Immatue CD8 DP T cells (HSA high) Semi-mature CD4 SP T cells (HSA high) Mature low high SP T cells (HSA low) 62.4 HSAIntro figure 1. Thymocyte subsets in the thymus. Thymocyte subsets inthe thymus include immature CD4+CD8+ DP T cells (HSA high), semi-mature CD4+ or CD8+ SP T cells (HSA high), and mature SP T cells (HSAlow). Immature DP T cells constitute the majority of thymocytes, and morethan 95% die by neglect. Only less than 5% go through positive selectionand differentiate into SP T cells. Semi-mature T cells are differentiatingfrom immature DP T cells to mature CD4+T cells. Among those, cellsexpressing TCR with too high avidity to self peptide-MHC complexes areeliminated by negative selection. Thymocytes that passed through negativeselection become mature T cells, and go into the circulation.
cortex Medulla Positive Selection DP HSA HSA Semi CD8 CD4 CD4 or CD8 TCR TCR Presentation peptide MHC One peptide-MHC complex 104 - 106 cells Cortical Thymic Epithelial Cells (cTECs)Intro figure 2. Positive selection in the cortex. Immature DP T cellsundergo positive selection, by the weak interaction of TCR with selfpeptide-MHC complexes expressed on cTECs. While being selectedpositively, immature DP T cells get into the medulla and become semi-mature T cells. One peptide-MHC complexes could generate about 104 -106 cells positively.cTECs: cortical thymic epithelial cells.
Semi HSA CD4 or CD8 TCR CD28 Negative selection Direct Indirect Direct Presentation Presentation Presentation peptide peptide peptide AIR MHC MHC MHC E Medullaly DCs DCs Thymic Epithelial Cells nesting in medulla from periphery (mTECs) BM-derived APCsIntro figure 3. Negative selection in the medulla. In negative selection,three types of APCs may play important roles. mTECs express selfantigens promiscuosly. In addition, they express a large part of TSAsdepending on Aire. Self-antigens expressed by mTECs are directlypresented to semi-mature T cells (left). However, because the capacity ofmTEC as a tolerance inducer is thought to be not enough, it is thought thatBM-derived APCs, especially DCs, play a key role for induction of negativeselection in aid of mTECs. DCs nesting in the medulla pick up antigensexpressed by mTECs and cross-present them to semi-mature T cells(center). Furthermore, circulating DCs home to the medulla from peripherywith peripheral antigens and present them directly (right).TSAs: tissue specific antigens. Aire: autoimmune regulator.
Weak TCR ligation Strong TCR ligation TCR signal TCR/CD28 signals Strong TCR signal TCR/CD28 signals TCRCD28CD4 Semi HSA no apoptosis apoptosis apoptosis apoptosis Fas independent Fas dependent Fas dependentIntro figure 4. Two types of apoptosis in semi-mature T cells.Apoptosis of semi-mature T cells is sorted into Fas dependent or not,based on the strength of TCR signal. When TCR signal is weak, the signalvia the TCR alone has no effect on the fate of semi-mature T cells,whereas TCR/CD28 signals induces apoptosis, independent of Fas (left). Incase of strong TCR signal, semi-mature T cells result in Fas-dependentapoptosis irrespective of CD28 signal, dependent on Fas (right).
Immature T cells Mature T cells TCR signal TCR/CD28 signals Strong TCR signal TCR/CD28 signals TCR CD28 TCR CD28 CD4CD4 CD8 DP HSA Naive no apoptosis apoptosis apoptosis activation Fas independent Fas dependent Intro figure 5. CD 28 signals in other T cell subsets. In immature T cells, TCR signal alone has no effect, whereas TCR/CD28 signals induce apoptosis, independent of Fas (left). In mature T cells, strong TCR signal alone induces Fas dependent apoptosis, but TCR/CD28 signals results in activation, such as proliferation and cytokine productions (right).
Immature T cells Semi-mature T cells Mature T cells TCR/CD28 signals TCR/CD28 signals TCR/CD28 signals TCR CD28 CD4 Naive apoptosis apoptosis activation Pro-apoptotic Pro-apoptotic Anti-apoptoticIntro figure 6. CD28 signals in each T cell subset. CD28 signals functionpro- or anti- apoptotic, depending on the differential developmental stagesof T cells. Immature DP T cells and semimature SP T cells (in case of weakTCR signal) results in apoptosis by TCR/CD28 signals (left and center). Onthe other hand, TCR/CD28 signals in mature T cells has anti-apoptoticfunctions (right).
PI3K (p85) SH3 RHO-GAP SH2 SH2 Grb2 SH3 SH2 SH3 Gads SH3 SH2 P/Q SH3 Tec PH TH SH3 SH2 Kinase Itk PH TH SH3 SH2 Kinase Lck SH3 SH2 KinaseIntro figure 7. CD28 signaling molecules. CD28 signaling moleculesinclude PI3K (p85), Grb2, and Gads as adaptor, and Tec, Itk, and Lck asprotein tyrosine kinases. These molecules possess SH2 and SH3 domainsrecuried for association with CD28.
CD28 p85 Itk Lck YMNM PXXP PXXP P Grb2 Gads Tec YMNM motif Two PXXP motifs p85 YXXM P Itk Grb2 Lck PXXP YXNX P Tec Gads with SH2 with SH3Intro figure 8. Cytoplasmic portion of CD28. The YMNM motif and twoPXXP motifs in the cytoplasmic portion of CD28 play critical roles in CD28signaling. Upon phosphorylation of Y in YMNM, p85 bind to YXXM motif,whereas Grb2 and Gads associate with YXNX motif. SH3 domain of Gadsalso interacts with the N- turminus of two PXXP motifs. Itk and Lck arerecruited to the N- and C- turminus of two PXXP motifs, respectively,through SH3 domain. Tec binds to CD28 through SH3 domain, and thisassociation requires two PXXP motifs.
+ & - controls YMNM mutants Proline mutantsWT YF nPA p85 Itk Lck Itk Lck p85 Lck YMNM PRRP PYAP FMNM PRRP PYAP pYMNM ARRA PYAPp Grb2 Gads Tec Tec Grb2KO NA cPA p85 Itk Lck p85 Itk p YMAM PRRP PYAP p YMNM PRRP AYAA Tec Grb2TM ML ncPA Itk Lck p85 p YMNL PRRP PYAP p YMNM ARRA AYAA Grb2 Gads Tec Grb2 Intro Figure 9. Mutant CD28 transgenic mice. Mutant CD28 molecules with different mutations in the CD28 cytoplasmic tail are below: CD28 with an unmutated cytosolic tail (WT); CD28 with a mutation in Y170F that abrogated p85, Grb2, and Gads bindings (YF); CD28 with a mutation in N172A that abrogated Grb2 and Gads bindings (NA); CD28 with a mutation in M173L that abrogated p85 binding (ML); CD28 with tail less form, lacking the entire region under the transmembrane (CD28-TM). CD28 with mutations in P175A and P178A (N-terminal proline residues) that abrogated Gads, Itk, and Tec bindings (nPA); CD28 with mutations in P187A and P190A (C-terminal proline residues) that abrogated Tec and Lck bindings (cPA); CD28 with mutations in P175A, P178A, P187A and P190A (both N- and C-terminal proline residues) that abrogated bindings of Gads, Itk, Tec, and Lck (ncPA). mutant CD28 were expressed under the control of human CD2 promoter and enhancer.