secrete IL-4 & IL-5 that induce switching to Ig isotypes (IgG4 in humans) effective against neutralization of extracellular pathogens. IgG4 in humans is not very effective at complement activation or ADCC but is good in controlling pathogens at mucosal sites where inflammation could be damaging. IL-4 also enhances isotype switching to IgE & plays important role in allergic reactions. IL-4 and IL-13 inhibit proinflammatory cytokine production downregulate NO production and decrease FcgR expression on macrophages deceasing ADCC. IL-4 upregulates MHC class II expression on APCs (macrophages, DCs & B cells) & thereby contribute to Th cell stimulation. IL-4 and IL-13 also enhance the humoral response by stimulating B cell proliferation.  IL-5 promotes the growth, differentiation and activation of eosinophils important for elimination of helminth worms. IL-3, IL-4 & IL-10 combine to promote the activation and proliferation of mast cells, effective against large worms. IL-10 acts as a brake on immune responses and balances stimulation exerted by other cytokines. ○ Inhibits the proinflammatory functions of macrophages. ○ Abrogates macrophae production of IL-12 and MHC class II. ○ Downregulates B7 expression on macrophages and DCs. Th17 Effector Functions Until recently, Th17 cells were thought to promote autoimmune diseases; however, this is unlikely their physiological function. Th17 cells are dominated by Il-17, IL-21, IL22, and IL-21 production. The massive inflammatory responses mounted by rhese cells are designed to protect mucosal surfaces against pathogens that resist assault by Th1 and Th2 cells. Pathogens that trigger strong Th17 cell-mediated responses include: ○ Bacteria such as borrelia burgdorferi and kelbsiella pneumonia. ○ Fungal species such as pneumocystitis carinii and candida albicans. IL-17 produced by Th17 cells induces nearby non-HSCs to produce a destructive, pro-inflammatory cytokine, such as TNF-alpha, IL-1, and IL-6. The Effector Cell Crossrefulation and Amplification Because of the cytokines they produce, various Th subsets can cross-regulare each other’s differentiation and activities. Th1 cells produce large amounts of IL-2, which promotes the proliferation of both Th1 and Th2 cells. However, IFN-gamma produced by Th1 cells: ○ Has a direct, anti-proliferative activity of Th2 cells and inhibits their differentiation. ○ Induces IL-12 production by macrophages which promotes Th1 differentiation. IL-4, IL-13, and IL-10 produced by Th2 cells: ○ Suppresses IFN-gamma and IL-2 production by Th1 cells. ○ Inhibits Th1 differentiation. ○ Downregulates macrophage IL-12 production. ○ IL-4 from Th2 cells promotes continued differentiation of Th2 subsets. IL-21 (not IL-17), produced by activated Th17 cells support continues Th17 differentiation. IL-21 also repressed the expression of FOXp3 that drives Treg differentiation. Nature of Th Responses Usually, an immune response has either a Th1, Th2, or Th17 phenotype, based on the predominant Th subset and cytokines observed in the host during that response.  An attack by intracellular pathogens facors Th1 response. Conversely, invasion by extracellular pathogens most often promote the development of Th2 response, or depending upon the specific invader, a Th17 response. Allergies are associated with a prevalence of Th2 cells, whereas Th1 cells dominate in transplant rejection. Th17 cells are associated with many autoimmune disorders. Despite these generalizations, the overall phenotype of immune response to a given pathogen can change with time, such as: ○ In malarial infection, both Th1 and Th2 responses are induced during the full course of infection. Tc Cell Differentiation and Effector Function Tc cell responses can occur in 5 stages: ○ Activation of naive Tc cell by a licenseed DC in a secondary lymphoid tissue. ○ Proliferation and differentiation of the activated Tc cell into daughter cells called pre-cytotoxic T lymphocytes. ○ Differentiation of pre-CTL in an inflammatory site into an armed CTL. ○ Activation of the armed CTL by encounter with specific non-self peptide presented by MHC class I on a target cell. ○ CTL-mediated destruction of the target cell as well as other cells displaying the identical pMHC. Target cells of CTLs include cells infected with intracellularly replicating pathogens, tumor cells, and foreign cells as part of a tissue transplant. An activated Tc cell has no lytic powers at all, only its mature CTL progeny develop cytotoxicity. Generation and Activation of CTLs Pre-CTLs leave the lymph node & travel to the site of pathogen attack. Mature CTLs contain cytotoxic granules in their cytoplasm & effector Tc generation is completed within 24-48hrs. Development of CTL response is reserved for the situations in which threat is actually present (inflammatory cytokines). Stimulation of TCR of an armed CTL rapidly increases the binding affinity of adhesion molecule pairs forming a bicellular conjugate. CTL delivers a ‘lethal hit’ of chemical mediators that causes target cell death. Unlike naïve T cells, to activate armed CTL, only engagement of a single TCR by a single specific pMHC is needed, & no costimulation is required. Mechanism of Target Cell Destruction The target cell destruction by CTLs can occur via the: ○ Granule exocytosis pathway. ○ FAS pathway. ○ Release of cytotoxic cytokines such as TNF and LT.  The pathway used depends on the nature of the attacking intracellular pathogen, but granule exocytosis accounts for the majority of target cell killing by CTLs. Granule Exocytosis Pathway After conjugate formation, cytoskeleton of the CTL reorganizes to bring cytotoxic granules to the site of CTL-target cell contact. The granules fuse with CTL membrane and their contents are directionally exocytosed towards the target cell membrane. Perforin & granzymes are major contents of these granules. Perforin is a pore-forming protein and the granzymes are a family of serine proteases. After entry into target cells, these proteins are immediately confined to endocytic system. Perforin then facilitates the release of granzymes from endolysosomal vesicles into cytoplasm of the target cell. Granzyme A initiates a caspase-independent pathway of DNA damage, while granzyme B triggers classical caspase-mediated apoptosis. Upon the degradation of its DNA and other important intracellular substrates, the target cell dies. This form of death is called ‘perforin/granzyme-mediated cytotoxicity’. FAS Pathway FAS is a transmembrane deth receptor that is widely expressed on mammalian cells. Naive Tc cells do not express FasL, but agdter activation and conjugate formation, FasL is expressed on the CTL surface. Engagement of Fas on a target cell by FAS ligand (FasL) expressed by an armed CTL results in the death of the target cell by apoptosis. Cytotoxic Cytokines CTLs can kill target cells by producing cytotoxic cytokines such as TNF and LT. Apoptosis is induced by binding of TNF and/or LT to TNF receptor 1 on the target cell surface. IFN-gamma prooduced by CTLs indirectly helps B cells in producing Abs which can increase ADCC and upregulates MHC class I. Dissociation of CTL After ~5-10 minutes after delivery of a lethal hit, adhesion molecules on CTL resume a low affinity conformation that allows the CTL to dissociate from the damaged target cell. The target cell succumbs to apoptosis within 3 hrs of dissociation. CTL commences synthesis of new cytotoxic granules and moves off to inspect other host cells. A single armed (and re-armed) CTL can attach to many host cells in succession, delivering lethal hits. How the CTL avoids self-destruction by its granules is a mystery. Termination of Effector T Cell Responses Th effector cells & CTLs are sustained by signals delivered by inflammatory cytokines (such as IL-12) & transcription factors (such as ID2 and BCL-3).  However, after the effectors have removed the threat, there is no further need for their presence. Continued exposure to inflammatory environment in the absence of Ag causes the effectors to downregulate IL-7R & IL-15R, reduction in their ability to receive survival signals. Three mechanisms then act in concert to further bias the balance of pro-apoptotic/anti-apoptotic gene expression and induce effector cell death: ○ Activation-induced cell death (AICD), ○ Cytokine “withdrawal” and ○ T cell clonal exhaustion. Memory T Cells For both CD4+ and CD8+ T cells, about 5-10% of the antigen specific progeny of T cells generated in a primary response survive AICD or IL-2 withdrawal. These cells give rise to, long-lived memory T cells which are central basis of vaccination. Memory T cells recognize same pMHC as naïve & effector T cells but have properties intermediate between them. Memory T cell are usually found in a resting state but can undergo self-renewal to ensure their long-term survival. Upon a second assault by the same pathogen, memory T cells mount a secondary response that is faster and stronger than the primary response. Memory T cells compared to naïve T cells differentiate at a significantly faster rate into effector cells when activated by Ag. Types of Memory T Cells There are at least 2 major types of memory T cells: ○ Effector memory T (Tem) cells. ○ \central memory T (Tcm) cells. These cells differ in some important properties. In the absence of specific antigen, Tem cells have a shorter lifespan than Tcm cells. Tcm cells express high levels of lymph node homing molecules CD62L and CCR7. ○ Thus, Tcm cells tend to migrate through lymph nodes and other secondary lymphoid tissues, thereby maintaining a long-term central reservoir of memory cells. In contrast, Tem cells express only low levels of CD62L and CCR7. ○ Thus, Tem cells mainly circulate through non-lymphoid tissues where pathogens are likely to attack for a second time. Tem cells constantly patrol the peripheral tissues and are able to migrate quickly into the site of infection. Memory T Cell Activation Memory T cells have less stringent activation requirement than naïve T cells.  Memory Th cells are dispersed at more anatomical sites than naive Th cells and can respond to pMHC presented by DCs, B cells and macrophages. Similarly, memory Tc cells can respond to infected host cells located almost anywhere in the body. Activation of both memory Th & Tc cells more closely resembles that of an effector T cells than a naïve T cells. Activation of memory T cells can occur at a very low concentrations of Ag with no/only minimal costimulation (if any) & duration of TCR signaling required is much shorter. Memory Tc cells do not require T cell help for activation. Upon activation both memory Th & Tc cells proliferate more readily & for longer period than their naive counterparts. Memory T Cell Differentiation and Life Span For differentiation, activated memory Th & Tc cells follow the same pathways as naïve Th & Tc cells but complete them more quickly (within 24 hrs as opposed to 4-5 days). Most memory T cells persist in the host for at least several months and often years (up to 50 years), greatly exceeding the longevity of both naïve and effector T cells. The maintenance of memory cells depends on IL-7 as it drives the expression of anti-apoptotic molecules that protect against AICD. ○ IL-2 and IL-15 also support their long-term survival. The length of life span of a memory T cell clone varies with the nature of Ag that initiated primary response as evident from the variability in immunization schedule of vaccines. Just one dose of some vaccines (polio) provides immunity for life, whereas booster doses of other vaccines (tetanus) must be given every few years to maintain protection. Lecture 23 Lymphocyte Tolerance in the Periphery The adaptive immune responses in peripheral tissues is tightly controlled in 2 ways: ○ Tolerance: prevents lymphocyte activation. ○ Immune regulation: controls actions of effector cells. Tolerance is manifested when the interaction between a mature peripheral lymphocyte and its scognate antigen does not result in activation of that lymphocyte. In peripheral tolerance, lymphocytes: ○ Fully undergo apoptosis. ○ Becomes functionally inactivated. This maintains tolerance to self-tissues Peripheral self tolerance is vital, as it prevents the activation of autoreactive lymphocytes which have escaped central tolerance mechanisms. Peripheral tolerance to innocuous (harmless) non-self antigens also exists & this helps to prevent inflammatory responses that would otherwise inflict unnecessary tissue damage.  Immune response of activated lymphocyte needs to be damped down to prevent/minimize collateral damage to surrounding healthy tissues. Regulation of any responses made to innocuous non-self entities, such as the commensal gut microbes or proteins in the food or the air we breath, is essential for normal health. Success in implementing both tolerance and regulatory mechanism ensures that the host focuses the power of the immune response on harmful non-self antigens. Failure to this paves the way to uncontrolled tissue damage and the potential development of autoimmune diseases. As BCRs & TCRs are randomly generated, a certain number of lymphocytes may bear receptors against self antigens. Most B cells with autoreactive BCRs undergo receptor editing in bone marrow during establishment of B cell central tolerance so that they do not recognize self antigens. Most T cells with potentially auto reactive TCRs are eliminated by deletion by negative selection during the establishment of ‘central T cell tolerance’ in the thymus. If an autoreactive lymphocyte is released into periphery because of failure of central tolerance mechanisms, then peripheral tolerance mechanisms attempt to ensure that auto reactive cell cannot be activated to attack self tissues. T Cell Tolerance in the Periphery DC-mediated tolerization: ○ All professional APCs (DCs, macrophages & B cells) play an important role in making a given protein antigenic, that is, able to bind lymphocyte antigenic receptors. ○ However, only DCs have the unique capacity to determine whether the interaction of a given pMHC with the TCR of a naive T cell will be immunogenic or tolerogenic. ○ This property of DC is dictated by the inherent nature of DC subtypes involved and the external influences acting on the DC in a given tissue environment. ○ Immature DCs are broadly distributed in the peripheral tissues & constantly take up antigens from their surroundings. ○ In the absence of pathogen attack or injury, these DCs only handles self or innocuous non-self antigens. ○ When DAMPs/PAMPs are not present, then immature DCs are not induced to mature. DC-Mediated Tolerization If an immature DC encounters a naïve T cell specific for an innocuous Ag, the DC exhibits tolerogenic properties & inactivates the T cell rather than activating it. Tolerogenicity of immature DCs helps to preserve peripheral tolerance to both self & non-self harmless antigens. Without signal 2, signal 3 can not be delivered & T cell is not activated despite receiving signal 1.  There are two main processes by which tolerogenic DCs inactivate naïve T cells: ○ Clonal deletion: The most important mechanism by which peripheral tolerance is maintained by clonal deletion of autoreactive Th cells. In this tolerogenic DCs usually induce apoptosis in autoreactive T cells. ○ Anergization: Those autoreactive Th cells that receive signal 1 alone from a tolerogenic DC but do not undergo apoptosis are ‘anergized’. An anergic Th cell can maintain its unresponsive state for up to severa months. T Cell Tolerance by Clonal Exhaustion Peripheral tolerance can also be invoked by the elimination of an entire T cell clone due to clonal exhaustion. In this situation, continuous exposure to an Ag forces the responding cells to proliferate and generate effectors so rapidly that they burn out without generating memory T cells. It is believed that tolerance to many self Ags that are present in the body in high abundance may be established this way very early in life. During embryogenesis, the presence of large amount of self antigen causes exhaustion of autoreactive clones that escaped central tolerance, ensuring peripheral tolerance to these self elements. B Cell Tolerance Autoreactive B cells that escape central B cell tolerance are controlled by peripheral tolerance mechanisms that differ slightly from those discussed for autoreactive T cells. If an autoreactive B cells encounters self antigen in the periphery, it receive signal 1 but still depends on an antigen specific Th effector cell to deliver signal 2 and 3. Thus, even if DAMPs/PAMPs are present, if the required Th cell has already been deleted, anergized or exhausted by central or peripheral tolerance, B cell can not be activated. Instead, the B cell is anergized & die by apoptosis within 3-4 days. This reliance of B cells on Th cell for activation allows the host to benefit, without undue risk of increased autoreactivity from somatic hypermutation of Ig genes. Sometime an autoreactive Th cell may be present in periphery & potential for activation of autoreactive B cell may exist. However, in these cases, in the absence of DAMPS/PAMPs DCs may delete or anergize the autoreactive Th cell rather activates it. Anergized autoreactive T cell does not deliver signal 2 to B cell & thus B cell is anergized & forced into apoptotic death.