T Cell Development II: Positive and Negative Selection

T Cell Development II: Positive and Negative Selection 8 88 The two phases of thymic development: - production of T cell receptors for antigen, by rearrangement of the TCR genes CD4 - selection of T cells that can interact effectively with self-mhc with moderate affinity 3 CD8 1 Second phase of thymic development: selection of T cells that can interact with self MHC and antigen The thymus subsets characterized by CD4 and CD8 expression This applies only to αβ TCR-bearing cells (>95% of T cells).(γδ T cells are not restricted to interactions with MHC class I or class II molecules) CD4 Single Positives Double Positives This phase of T cell development consists of two steps: positive selection (TCR that can interact with self-mhc) negative selection (eliminate self-reactive cells that are strongly stimulated by MHC + self) Double Negatives CD8 Single Positives 1

MHC Expression Cells MHC Class I MHC Class II T cells +++ - B cells +++ +++ Macrophages +++ +++ Dendritic cells +++ +++ Thymic Epithelia + +++ Neutrophils +++ - Hepatocytes + - Kidney + - Muscle +/- - Red blood cells - - APCs Interaction with Cortical epithelial cells --> Positive Selection Positive selection takes place in the cortex of the thymus lobules: Positive Selection Positive selection refers to the selection of thymocytes that are able to bind to, and interact with, self-mhc molecules present on thymic cortical epithelial cells In positive selection developing thymocytes continue to live if they bind MHC well enough to receive a signal through their TCR. If they don t bind they die by neglect (about 95% of them). Positive selection leads to MHC restriction TCR α chain rearrangements can continue during positive selection --> Why? 2

TCRα locus-- replacement rearrangement The expression of either CD4 or CD8 is determined during positive selection Vα Jα Cα Those cells that have a TCR that binds to MHC class I end up as CD8 single-positive cells Those cells that have a TCR that binds to MHC class II end up as CD4 single-positive cells The expression of either CD4 or CD8 is determined during positive selection Recruitment of the CD4 or CD8 coreceptors induces distinct signals that enable the differentiating cell to couple its TCR specificity with a program of maturation to an appropriate effector lineage Selects for the useful Ignores the useless The signal delivered by engagement of the CD4 coreceptor and the TCR appears to be stronger and/or longer lasting than that delivered through engagement of the CD8 coreceptor and the TCR. Destroys the harmful 3

Positive Selection Selection of thymocytes that interact with self-mhc molecules leads to MHC restriction Thymocytes that do not interact with self MHC die by neglect. Positive selection is mediated by the interactions of thymocytes with thymic cortical epithelial cells. Thymic cortical epithelial cells express both MHC class I and MHC class II molecules, complexed with selfpeptides. Antigen (male H2b cells) Female H2b mouse plate at 1 cell/well T cell receptor transgenic mice: 9 days T cells + DCs + ovalbumin Test for antigen specificity grow and expand clone the TCR alpha and beta chain genes from the T cell clone Rest and restimulate with Ag and cytokines, rest, repeat T cell receptor alpha- & beta-chain genes specific for MHC class I or MHC class II H-2 b restricted anti-h-y TCR transgenes analyzed in a H-2 b female Anti HY transgenic mouse All T cells will express the same TCRαβ receptor TCR specificity shunts development in the direction of CD8 SP cells 4

Presence of a second MHC haplotype doesn t prevent survival and selection Non-selection: H-2 b restricted anti-h-y TCR transgenes analyzed in a H-2 d female If TCR can t recognize self-mhc peptide complex, it dies by neglect. CD4/CD8 FACS profile of thymocytes from a MHC-I deficient mice Negative Selection Wild type MHC-I Deficient Thymocytes undergo negative selection in the medullary region: 5

Negative Selection Negative selection refers to the elimination of those thymocytes that bind to self-mhc molecules + self with high affinity. In negative selection developing thymocytes die if they bind MHC + self peptides too well (strongly enough so that they would be activated by this interaction, via signaling 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 the APCs? (Also to a lesser extent interaction with medullary epithelial cells) Thymocytes that bind to self + MHC too strongly are eliminated as possibly self-reactive cells, and undergo apoptosis. --> Why? Some T cells are reactive with self molecules that are not expressed in the thymus --> problem? What about genes that are not expressed in the thymus? How do you get rid of T cells that recognize peptides derived from tissue specific proteins? Pancreas, eye, heart, intestine, etc APECED (autoimmune polyendocrinopathy-candidiasisectodermal dystrophy): a human autosomal recessive disorder --> mutations in AIRE Medullary thymic epithelial cells express many unexpected tissue specific proteins. Expression of these genes requires a transcription factor named Aire. Peptides from these proteins are presented to developing thymocytes and mediate negative selection of potentially self-reactive TCRs. 6

Negative selection: H-2 b restricted anti-h-y TCR transgenes analyzed in a H-2 b male Selects for the useful Ignores the useless Destroys the harmful High affinity binding of DP TCR+ T cell to self-peptide MHC complex leads to negative selection. SP cells which do exist are MHC class II restricted CD4+ cells. How does TCR signaling lead to either positive or negative selection? How does TCR signaling lead to either positive or negative selection? Different peptides will interact with a given TCR with different affinities The combination of this particular affinity and the number of peptides equals the avidity of the TCR for that particular peptide. The avidity between a particular TCR and different peptides will result in distinct signals. These signals lead to unique developmental outcomes positive vs. negative selection How exactly these differences are translated into different outcomes remains controversial and an area of intense research 7

Fetal thymic organ culture Fetal mouse Thymic lobes Anti-OVAp TCR transgenic mouse (class I restricted TCR) OT-1 TCR transgenic mice (anti- Ova-peptide + K b ) - peptide + peptide Incubate 5-6 days Dissociate thymic lobes and analyze cells Variants of Ova (SIINFEKL) + K b Thymic selection Hoquist & Bevan Peptide Sequence β2-microglobulin-deficient β2m-/- β2m+/- TCR 40 kd GAYEFTTL transgenic mouse. None None CDC ASYEFTQL None None PolySER SSYSYSSL Add different None peptides + None β2 microglobulin P7 SIINFEPL Weak to thymic Pos. organ None culture V-OVA RGYNYEKL and assay Positive for selection. None K1 KIINFEKL Positive Negative R4 SIIRFEKL Positive None E1 EIINFEKL Selection Positive correlates with Negative TCR affinity OVAp SIINFE KL for that peptide/mhc Negative complex. Negative Low affinity peptides induce positive selection in FTOCs Positive selection 8

Variants of Ova (SIINFEKL) + K b Thresholds for positive and negative selection Thymic selection Peptide Sequence β2m-/- 40 kd GAYEFTTL None CDC ASYEFTQL None PolySER SSYSYSSL None P7 SIINFEPL Weak Pos. V-OVA RGYNYEKL Positive K1 KIINFEKL Positive R4 SIIRFEKL Positive E1 EIINFEKL Positive OVAp SIINFE KL Negative Summary The avidity of the interaction between a peptide and a TCR determines the outcome for cells expressing that TCR Low avidity peptides lead to death by neglect Intermediate avidity peptides lead to positive selection High avidity peptides lead to negative selection Alloreactivity Reaction of T cells against target cells from genetically non-identical individual of same species. VERY high frequency of allo-reactive T cells in unperturbed mice and humans (~1 in 10 T cells)!!! Why? 9

Alloreactivity= Cross-reactivity Repertoire selected for a certain range of affinity to self-mhc. T cell repertoire has inherent MHC reactivity. Alloreactive cells have not undergone negative selection against foreign MHC-peptide reactivity. Responder cells (viable splenocytes; will divide if they recognize foreign MHC+Peptide Mixed lymphocyte reaction (MLR) Incubate mixture of cells and measure proliferation of responders Tester cells (irradiated splenocytes; will not divide in culture) Mixed lymphocyte reaction (MLR) Skin transplants require MHC identity Parental recipient Skin graft donor F1 hybrid recipient Responder Splenocytes Irradiated Tester Proliferation H-2 b H-2 d ++++ H-2 d H-2 b ++++ H-2 bxd H-2 b - H-2 b H-2 bxd ++++ b/b k/k b/b k/k b/b b/b Parent k/k Parent b/k b/k McDevitt s and Gorer & Snells experiments mapping the genetics of transplantation led to the discovery of the MHC k/k b/k progeny b/k 10