Medical and Veterinary Notes 8-9 PDF

Self–MHC restriction of TCR - In 1974, Zinkelnagel and Doherty showed that o Antigen (Ag) recognition by T cell is not specific for the Ag alone rather it is specific for the Ag associated with MHC molecule. - T cells recognize the Ag only when it is...

Self–MHC restriction of TCR - In 1974, Zinkelnagel and Doherty showed that o Antigen (Ag) recognition by T cell is not specific for the Ag alone rather it is specific for the Ag associated with MHC molecule. - T cells recognize the Ag only when it is presented by self-MHC molecule. - ‘Self-MHC restriction’ phenomenon differentiates recognition of Ag by T cells and B cells. - Doherty and Zinkelnagel were awarded Nobel Prize for this work in 1996. Diagram - Recognize antigen get activated activation process complex - Once activated kill infected cell - Ctl will kill them based on antigen presenting cell - T cell recognizes it kills cells - Target cell is infected (a) - Cell specific and recognizes then kills - Specific peptides are necessary (b) has the wrong peptide so no killing - Specific cell (needs self MHC) is also necessary for proper killing (c ) Model for MHC restriction of TCR - Two models were proposed - 1. The dual receptor model: Two separate TCR; one for antigen (Ag) and one for MHC molecule. - 2. The altered-self model: Single TCR recognizes an alteration in self-MHC molecules induced because of association with foreign Ag. o Self MHC changes from Ag - Kappler and Marrack showed that specificity for both Ag and MHC molecule resides in a single receptor. - At present, ‘altered-self model’ is an accepted model for MHC restriction of TCR. - Recognizes antigen with MHC receptor and only if presented by self molecule - MHC can alter shape self MHC molecule is altered by peptides sitting on it - Altered self model is correct T Cell Receptor (TCR) - Responsible for Ag recognition by T cells. - Expressed on most thymocytes and all mature T cells. - Resembles B cell receptor (BCR) in many ways.  - Like B cells, T cells possess an Ag 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 Igα & Ig-β of BCR, TCR is associated with a signal–transducing complex called CD3. - Recognize only one antigen one cell expresses one - Heavy and light chain recombination VDJ recombination in heavy chain VJ recombination in light chain o Similar type of recombination in T cells o B cell cant pass signal by self phosphorylation has to happen T Cell Receptor (TCR) - TCR also differs from BCR (Ig) in many ways. o The spectrum of Ags recognized by TCR is much more restricted than BCR. o TCR is membrane-bound and in contrast to BCR (Ig), it does not occur in a soluble form, so T cells do not secrete their TCRs. o In contrast to BCR, most TCRs except few are not specific to antigen alone rather they are specific to Ag combined with MHC molecule. o A small percentage of T cells recognize lipid or unprocessed Ags that may or may not be associated with MHC-related molecules. Basic BCR Structure - Light and heavy chain o Hinge region move light chains - Antigen binding molecule regions o Constant region o Fc region ▪ Crystalizes - Antigen binds to variable region o Fab/ Vh Vl - Region 1 2 and 3 o CDR TCR Structure - Two types of T-cell receptors: αβ TCR and γδ TCR. o Made of alpha beta and gamma delta  ▪ Alpha and beta combine and gamma and delta combine Is set can not combine randomly ▪ Alpha bata make up 90% and are in secondary lymphoid tissue - Mutually exclusive expression of both TCRs so no αδ, or γβ T cells exist. Both αβ- and γδ T-cells develop independently. - The αβ T-cells function in adaptive immunity but γδ T-cells participate in innate immunity. Basic TCR Structure - TCRs are transmembrane and insoluble proteins. - Member of Ig superfamily. - ~10,000- 30,000 TCR molecules on the surface of a T cell. TCR Structure - Unlike mIg, TCR is a heterodimeric glycoprotein with a single Ag binding site & resembles an Ig Fab fragment. - Amino acids in Ag binding site establish contacts with the antigenic peptide and MHC molecules to which it is bound. - Unlike Ig molecule which can undergo isotype switching, a TCR’s ‘C’ region is fixed for the life of a given T cell clone. - Short connecting sequence located between TCR ‘C’ domain and transmembrane domain is analogous to the Ig hinge. Crystal Structure - V domains in both α & β chains contain four complimentarity determining (CDRs) or hypervariable (HV) regions: CDR1, CDR2, CDR3 and HV4 which are equivalent to present in Ig light (L) and heavy (H) chains. - The various CDRs are involved in peptide-MHC (pMHC) recognition. - HV4 is a region of amino acid variability but it does not contact the peptide within the pMHC complex directly. Crystal structure - The V domains of the TCRγ and δ chains also contain CDR1, CDR2, CDR3 and HV4 regions. The TCR-CD3complex  - Immunoreceptor tyrosine-based activation motifs (ITAMs) interact with tyrosine kinases and play important role in signal transduction. The CD3 complex: TCR-CD3 - The cytoplasmic tails of TCR chains are too short for signal transduction. - Like Igα and Igβ chains in B cells, additional proteins (CD3 dimers) are responsible for signaling through TCR after Ag interaction. - CD3 dimers do not affect the interaction with Ag. - CD3 complex contains three heterodimeric proteins made up of variable combination of five ITAM-containing invariant polypeptide chains (epsilon, delta, zeta, eta and gamma) that associate to form three dimers. - TCR non-covalently associates with CD3, forming a TCR-CD3 complex or TCR complex. The CD3 complex: TCR-CD3 - CD3 complex has two major functions: - 1. The CD3 chains are required for intracellular signaling. o Upon engagement of TCR by pMHC, tyrosine residues in the CD3 ITAMs are phosphorylated by an intracellular signaling kinase called Lck o Additional signaling kinase are then recruited to propagate the signaling cascade. - 2. CD3 expression is required for the surface expression of αβ or γδ TCR o In the endoplasmic reticulum (ER), TCR physically associates with CD3 complex and finally transports to the T cell surface. The CD4 and CD8 coreceptors - TCR binds to Ag-MHC complex. - Additional accessory membrane molecules also play important role in Ag recognition and T cell signal transduction/activation. - The most important of these are CD4 and CD8 molecules. These are considered coreceptors. - Despite equivalent functions, CD4 & CD8 proteins show little similarity in either structure or amino acid sequence. - Some intraepithelial T cells express a CD8αα homodimers. Interact with Lck. The CD4 and CD8 coreceptors - Mature αβ T cells are either CD4+ or CD8+ cells and in humans, about 2/3 are CD4+ and 1/3 are CD8+.  - Most mature γδ T cells express neither CD4 nor CD8 but some γδ T cells in gut are CD8+. - CD4 and CD8 are called ‘coreceptors’ as one of these proteins colocalizes with TCR and binds to the same MHC molecule on the APC/target cell that is engaged by the particular TCR. - CD4 and CD8 molecules bind to conserved regions (outside the peptide binding groove) of MHC class II & class I molecules respectively. - Coreceptor binding does not depend on the identity of the antigenic peptide by TCR. The CD4 and CD8 coreceptors - CD4 +ve T cells are largely helper T cells (Th cells) while CD8 +ve T cells are largely cytotoxic T cells (CTLs). - CD4 and CD8 have two major functions: o 1. Stabilization of TCR-pMHC binding by the interaction of CD4 with MHC class II and CD8 with MHC class I. o 2. Recruitment of Lck to the TCR-CD3 complex. - Although neither CD4 nor CD8 is absolutely required for the initial engagement of TCRαβ by pMHC, their binding to MHC molecule greatly enhances TCR-pMHC binding. - CD4 or CD8 binding to MHC molecule brings Lck into close proximity of CD3 chains and Lck then phosphorylate ITAMs & leads to T cell activation. The CD4 and CD8 coreceptors The binding by the coreceptors to MHC molecules stabilizes the interaction between T cell & APC/target cell. TCR genes - TCRa, b, g and d polypeptide chains are encoded by TCRA, TCRB, TCRG and TCRD loci, respectively. - Note that in both mouse and human, TCRA and TCRD loci are present on chromosome 14 and TCRD locus is nested within TCRA locus. - Alpha and delta are on same chromosome and same locus TCR genes - Similar to Ig, each TCR chain has a variable (V) & a constant domain (C) encoded by V and C exons. - Similar to Ig heavy chain, TCR V exon is made up of small V, D and J gene segments in TCR B and TCR D loci that are assembled at the DNA level by V(D)J recombination. - Analogous to Ig light chain, the TCRA and TCRG loci contain V and J segments but no D segment. - Beta and Delta V(D)J - Alpha and Gama VJ TCR genes - Only one Ca exon in TCRA in both mice & humans. - Two Cb exons in TCRB in both mice & humans but these are not functionally different as in T cells there is no mechanism analogous to isotype switching in B cells. - Two Cg exons in humans TCRG. - Three functional Cg exons in mouse TCRG (Cg1, 2 & 4) (Cg3 is a pseudogene). - One Cd exon in TCRD in both mice and humans. - TCR genes - Although TCRD has many similarities to Ig heavy (Igh) chain, it also exhibits some startling differences. - 1. In both mice and humans, TCRD is nested within the TCRA locus and this prevents the expression of both TCRD and TCRA on the same T cell. - 2. In addition to use of one Dd segment to create a VDJ exon, multiple Dd gene segments can be used in tandem to create a VDDJ or VDDDJ exon which dramatically enhances the junctional diversity found in TCRd chains. TCR genes Genomic organization of the human TCR loci Genomic organization of the mouse TCR loci Order of gene rearrangement - TCR genes are in the germline configuration when T cell progenitor leaves the bone marrow and enters the thymus. - In the thymus, immature thymocyte rearranges its TCR genes & become either an ab- or a gd T-cell depending upon the signals it receives both through its TCR and from the local environment. Order of gene rearrangement - TCRA and TCRB rearrangement: o Irrevocable commitment of a thymocyte to TCRab lineage depends upon V(D)J recombination resulting in a functional TCRb gene. o TCRB locus rearranges prior to the TCRA locus. o Simultaneously in TCRB on the maternal & paternal chromosomes, V(D)J recombination first joins a Db gene segment to a Jb segment, and then a Vb segment to DbJb. o D and J always combine first TCRA and TCRB rearrangement - The functionality of the completed TCRb gene is tested via formation of ‘pre- TCR’, a signaling complex (TCRb chain, a surrogate TCRa called ‘pre-T alpha’ chain and CD3 chains).  - Successful intracellular signaling through Pre-TCR indicates that candidate TCRb protein is functional and TCRb rearrangement is successful. - The assembly of a functional TCRb gene on one chromosome suppresses its rearrangement on other chromosome and this is called ‘allelic exclusion’. - In a thymocyte if TCRb rearrangement is unsuccessful on both chromosomes, then cell dies by apoptosis instead of rearranging its TCRa gene or becoming gd T cell. - More than 90% of cells die in thymus TCR and TCRB rearrangement - If a functional TCRb gene is produced, then V and J gene recombination of TCRA starts on both chromosomes. - If a productive TCRa chain gene rearrangement takes place on either chromosome, TCRa chain can combine with TCRb to produce a functional surface TCRab. - If TCRA rearrangement fails on both chromosomes, the cell dies by apoptosis. TCRA and TCRB rearrangement - In thymocytes which finally become gd T cells, rearrangement of genes starts simultaneously but independently in TCRG and TCRD loci on both chromosomes. - In these cells although TCRA locus physically surrounds TCRD, TCRA does not undergo rearrangement. - VJ joining of TCRg gene segments occurs in a usual manner. - TCRd D gene segments can be combined with each other to form tandem D-D or D-D-D units. o Can have more than one D region combine together - The D, D-D, or D-D-D entities are joined to Jd and then finally to Vd to complete the V exon.  - T cell will either be alpha gama or beta delta 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 & the TCR genes in developing B cells. V(D)J recombination - Similar to BCR genes, the TCR genes are flanked by the same 12-RSS and 23- RSS sequences. - In TCRB locus (see fig 8.8), RAG recombinases follow the same 12/23 rule to juxtapose only those RSSs that are not of the 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 facilitate the tandem joining of D segments prior to addition of the Jd segment (it happens less frequently in TCRB locus). V(D)J recombination - Can have D-D or D-D-D combinations - In T cell receptors - Increases diversity - Genes combine - Antibody and t cell similar process - T cell receptor expressed successively then no antibody expressed - Can only have one expressed at a time - Have to combine in certain ways - Multiple V and D region genes - D combines with V first - Get a DJ portion with other pieces attached - The extra portion is deleted - Connect the V region - Have completed gene recombination TCR gene transcription & 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 ER, disulfide bonding links TCRa & b chains or TCRg & d chains which later associate with CD3 molecule & appear on the cell surface as TCR complex. TCR diversity - The isotype switching and somatic hypermutation mechanisms which create diversity in Ag-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: o 1. Multiplicity of germline segments o 2. Combinatorial diversity o 3. Junctional diversity o 4. The ab or yS chain pairing. Multiplicity and combinatorial 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. Multiplicity and combinatorial joining of germline gene segments - 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 TCRb (less) and TCRd 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. Multiplicity and combinatorial joining of germline gene segments - Possible combinations: - Mouse TCRa 84Va x 38Ja x 1Ca =3192 - Mouse TCRb 22Vb x 2Db X 11Jb x 2Cb =968  - Mouse TCRg 7Vg x 4Jg x 4Cg =112 - Mouse TCRd 15Vd x 2Dd X 2Jd x 1Cd =60 - More diversity in TCRab than in TCRgd. - Note: You do not need to remember all these numbers but understand the concept. 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 Dd segment may be included in tandem in a TCRd 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 TCRd chains to the TCR repertoire. Chain pairing - The random pairing of TCRa and b chains (or TCRg and d chains) also contributes to TCR repertoire. - The total number of possible ab heterodimers approaches 10^18 in humans and 10^15 in mice. - These numbers compare to the 1011 specificities estimated for the Ig repertoire. - Due to death of T cell clones before they ever meet their Ag as well as the processes of central and peripheral tolerance, a human & mouse has ~2x107 and 2x106 functional ab T cell clones respectively. TCR-Antigen interaction - The interaction between a TCRab TCR and its pMHC epitope underlies the fundamental aspects of the cell mediated adaptive immune response. - 1. The strength of binding between a thymocyte’s TCR & various pMHCs encountered in the thymus determines positive or negative selection of thymocytes. - 2. The strength of binding between a mature ab T cell’s TCR and pMHC presented by APC in the periphery determines whether the T cell will –  o Be activated to proliferate & differentiate into effector cells or o – Become anergic (non-responsive). TCR-Antigen interaction - Both the TCRa and b chains are usually involved in binding to both the MHC molecule and the peptide, and this binding occurs virtually simultaneously. TCR-Antigen interaction - Understand the basic concepts in these pictures. - Fig 9.6: Important adhesion contacts between Human T cells and DCs. TCR-Antigen interaction - The area of contact between TCR & pMHC is relatively small and only a few peptide residues generally make contact with a TCR. - The binding affinity of a TCR (K=~ 5x105 M-1) is significantly lower than that of Ab ((K= 107-1011 M-1) for its antigen. - Allows for more binding TCR-Antigen interaction - Relatively modest affinity of TCR has many implications: - 1. The initial contact between T cells & APCs/target cells is established not by TCR-pMHC interaction but rather by binding of complementary pairs of adhesion molecules. - 2. The contact between CD4/CD8 & MHC molecules is also important in holding cells together. - 3. TCR can bind with varying affinities to a broad range of pMHCs and such promiscuity facilitates thymic selection Comparison of B and T cell development  - B and T lymphocytes are derived from same hematopoietic progenitors, but their development differs in many ways. - 1. Thymus is required for the generation of T cells but not for the B cells. B cells develop in the bone marrow. - 2. Naïve B cells are produced throughout the life. In contrast, thymus involutes around puberty and so the production of new naïve T cells is sharply reduced after puberty. - 3. 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. - 4. TCR unlike BCR is fixed for life of the T cell and cannot undergo the somatic hypermutation like BCR in B cells. - 5. Most B cells result from a single developmental program, but functional T cells can result from many different pathways (αβ/γδ T cells, Th/Tc cells & T regulatory cells). Thymus - Concentric layers of degenerating epithelial cells- growth and learning - T cells developing in the thymus are called ‘thymocytes’. - A flat, bilobed structure above the heart. - A site of T-cell development & maturation. Thymus - In ‘nude mice’ thymus fails to develop. o Mutation in stromal cells so t cells do not double up properly - The ‘nude mutant’ 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 & this plays 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. - It may lead to abnormal T cell numbers but not always. - No stromal cells no doubleupment Colonization of thymus - The fetal thymus is ‘colonized’ or ‘seeded’ with hematopoietic progenitors which then proliferate and mature in the thymus into functional naïve T cells.  - T cells like other hematopoietic cells are derived from HSCs which are present in fetal liver in a fetus & bone marrow in adolescent/adult individual. Colonization of thymus - Proliferating HSCs differentiate into MPPs. They further differentiate into CMP, CLP & MCP then leave bone marrow or liver & enter the blood circulation. - Circulating CLPs give rise to a slightly more differentiated progenitor called the ‘NK/T precursor’ that can generate NK & T cells but not B cells. Colonization of thymus - Upon entering thymus, a NK/T cell precursor differentiates into αβ or γδ T cells, lymphoid DCs or NK/NKT cells based on the cytokines & 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 γδ thymocytes but subsequent waves of NK/T precursors just before birth & shortly thereafter give rise to both αβ & γδ thymocytes. Colonization of thymus - After birth, NK/T precursors destined to become T cells are more biased towards αβ T cell lineage such that γδ 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: - 1. Double negative phase (DN, DN1-DN4): express neither CD4 nor CD8.  - 2. Double positive (DP) phase: express both CD4 and CD8. - 3. Single positive (SP) phase: express either CD4 or CD8 but not both. Thymocyte maturation in the thymus - Once SP thymocytes emerge from thymus & enter the circulation & secondary lymphoid organs, they are considered mature naïve CD4+ or CD8+ peripheral T cells. - Several selection processes occur during these transitions. The thymic environment - The development of thymocytes 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 and thymic fibroblasts. - Thymic DCs, cTECs & mTECs are vital for the establishment of T cell central tolerance during the DP phase. The thymic environment - Expression of ‘Notch1’ on thymocytes directs them to T cell lineage & thus it is a key protein in T-lineage specification. - cTECs & 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. - Continued Notch1 signaling is required to sustain the survival of thymocytes until they pass through the DN stage. Thymic microenvironment & location of developing thymocytes - Thymic fibroblast secrete extracellular matrix components (i.e. collagen) which help in concentrating cytokines crucial for thymocytes’ development and in controlling thymocytes adhesion to stromal cells. The DN phase - The DN phase thymocytes in addition to CD4 and CD8 are also negative for TCR expression, cannot bind pMHC and do not carry out effector function. - Four subsets DN1-DN4. - In mice DN1-DN4 subsets are distinguished from each other by their expression of the surface markers: o 1. C-kit: a cytokine receptor o 2. CD44: an adhesion protein o 3. CD25: α chain of the IL-2 receptor. - In human DN1-DN4 subsets are distinguished from each other by their expression of the surface markers: o 1. CD34: an adhesion protein. o 2. CD38: an adhesion protein. o 3. CD1a: an MHC like protein. The DN phase - DN1 subset o TCR genes in germline configuration & reside in thymic cortex. o cTECs supply stem cell factor (SCF) that binds to c-kit on DN1 cells and delivers a survival signal. o Transcription factor GATA-3 is vital for the generation of DN1. - DN2 subset o Murine DN2 are known as Pro-T cells (progenitor T cells). o Present primarily in the outer cortex and TCR genes remain in germline configuration. o Commence expression of the CD3 chains. o Under the influence of IL-7 and SCF, DN2 thymocytes start to proliferate rapidly. DN3 subset - DN3 cells stop proliferating & remain in 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 αβ and γδ T cells. - TCRG, TCRD and TCRB loci commence V(D)J recombination with concomitant upregulation of RAG and TdT.  - DN3 cells destined to become αβ T Cells express a functional ‘Pre-TCR complex’ that allows them to determine if a functional TCRβ chain has been produced. - Successful rearrangement at TCRβ 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 αβ T cell lineage and express a diverse repertoire of TCRβ chains. DN3 thymocytes - In DN3 thymocytes that generate αβ T cells, the TCRβ locus is first to undergo VDJ recombination. - DN3 thymocytes that have successfully arranged their TCRβ chains undergo ‘β- selection’, and cells that survive β selection are said to have passed the ‘pre-TCR checkpoint’. - This results in proliferation & differentiation of DN3 cells. - This process involves the expression of a glycoprotein known as ‘Pre-Tα chain’ on the DN3 cells which act as a surrogate for the real TCRα chain which yet to rearrange. - Pre-TCRα 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’ & acts as a sensor by initiating signal transduction. Maturation to DN4 stage. - If the TCRβ rearrangement on both chromosomes has been unsuccessful, the cell neither attempts to rearrange its TCRA genes nor becomes a γδ T cell; instead, it dies by apoptosis. - Only 10% of DN3 thymocytes successfully rearrange their TCRβ genes, are βselected 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 (Fig 9.2). - DN4 cells contain a functionally rearranged TCRβ gene. - DN4 cells downregulate their expression of CD25, RAG and TdT. - DN4 cells start expressing very low level of CD4 and CD8. Markers characterizing the phases of αβ T cell development in mouse The double positive (DP) phase - In both humans and mice, the DP phase of αβ T cells development is dominated by the thymic selection processes that shape the mature αβ T cell repertoire. - CD4 and CD8 expression levels are upregulated & they play important roles in directing thymocyte development. - DP thymocytes move towards the thymic medulla. The double positive (DP) phase - TCRαβ Pool expansion & TCRA locus rearrangement: - 1. DP thymocytes receive signals through Pre-TCR for proliferation. - 2. V(D)J recombination in both TCRA loci start (delete TCRD loci). - 3. Newly synthesized TCRα chains combine with TCRβ chains to form complete TCRαβ heterodimers. - 4. 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 & express a TCR that includes an invariant TCRα chain (thus also called iNKT cells). - Check points of T cell development. 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 & negative selection events allows only self MHC-restricted & nonselfreactive T cells (tolerant to self) to mature & leave thymus. - Finally, functionally distinct mature CD4+ and CD8+ subpopulations that exhibit class II and class I MHC restriction respectively exit thymus. The Thymus as a Testing Ground for T cells - Failure to successfully rearrange TCR genes, to pass positive selection, or to pass negative selection results in cell death. Fig 9.5. Affinity/Avidity Model of thymic selection. - 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 & die by apoptosis in the thymus. or ‘neglect’ Positive selection - Thymic stromal cells, including epithelial cells, macrophages & dendritic cells play essential roles in positive and negative selection. - These cells express MHC class I and high levels of MHC II. Positive Selection - Takes place in the CORTEX of the thymus (takes place first) - T cells which recognize self-MHC with low or intermediate affinity receive a “survival/protective” signal from specialized APCs in the thymus (cortical Thymic Epithelial Cells-cTECs) 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 after selection. Negative selection - Negative selection which is also called ‘Central tolerance’ takes place in the medulla of thymus. - AIRE+ (autoimmune regulator) medullary thymic epithelial cells (mTECs) Negative Selection - In negative selection, T cells which demonstrate too high an affinity for self-MHC molecules alone or selfantigen presented by self-MHC by medullary thymic epithelial cells (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 on 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 organization & gene expression that result in - 1. silencing of a coreceptor gene (CD4 or CD8) and - 2. expression of gene associated with a specific lineage function.  - The mechanism which regulates this is not fully understood but is likely linked to the specificity of the TCR for either Class I or Class II MHC. Single positive (SP) CD4+ and CD8+ cells - 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 rid of cells which express a high affinity for self-antigens. - However, negative selection in thymus is not perfect because of two reasons: -  Thymocytes which have low affinity to self-antigens do escape the negative selection. -  Thymocytes have not browsed the right ‘tissuespecific antigen/MHC combination. - Body has evolved many other mechanisms to avoid autoimmunity -.  T regulatory cells (Treg): these cells negatively regulate immune responses. -  Peripheral mechanism of tolerance mediated through T-cell anergy (will be discussed in later lectures). T cell - Produces cytokines o T helper cells - Used in recognition T-cell Activation - Similar to B cells, the complete activation of naïve T cells generally requires 3 signals: - 1. Engagement of the antigen receptor(TCR) by antigen  - 2. Costimulation, and - 3. Receipt of cytokines - However, these signals differ slightly between B and T cell activation and between naïve Th and Tc cell. - Additional differences in the activation of effector and memory T cells also exist. - To activate T cells you need a very high strong concentration to begin - Dendritic cells take vaccine engulf and process it then move it towards lymph node Meeting of naïve T cells and DCs - Activation of most naïve cells happens in the paracortex of the lymph node (LN) where Ag loaded mature DCs congregate and naive T cell recirculate. - Immature migratory & lymphoid resident DCs capture the materials from their microenvironment including foreign substances and pathogens. - Soluble antigen gets processed by dendritic cells Meeting of naïve T cells and DCs - In case of infection & inflammation, pro-inflammatory cytokines & DAMPs/PAMPs induce the maturation of DCs. - Maturing migratory DCs enter LN via an afferent lymphatic & settle in the paracortex surrounding the high endothelial venules (HEVs). Resident DCs are already present there. - Mature DCs process their captured Ags & display antigenic peptides on MHC II via exogenous processing & on MHC I via cross presentation. - Find antigen stay in one spot of not found in a constant circulation - Can do cross presentation antibody on inside and outside of cell Meeting of naïve T cells and DCs - If a DC is infected by a pathogen, intracellular Ags may also be processed via endogenous pathway and presented on MHC I or displayed on MHC II by autophagy. - Naïve T helper (Th) and Cytotoxic T cells (Tc) circulate in the blood & throughout the secondary lymphoid tissues such as LN. Meeting of naïve T cells and DCs - Mostly a naïve T cell enters the node via its HEVs & inspects the pMHCs displayed by mature DCs in the vicinity of these vessels.  - T cells ‘crawls’ slowly over the surface of a DC in a process facilitated by several adhesion molecules pairs which hold the T cells & DC together for pMHC screening. - pMHCs that are bound with sufficient affinity/avidity by TCR have the potential to activate the T cell. - Important adhesion contacts between Human T cells and DCs. 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 naïve 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 & CD8. - The additional intracellular signaling enzymes are then recruited to cytoplasmic tails of CD4 & CD8 & CD3 chains. - Together, these enzymes mediate a cascade of chemical reactions that leads to activation of many other enzymes. - When this activation cascade occurs for multiple TCRs, the TCR receives signal 1. Signal 1 for T cell activation - Because of low affinity, a single pMHC engagement with TCR does not engage a single TCR long enough to achieve complete activation of a naïve T cell. - Similarly, transient interaction between a few pMHC-TCR pairs is also not sufficient. - Sustained interaction between naïve T cell & DC for many hours is needed for the proper & sufficient T cell activation. - TCRs and pMHCs required for sustained signaling are gathered together by the formation of an ‘immunological synapse’ between T cell & DC interface. - Both cells undergo rearrangement of their actin cytoskeletons & polarization. TCR-mediated signaling - Formation of an IS is not required for the initiation of TCR signaling, but sustained T-cell activation is more effective if an IS forms. (IS) Initiation Cell membrane antigen molecules T cell receptor in lipid rafts Signal 1 for T cell activation - Immunological synapse results in the formation of three concentric rings, each containing various signaling, adhesion & cytoskeletal molecules that cluster around the TCR-pMHC pairs. - Inner ring: is called central supramolecular activation cluster (cSMAC) and it is composed of aggregated TCRs & costimulatory molecules. - Middle ring: peripheral supramolecular activation cluster (pSMAC) contains signaling adaptor talin & large number of integrins & other adhesion molecules. o Adaptor molecule - Outer ring: is called distal supramolecular activation cluster, (dSMAC) which mainly contains actin-based cytoskeletal structures and large proteins. (Fig 9.8) - Three signal model of naïve Th and Tc cell activation. Signal 2 for T cell activation - In most cases, the engagement of TCRs by pMHCs is not sufficient to fully activate a naïve Th or Tc cell, and signal 2 in the form of costimulatory 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 B7 (CD80 or CD86) on the surface of DC to convey the signal 2 to T cell. Signal 2 for T cell activation  - Initially DC does not express optimal level of B7. However, delivery of signal 1 to T cells & initial CD28 binding to B7 upregulate the expression of CD40L on T cells. - Once CD40L on Th cell engages CD40 expressed by DC, the DC greatly increases its B7 expression and its binding to CD28 on Th cell & as result vigorous signal 2 is delivered. - Delivery of Signal 2 enhances the activating intracellular signaling induced by signal 1. Signal 2 for T cell activation - Tc cells upregulate CD28 but most Tc do not express CD40L after receiving signal 1. Thus, Tc cells cannot induce a DC to initiate CD40 signaling & upregulate B7 expression. - Instead, Tc cells rely on CD28 engagement resulting from interaction with a DC that already expresses B7 due to a previous interaction with Ag-activated Th cell. - It is believed 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 both Th and Tc cells, CD28 signaling lowers the T cell activation threshold necessary to activate new gene transcription and push it to proliferate and differentiate. Signal 2 for T cell activation ON EXAM - In the absence of CD28 costimulation (signal 2), naïve T cells are anergized instead of activated and fail to respond to pMHC. This is a very important concept in T cell biology. - Costimulation via CD28/B7 interaction (signal 2) has several molecular effects: - 1. IL-2R expression is induced on T cell surface, allowing the cell to receive signal 3. - 2. Th cells start to secrete large quantities of IL-2 and other important cytokines and chemokines. - 3. The expression or upregulation of additional costimulatory and regulatory molecules is induced in both Th and Tc cells. - 4. 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 two most important negative regulators of T cell activation are PD-1 (programmed death-1) and CTLA-4 (cytotoxic T lymphocyte associated molecule 4). - Important regulators Negative regulation of signal 2 - 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. Negative regulation of Signal 2 - 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 & CTLA-4 Question on EXAM!! Where is CD28? - 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 & activated B cells. - CD28 and CTLA-4 (CD80 or CD86) 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’. Signal 3 for T cell activation - 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. T helper (Th) cell differentiation - When a naïve Th cell is fully activated, it start producing copious amount of IL-2 and proliferate vigorously. The progeny generated are called Th0 cells.  - After 48-72 hrs 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 are Th9, Th22 & follicular Th (fTh) cells. - Th0 cells can also generate ‘induced regulatory T cells’ (iTreg) which play important role in peripheral tolerance. T helper (Th) cell differentiation - Type of effector Th subset generated from Th0 cell is determined by o 1. Cytokines & other factors present in the immediate microenvironment. o 2. The nature of the DC by which the naïve 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 & deliver signals that direct Th cell differentiation such that Th effectors suited for eliminating the pathogen are produced. T helper (Th) cell differentiation - 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 & 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 naïve T cells) by an APC activate these Th effectors & cause them to secrete subset- specific panels of cytokines and mediate the effector function. Th effector cell differentiation & functions Fig 9.9. - These differentiation paths are not fixed for life of a T cell clone, & one type of effector can become another type if circumstances change & its transcription program shifts in response. - Read details from the text. - Get signal 1 then 2 then 3 - Differentiate into different effector cells - REMEMBER CYTOKINES - STAT4 lymph node or tissue where antigens are present must be activated again by antigen upon activation expresses T-bet making them Th1 making IFNy (mediate cell immunity macrophages and dendritic cells activated.) can also produce IL-12 and IL-27 - IL-4 (important for isotype switching) leads to production of STAT6 which when activated again and expresses GATA-3 making them Th2 cells. The cells produce IL-4 IL-5 and IL-13 - 3 cytokines needed for STAT3 IRF4 and when they are activated again becomes a RORyt which is a Th17 cell producing IL-17 IL-22 IL-21 and IL-26 - STAT5 which when activated again and produces Foxp3 and is called a iTreg cell which produces IL-10 and TGFB for peripheral tolerance o iTreg T regulatory cell Th1 Cells - Intracellular pathogens such as viruses and intracellular bacteria trigger macrophages and DCs to produce IFN-g, IL12 and IL-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 & activate transcription factor T-bet. - T-bet drives IFN-g production. - Th1 cells oppose Th2 cells - Most extracellular pathogens do not induce IL-12 production by macrophages & DCs. - Instead, they stimulate an unknown cell type (might be a mast cell or NKT cells) 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 Ag & activate 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 ‘crossregulate’ each other. - The cytokines they secrete typically enhance their own differentiation & expansion & inhibit commitment to other helper T-cell lineage. - This effect is known as cross-regulation. - This is particularly true of Th1 & Th2 pair as well as Th17 & iTreg pair. - IL-4 & IL-5 produced by Th2 cells suppress the expansion of TH1 population. - IFN-y produced by Th1 cells inhibits the expansion of TH2 population. Th17 cells - Th17 effector cells counter infections of the skin and mucosae (particularly lung & intestine) that are initiated by certain species of extracellular bacteria and fungi. - Th0 cells exposed to a combination of immunosuppressive cytokine TGF-b 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. Th17 cells  - In the inflammatory site, antigen-stimulated Th17 effectors activate transcription factor RORgt that drives production of IL-17, IL-21, IL-22 and IL-26. - Both IFN-g (produced by Th1) and IL-4 (produced by Th2) suppress TH17 differentiation. - Th17 cells play important role in autoimmune and/or autoinflammatory diseases in both human and mice. Induced regulatory T cells (iTreg) cells - Th0 cells can differentiate into ‘induced regulatory T cells’ (iTreg) cells if exposed to TGFb plus IL-2. - Exposure to these cytokines (TGFb plus IL-2) 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 & TGFb. - Both these cytokines (IL-10 & TGFb) then act on other effector T cells to curtail their responses. Induced regulatory T cells (iTreg) cells - IL-6 and IL-21 are potent repressors of TGFb-driven Foxp3 expression. - Thus, Th0 cells exposed to TGFb are induced to become Th17 cells rather than iTreg if IL-6 and/or IL-21 is also present. - The balance between the generation of Th17 cells and iTreg cells is fine tuned by the surrounding environment Activation of Th effector cells - Localization: o After differentiation, Th effector cells may remain in the LN to provide T cell help to naïve Tc cells in the paracortex and naïve B cells in the primary follicles. o Alternatively, Th effector cells may leave LN and go to other tissues (under the skin & mucosa) or site of inflammation. o All Th effector cells initially express CCR7 which permit the migration of the effector T cells from paracortex to the primary lymphoid follicles where naïve B cells are present. Activation of Th effector cells  - Localization: o 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. Activation of Th effector cells - Interaction with APCs: o Effector Th cells encountering with APCs either in the LN or at the site of attack are activated essentially in the same way as naïve T cells but with some important differences. o Compared to naïve T cells, effector Th cells express higher levels of adhesion molecules which facilitates rapid and more effective TCR triggering. o Thus, effector T cells are activated by significantly lower quantities of antigen/pMHC compared to naïve cells. ▪ If binding is effective only need small amount of antigen Activation of Th effector cells - Interaction with APCs: o For Th effector cells activation, far less costimulation by APC is required. o Thus, these cells respond efficiently to pMHC presented by DCs, macrophages or B cells or by non-hematopoietic cells such as gut or skin epithelial cells. o In general, B cells are the principal APCs presenting Ag to Th2 cells whereas macrophages predominate as APCs in interaction with Th1 and Th17 cells. ▪ B cell itself can present Activation of Th effector cells - Differential costimulatory requirements: o While CD28-B7 is the major costimulatory mechanisms for naïve T cell activation, effector T cells appear to require only low level of CD28-B7 costimulation for activation.  o CD28 signaling downregulates the expression of chemokine receptors & this prevents the effector cells from migrating away from the site where Ag has been encountered. Activation of Th effector cells - Differential costimulatory requirements: o Two supplementary costimulatory pairs important for effector T cells activation are OX40-OX40L & ICOS-ICOSL. o In Th1 responses, OX40 expressed on a Th1 cell surface binds to OX40 ligand (OX40L) expressed on APCs. o Similarly, inducible costimulatory (ICOS) molecule, which is upregulated on Th2 and TH17 cells only after activation, binds to ICOS ligand (ICOSL) expressed on APCs. o ICOS is rarely expressed by Th1 effectors. Superantigens - Bypass normal immune recognition requirement by nonspecifically ligating TCR and MHC simultaneously. - Are viral or bacterial proteins that bind simultaneously to the vb domain of TCR & to the a chain of MHC class II. - This produces an activating signal that induces T-cell activation & proliferation; however, this does not bypass the need for costimulation. - Two types: - 1. Endogenous superantigens: are cell-membrane proteins encoded by certain viruses that infect mammalian cells e.g. Minor lymphocyte stimulating (Mls) determinants from mouse mammary tumor virus (MTV). - 2. Exogenous superantigens: soluble proteins secreted by bacteria e.g. variety of exotoxins secreted by Gram positive bacteria such as o Staphylococcal enterotoxins o Toxic-shock syndrome toxin (TSST1) o Exofoliative-dermatitis toxin (ExFT) o Mycoplasma-arthritidis toxin (MAS) o Streptocccal pyrogenic exotoxins. - T cell activation by superantigens is polyclonal & results in overproduction of Th- cell cytokines leading to systemic toxicity - Chains need to interacrt - Ligate two chains signal goes through - Cytokines are important  - Over produced they can become toxic - Overproduction of cytokines leads to systemic toxicity Th1 effector functions - Supply help to Tc and B cells and provide cell mediated and humoral defense against intracellular pathogens. - Play important role in delayed type hypersensitivity (DTH). o Allergies - Secrete a panel of cytokines dominated by IL-2, IFN-g and lymphotoxin (LT) (sometime called TH1 cytokines). - IL-2 drives T & B cell proliferation & enhances reactive O2 species intermediates (ROI) production by macrophages. - IFN-g and LT activate macrophages, increase phagocytosis and upregulate nitric oxide (NO) production. - IFN-g increases NK cells and macrophages expression of high affinity FcgR molecules that promote ADCC. o Bind to gammagobulins o Th1 cells have many functions o Isotype switching between cells Th1 effector functions - IFN-g promotes the isotype switching of B cells to IgG1 and IG3 in humans and IgG2a & IgG3 Abs in mice which are effective against intracellular pathogens. - IgG1 & IgG3 Abs in humans are best suited for opsonization, phagocytosis & complement activation. o Increase activity - IgG1 & IgG3 Abs bind with high affinity to FcR on NK cells, macrophages & other phagocytes further increasing 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 IL2 and by providing CD40/D40L contacts for DC licensing. o Important 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 (sometime called Th2 cytokines). o Help b cells to isotype switch  - 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. o Not very important in inflammation can downregulate it - 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. Th2 effector functions - 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 & 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. – Abrogate macrophage 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, IL-22 & IL-26 production. - The massive inflammatory responses mounted by these cells, are designed to protect mucosal surfaces against pathogens that resist assault by Th1 and Th2 cells. Th17 effector functions - Pathogens that trigger strong TH17 cell-mediated responses include - Bacteria such as o Borrelia burgdorferi and  o Klebsiella pneumoniae - Fungal species such as o Pneumocystitis carinii and - Candida albicans. - IL-17 produced by Th17 cells induces nearby nonhematopoietic cells to produce destructive proinflammatory cytokines such as TNF-alpha, IL-1 and IL-6 The effector cell cross regulation & amplification - Because of the cytokines they produce, various Th subsets can cross-regulate each other’s differentiation & activities. - TH1 cell produce large amount of IL-2 which promotes the proliferation of both Th1 and Th2 cells. - However, IFN-g produced by Th1 cells o has a direct anti-proliferative activity on Th2 cells & inhibits their differentiation. o Induces IL-12 production by macrophages which promotes Th1 differentiation. The effector cell cross regulation & amplification - IL-4, IL-13 and IL-10 produced by Th2 cells o IL-4 most important o Suppress IFN-y and IL-2 production by Th1 cells. o Inhibit Th1 differentiation o downregulate macrophage IL-12 production o IL-4 from Th2 cells promotes continued differentiation of Th2 subsets. - IL-21 (not IL-17) produced by activated Th17 cells support continued Th17 differentiation. o Produced by th17 cells o Helps keep them in group - IL-21 also represses 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 favors Th1 response.  - Conversely, invasion by extracellular pathogens most often promotes the development of Th2 response, or depending upon the specific invader, a Th17 response. Nature of Th responses - 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 generalization; however, the overall phenotype of an immune response to a given pathogen can change with time e.g. o In malarial infection both Th1 and Th2 responses are induced during the full course of infection. - Depending on what stage od disease you are on may see Th1 or Th2 depends on stage for immune response Overview: Tc cell differentiation & effector function - Cytotoxic T cell (Tc) responses can occur in 5 stages: - 1. Activation of naive Tc cell by a licensed DC in a secondary lymphoid tissue. - 2. Proliferation & differentiation of the activated Tc cell into daughter cells called ‘Pre-cytotoxic T lymphocytes’ (pre-CTLs). o Do not become effector cells they are pre-ctl cells - 3. Differentiation of a pre-CTL in an inflammatory site into an ‘armed’ CTL. o Start to have arms that are the weapons used to kill - 4. Activation of the armed CTL by encounter with specific non-self peptide presented by MHC Class I on a target cell. o Once armed antigen needs to come and tell what cell to kill - 5. CTL-mediated destruction of the target cell as well as other cells displaying the identical pMHC. Can then kill the cell o Tc cell ▪ Licensed dc cells ▪ Get IL2 donated do not produce ▪ In a pre-active phase ▪ Kill cells so require more steps Overview: Tc cell differentiation & effector function - Target cells of CTLs include cells infected with intracellularly replicating pathogens, tumor cells & foreign cells as part of a tissue transplant. o Kill foreign cells in not part of MHC  o Kills cells in organ transplants o T cell gets activated then pre cytotoxic lymphocyte they do not have the power to kill need to get armed first - An activated Tc cells 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 - Produced by activated macrophages and DCs. - Activation of CTL Mechanism of target cell destruction - 3 different pathways - Depends on pathogen Mechanism of target cell destruction - The target cell destruction by CTLs can occur via the - 1. Granule exocytosis pathway o Granules formed after activation o Certain type of enzymes inside o When cell comes inContact they srat to get thrown o Fuse with membrane and granules are fused with membrane - 2. Fas pathway and/or o Cell will die by apoptosis o Fas ligand expressed on the CTL surface o Interacts with Fas protein o Indices apoptosis - 3. Release of cytotoxic cytokines such as TNF and LT. o Produce cytokines o On target cells there is cytokine receptors o When binding occurs apoptosis is induced - 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 CTLtarget 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. o Both are inside granules o Enter the cell o Perforin makes holes expelling cell content (pore forming) o Granzymes induce apoptosis Granule exocytosis pathway - 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 caspasemediated 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’. CTL Killing Mechanism Fas Pathway - Fas is a transmembrane ‘death receptor’ that is widely expressed on mammalian ells. - Naïve Tc cells do not express FasL but after 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 (TNFR1) on the target cell surface. - IFN-y produced by CTLs indirectly help B cells in producing Abs which can increase ADCC and upregulate 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 o Want to downregulate when not needed o Certain molecules do this - 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: - 1. Activation-induced cell death (AICD), - 2. Cytokine “withdrawal” and - 3. T cell clonal exhaustion. Memory T cells - Remember and mount greater immune response - Between naïve and effector 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 cells - 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 & 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. - Helps with isotype switching - Second response is faster and stronger Types of memory T cells - There are at least two major types of memory T cells:  o Effector memory T (Tem) cells (shorter life span than Tcm) and o Central memory T (Tcm) cells. - These cells differ in some important properties. - In the absence of specific Ag, Tem cells have a shorter life span than Tcm cells. - Tcm cells express high levels of LN homing molecules CD62L and CCR7. - Thus Tcm cells tend to migrate through LNs & other secondary lymphoid tissues, thereby maintaining a long-term central reservoir of memory cells. Types of memory T cells - In contrast, Tem cells express only low level of CD62L and CCR7. - Thus Tem cells mainly circulate through non-lymphoid tissues where pathogens are likely to attack a second time. - Tem cells constantly patrol the peripheral tissues and are able to migrate quickly into site of infection. Memory T cell activation - Memory T cells have less stringent activation requirement than naïve T cells. o Activated easier than naïve T cells o Activated by many antigen presenting 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. Memory T cell activation - 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. Memory T cell differentiation and life span - 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. Lymphocyte tolerance in the periphery - The adaptive immune responses in peripheral tissues is tightly controlled in two ways. - 1. Tolerance: prevents lymphocyte activation. - 2. Immune regulation: control actions of effector cells. - Get rid on many cells during development - Central tolerance in thymus - Peripheral tolerance Lymphocyte tolerance in the periphery - Tolerance: is manifested when the interaction between a mature peripheral lymphocyte & its cognate Ag does not result in activation of that lymphocyte. - In peripheral tolerance, lymphocyte - either undergoes apoptosis - or is functionally inactivated (i.e. lymphocyte has been ‘tolerized’). - This maintains tolerance to self-tissues. - Peripheral ‘self tolerance’ is vital as it prevents the activation of autoreactive lymphocytes that have escaped central tolerance mechanisms. Lymphocyte tolerance in the periphery - Peripheral tolerance to innocuous (harmless) non-self Ags 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. - B7 important for activation - Harmful antigens are recognized in body and danger is sensed then second signal can come in - Want to down regulate immune system Lymphocyte tolerance in the periphery - Success in implementing both tolerance and regulatory mechanism ensures that the host focuses the power of the immune response on harmful non-self Ags. - Failure to this paves the way to uncontrolled tissue damage and the potential development of autoimmune diseases. Lymphocyte tolerance in the periphery - As BCRs & TCRs are randomly generated, a certain number of lymphocytes may bear receptors against self Ags. - 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 Ags. - 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: o 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. o 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. o 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. o Need something dangerous for DC to become mature T cell tolerance in the periphery - DC-mediated tolerization: o Immature DCs are broadly distributed in the peripheral tissues & constantly take up Ags from their surroundings. o In the absence of pathogen attack or injury, these DCs only handles self or innocuous non-self Ags. o 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 Ags. - Without signal 2, signal 3 can not be delivered & T cell is not activated despite receiving signal 1. DC-mediated tolerization - There are two main processes by which tolerogenic DCs inactivate naïve T cells: - 1. Clonal deletion: o The most important mechanism by which peripheral tolerance is maintained by clonal deletion of autoreactive Th cells. o In this tolerogenic DCs usually induce apoptosis in autoreactive T cells. - 2. Anergization: o Those autoreactive Th cells that receive signal 1 alone from a tolerogenic DC but do not undergo apoptosis are ‘anergized’. o An anergic Th cell can maintain its unresponsive state for up to several month o Cells stay for a long time o Wont get activated for several 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 & 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 Ag 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 Ag 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. B cell tolerance - 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 activate it - Anergized autoreactive T cell does not deliver signal 2 to B cell & thus B cell is anergized & forced into apoptotic death Cd40 used for isotype switching