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Specificity, Diversity, and Self-tolerance of T-Cell Receptors by Thymic Selection Andrej Kosmrlj Abhishek Jha Mehran Kardar Eric Huseby Arup K. Chakraborty T Cells orchestrate adaptive immunity, via Receptors that recognize short peptides from pathogen proteins T Cell TCR pMHC Antigen Presenting Cell T Cells Receptors (TCRs) must be: Self-tolerant, i.e. weakly binding to endogenous pMHC, to avoid autoimmune diseases, e.g. Multiple sclerosis, diabetes, … pMHC Diverse, to recognize an evolving landscape of pathogens. Specific/degenerate, to lock on specific pathogen, and tolerate its mutants. What is the shortest peptide length that ensures self-tolerance? n*= 5.3 Length of peptide, n In principle all human peptides can be recognized from sequences of length 5-6. What is the physical mechanism that enables recognition of self-peptides? T Cells are designed in the Thymus where a diverse repertoire of thymocytes is culled by encounters with self pMHC. Positive selection TCR must bind sufficiently strongly to at least one self pMHC (implicated in MHC restriction, and sensitivity). Negative selection TCR must not bind any self pMHC too strongly (deleting autoimmune TCR). Model for TCR selection in the Thymus conserved variable TCR {L} E N 10 U L , J i 1 pMHC {J} MHC i i MiyazawaJernigan peptide Surviving T cells: E > EP for at least one peptide, E<EN for all peptides + ✓ -- E Diversity The selection process biases the composition of amino-acids in mature TCRs: Negative selection leads to a slight preference for weak amino-acids. Frequency of TCR amino-acids from known TCR-pMHC crystal structures (Thermophiles used for ordering, Shakhnovich et al., PLOS, 2007) Frequency in TCR/Frequency in proteome (MJ used for ordering) STRONG WEAK Specificity Eric S. Huseby et al, Cell (2005); Nature Immunol. (2007), compared the T cells of normal mice, with mice genetically engineered to present only one type of peptide in their thymus. T cells selected in the thymus are challenged with an antigenic peptide, and reactive T cells identified. Does a reactive T cell remain reactive upon mutating the peptide’s amino acids? If mutations to an amino acid destroy reactivity with at least half the T cells, the amino-acid is labeled a “hot spot”. • Main results: – Single peptide selection: few hot spots – cross-reactive T cells – Many peptide selection: many hot spots – specific T cells • Specificity to antigen peptide: – Single peptide: mutations don’t matter – cross-reactive T-cells – Many peptides: mutations destroy reactivity – specific T-cells frequency Numerical results for hot-spots† mirror the experimental situation † Hot-spots are defined as locations along the sequence, where mutations of a peptide amino acid destroy reactivity with more than half the reactive T cells Frustration during negative selection constrains TCR sequences One peptide T C R Ep < E < EN selected Frustration during negative selection constrains TCR sequences One peptide T C R EN i U (li , ji ) E p selected Frustration during negative selection constrains TCR sequences One peptide T C R EN i U (li , ji ) E p selected Many peptides T C R E > EN negatively selected Optimizing interactions with one peptide can lead to “bad” interactions with another – FRUSTRATION. Positive selection does not involve frustration. Specificity, Diversity, and Self-tolerance of T-Cell Receptors by Thymic Selection Andrej Kosmrlj Abhishek Jha Mehran Kardar Eric Huseby Arup K. Chakraborty Frustration during negative selection constrains TCR sequences M e P (l ) (1 ( E (l , j ) E N ) p( j ) j 1 M ~ exp ln( 1 ( E (l , j ) E N )) j 1 exp M ln( 1 ( E (l , j ) E N )) 1 (E ) ~ exp exp( bE ) I 10 E (l , j ) U (l , j ) 1 Frustration during negative selection constrains TCR sequences Solution: Prob. that a TCR is selected 10 20 P (l ) exp M hij i 1 j 1 (h11 h12 h13 ..)( h21 h22 ..)...( h20,1 h20, 2 ..) escape hij exp bU (li , j ) p( j ) negative selection lead to choice of TCR amino acids which minimize interactions with other amino acids in En peptides. Frequency in TCR/Frequency in proteome COMPUTATIONAL RESULTS strong weak Robust to variations in potential AMINO ACID FREQUENCIES FROM TCR-PMHC CRYSTAL STRUCTURES Frequency in TCR/Frequency in proteome (Thermophiles used for ordering, Shakhnovich et al., PLOS, 2007) STRONG WEAK Distribution of single site contact energies for selected T cell-antigenic peptide interactions Increased number of moderate interactions Decreased number of strong interactions strong weak Selection with many peptides: TCR sequences contain amino acids that interact with Ag peptide amino acids moderately Modest interactions lead to specificity: Weak multivalent interactions stabilize the interface, making each interaction important for recognition Selection against one peptide – only few important sites T C R How much Free Energy of Binding is due to each amino acid for specific versus degenerate TCRs? P-1E P-1E B3K 506 TCR C57BL/6 derived MHC + peptide specific P2Q P3K P8K P5K YAe62.8 TCR IAb-SP derived MHC + peptide degenerate IAb + 3K KD G IAb + 3K KD G Mutation M kcal/mol Mutation M kcal/mol WT P-1 A P2 A P3 A P5 A P8 A 7 26 278 > 550 > 550 92 0.0 0.7 2.2 > 2.6 > 2.6 1.5 WT P-1 A P2 A P3 A P5 A P8 A 8 9 56 62 > 550 7 0.0 0.0 1.1 1.2 > 2.5 -0.1 TCR sequences are specific, but diverse One type of sequences are selected – ones with a predominance of weak amino acids + + + + - + + + + + - + + + TCR sequences are cross-reactive (degerate) Several antigenic peptides composed of sufficient number of strong amino acids can interact productively with a given TCR ADAPTIVE IMMUNITY IN HEALTH AND DISEASE Flexible system to combat diverse pathogens Mis-regulation leads to autoimmune diseases Multiple Sclerosis Diabetes The challenge: develop principles that govern the emergence of an immune response or autoimmunity and design rules for therapies The problem: underlying mechanisms characterized by cooperative dynamic processes involving many components and a spectrum of length/time scales T CELLS RECOGNIZE SHORT PEPTIDES DERIVED FROM PATHOGEN’S PROTEINS Extraordinary Sensitivity of T cells for Antigen mixture of self (En) and antigenic (Ag/agonist) As few as 3 Ag molecules in a sea of 30,000 En can activate a T cell (Nature, 2002) How does this exquisite sensory apparatus work without frequent “noise”-induced autoimmune responses? T cell sensitivity to Ag pMHC is predicated upon degenerate weak interactions with En pMHC. With Mark Davis’ lab. (Nature Imm., 2004; Nature, 2005; PNAS, 2007; unpublished) INFLUENCE OF PARAMETERS IN MODEL Parameters: Ec, EN, Ep Ec-EN is large (conserved TCR-MHC interactions are very weak) Ec Ep Ec Ep EN EN Small gap Large gap Positive selection limiting; These are the TCRs that are not positively selected (MHC restriction); one or many types of peptides lead to similar consequences Ec-EN is small (conserved TCR-MHC interactions are very strong) Ec EpEN Ep Ec EN Small gap Large gap Negative selection very easy; For many types of peptides almost all T cells are negatively selected. An approach at the intersection of disciplines Life Sciences Physical Sciences Engineering sciences Theory/computation statistical physics chemical kinetics Experiments genetics biochemistry imaging THYMIC SELECTION THRESHOLDS Palmer lab, Nature (2006) strongest weakest Sharp boundary separates positive and negative selectors MOLECULAR MECHANISM (w/A.Weiss’ lab.) A MEMBRANE-PROXIMAL SIGNALING MODULE IMPLICATED (with Jayajit Das, Ashok Prasad; Jeroen Roose, Art Weiss@UCSF) A positive feedback loop results in digital signaling and a sharp threshold Cell, PNAS, in review (2008) SHORT PEPTIDES ARE SUFFICIENT TO RENDER T CELLS SELF-TOLERANT AND REACTIVE TO FOREIGN SHORT PEPTIDES ARE SUFFICIENT TO RENDER T CELLS SELF-TOLERANT AND REACTIVE TO FOREIGN Longer peptides (length = n) will enable sampling of more distinct peptides ~ 20n # Unique peptides of length n SHORT PEPTIDES ARE SUFFICIENT TO RENDER T CELLS SELF-TOLERANT AND REACTIVE TO FOREIGN 8 10 7 10 6 10 5 10 4 10 3 10 2 10 n=5.3 1 10 5 10 15 20 25 Length of peptide, n Condition for plateau: 20 N ; n Total # of unique peptides of size, n ln N n ln 20 Total size of human proteins ACKNOWLEDGMENTS Group members: Collaborators: J. Das, A. Prasad, M. Artomov, C. Govern, H. Zheng, A. Jha, J. Locasale, K. Fowler, M. Wolfson, A. Prabhakar, M. Yang, F. Liang, A. Kosmrlj. M. Davis, A. Shaw, P. Allen, A. Weiss, J. Roose, H. Ploegh M. Dustin, M. Kardar, A Perelson, J. Chen, U. von Andrian, H. Eisen, V. Kuchroo, E. Palmer, E. Huseby. Funding: NIH Immune Response Consortium Is there an optimal peptide length? Fraction of Reactive T Cells 0.05 Constant Negative Selection Threshold E=E ENegENeg=0.4 0.04 Most TCR negatively selected 0.03 0.02 0.01 0.00 0 5 10 15 Length of Peptide n 20 25 IMPORTANCE OF PEPTIDE IN ALLO REACTIVITY DEPENDS UPON THE ALLO MHC Bigger change Modest change Theoretical models and experimental tests (Nature Imm. (2004); Nature (2005); PNAS (2007); unpublished) T cell sensitivity to Ag pMHC is predicated upon degenerate weak interactions with En pMHC that are tuned in the thymus.