1T Cell Development II: Positiveand Negative SelectionCD4CD888813- production of T cell receptors for antigen, byrearrangement of the TCR genes- selection of T cells that can interact effectivelywith self-MHC with moderate affinityThe two phases of thymic development:Second phase of thymic development: selection of T cells that can interact withself MHC and antigen• This applies only to αβ TCR-bearing cells (>95% of Tcells).(γδ T cells are not restricted to interactions with MHCclass I or class II molecules)• This phase of T cell development consists of two steps:– positive selection (TCR that can interact withself-MHC)– negative selection (eliminate self-reactive cells that arestrongly stimulated by MHC + self)!Double PositivesCD8 Single PositivesCD4 Single PositivesDouble NegativesThe thymus subsets characterized by CD4 and CD8expression2Cells MHC Class I MHC Class IIT cells +++ -B cells +++ +++Macrophages +++ +++Dendritic cells +++ +++Thymic Epithelia + +++Neutrophils +++ -Hepatocytes + -Kidney + -Muscle +/- -Red blood cells --MHC ExpressionAPCs• Positive selection refers to the selection of thymocytesthat are able to bind to, and interact with, self-MHCmolecules present on thymic cortical epithelial cells• In positive selection developing thymocytes continue tolive if they bind MHC well enough to receive a signalthrough their TCR. If they don’t bind they die by neglect(about 95% of them).• Positive selection leads to MHC restrictionInteraction with Cortical epithelial cells--> Positive SelectionPositive selection takes place in the cortex of the thymus lobules:Positive Selection! TCR α chain rearrangements can continue duringpositive selection --> Why?!!3TCRα locus-- replacementrearrangementCαJαVαThe expression of either CD4 or CD8 isdetermined during positive selection•Those cells thathave a TCRthat binds toMHC class Iend up as CD8single-positivecells•Those cells thathave a TCRthat binds toMHC class IIend up as CD4single-positivecells•Recruitment of the CD4 or CD8 coreceptors inducesdistinct signals that enable the differentiating cell to coupleits TCR specificity with a program of maturation to anappropriate effector lineage•The signal delivered by engagement of the CD4 coreceptorand the TCR appears to be stronger and/or longer lastingthan that delivered through engagement of the CD8coreceptor and the TCR.The expression of either CD4 or CD8 isdetermined during positive selectionIgnores the uselessSelects for the usefulDestroys the harmful4• Selection of thymocytes that interact with self-MHCmolecules leads to MHC restriction• Thymocytes that do not interact with self MHC die byneglect.• Positive selection is mediated by the interactions ofthymocytes with thymic cortical epithelial cells.• Thymic cortical epithelial cells express both MHC class Iand MHC class II molecules, complexed with self-peptides.Positive Selectionplate at 1 cell/wellgrow and expandTest for antigenspecificityclone the TCR alpha and beta chain genesfrom the T cell cloneAntigen (male H2b cells)T cells + DCs + ovalbuminRest and restimulatewith Ag andcytokines, rest,repeat9 daysT cell receptor transgenic mice:Female H2b mouseT cell receptoralpha- & beta-chain genesspecific for MHC class I orMHC class IIAll T cells will express the same TCRαβ receptorAnti HY transgenic mouseH-2b restricted anti-H-Y TCRtransgenes analyzed in a H-2b femaleTCR specificity shuntsdevelopment in thedirection of CD8 SPcells5Presence of a second MHChaplotype doesn’t preventsurvival and selectionNon-selection: H-2b restrictedanti-H-Y TCR transgenesanalyzed in a H-2d femaleIf TCR can’t recognizeself-MHC peptidecomplex, it dies byneglect.CD4/CD8 FACS profile of thymocytes from a MHC-I deficient mice Wild type MHC-I DeficientThymocytes undergo negative selection in the medullary region:Negative Selection6• Negative selection refers to the elimination of thosethymocytes that bind to self-MHC molecules + selfwith high affinity.• In negative selection developing thymocytes die if theybind MHC + self peptides too well (strongly enough sothat they would be activated by this interaction, viasignaling through their TCR).Negative Selection• There, Thymocytes interact with antigen-presenting cells(dendritic cells, macrophages) that express self-antigens +MHC class I or MHC class II molecules. --> Origin of theAPCs? (Also to a lesser extent interaction with medullaryepithelial cells)• Thymocytes that bind to self + MHC too strongly areeliminated as possibly self-reactive cells, and undergoapoptosis. --> Why?• Some T cells are reactive with self molecules that are notexpressed in the thymus --> problem?Negative SelectionWhat about genes that are not expressed in the thymus?How do you get rid of T cells that recognize peptidesderived from tissue specific proteins?Pancreas, eye, heart, intestine, etc…Medullary thymic epithelial cells express manyunexpected “tissue specific” proteins.Expression of these genes requires a transcriptionfactor named Aire.Peptides from these proteins are presented todeveloping thymocytes and mediate negativeselection of potentially self-reactive TCRs.APECED (autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy): a human autosomal recessivedisorder --> mutations in AIRE7Ignores the uselessSelects for the usefulDestroys the harmfulNegative selection: H-2b restricted anti-H-YTCR transgenes analyzed in a H-2b maleHigh affinity binding of DP TCR+ T cell to self-peptide MHC complex leads to negativeselection.SP cells which do exist are MHC class II restricted CD4+ cells.How does TCR signaling lead to eitherpositive or negative selection?Different peptides will interact with a given TCR with different affinitiesThe combination of this particular affinity and the number of peptidesequals the avidity of the TCR for that particular peptide.The avidity between a particular TCR and different peptides will resultin distinct signals.These signals lead to unique developmental outcomespositive vs. negative selectionHow exactly these differences are translated into different outcomesremains controversial and an area of intense researchHow does TCR signaling lead to eitherpositive or negative selection?8Fetal thymic organ cultureFetal mouseIncubate 5-6 days- peptide + peptideThymic lobesDissociate thymic lobes and analyze cellsAnti-OVApTCR transgenicmouse (class Irestricted TCR)OT-1 TCR transgenic mice(anti- Ova-peptide + Kb)Variants of Ova (SIINFEKL) +