David ShabtaiRotation in Mike Dustin’s LabSummer 2005

Review of Pertinent Papers:

Poor immunogenicity of a self/tumor antigen derives from peptide-MHC-I instability and is independent of tolerance.

Yu Z, Theoret MR, Touloukian CE, Surman DR, Garman SC, Feigenbaum L, Baxter TK, Baker BM, Restifo NP.J Clin Invest. 2004 Aug;114(4):551-9. PMID: 15314692

The protein gp100[209-217] cannot induce an effective immune response because it dissociates too quickly from the cognate MHC receptor.

Engineered two mice:o TCR with mouse constant and CD3 regions but human

variable regions; as well as human MHC class I HLA-A2 on a mouse MHC class I K[b]alpha3 domain – this created a mouse that could recognize a human peptide with human variable regions on both the TCR and MHC but still allowed for signaling through the standard mouse pathways. The mouse naturally expresses gp100 in melanocytes.

o This mouse had the same T cell engineering but did not express normal gp100 in the rest of its body. A section of the neomycin gene was inserted into the gp100 gene so that the T cells never encountered the gp100 epitope for which they are specific – there is no possible tolerance or other form of self negative selection (they call it the KO mouse).

They are trying to determine if the gp100 can somehow signal tolerance so that you can have specific T cells for gp100 that are ineffective for some reason. These mice should not have this problem since they never encountered self gp100.

Engineered two proteins for immunizing these animals:o The cognate sequence of gp100 residues 209-217.o A modified protein with a meth at position 2 instead of a

thr.o Once activated, a T cell cannot distinguish between the

two sequences and will react to both in the context of MHC.o The only difference is in the ability of each peptide to

activate the T cells in the context of MHC.

“Despite large numbers of circulating antigen-specific CD8 T cells and the expression of target antigens in normal melanocytes and other pigmented cells in eyes and brain, no significant changes in the color or appearance of hair, skin, or eyes were observed … compared with nontransgenic littermates.” They try to show that “self-tolerance mechanisms partially abrogate the early T cell activation events.”

The native epitope could not immunize either mouse, whereas the modified protein immunized both.

The modified peptide dissociated from the MHC complex with a half life of 27 hr., while the native peptide had a much shorter half life of 3.7 hr.

“Thus the increased affinity resulting from the position 2 modification was predominantly due to slower peptide dissociation.”

Tumor suppression:o Modified peptide with IL-2, but not with the native peptide.o APC pulsed with either peptide and then incubated for 24

hr. before coincubating with T cells – only modified protein was able to induce tumor regression.

o Assume that the time delay between APC ingestion / display of the peptide on the MHC until in arrives in the lymph nodes to present to the T cells is critical since the native protein will dissociate before it ever reaches the presentation stage.

o In discussion: “In addition, we have evidence that IL-2, which has been shown to reverse T cell anergy, can restore the responsiveness of transfected-mouse T cells from WT mice to self antigen stimulation to the same level as that of [cells] … from KO mice.”

Perhaps it would have been important to show that?

Recombinant virus vaccination against "self" antigens using anchor-fixed immunogens.

Irvine KR, Parkhurst MR, Shulman EP, Tupesis JP, Custer M, Touloukian CE, Robbins PF, Yafal AG, Greenhalgh P, Sutmuller RP, Offringa R, Rosenberg SA, Restifo NP.Cancer Res. 1999 Jun 1;59(11):2536-40.

This seems to be the prelude to the 2004 paper. It shows that immunizing with the native gp100 produces no response; only immunization with the modified protein has an effect.

Immunization is not possible with the full length protein; it can only be accomplished with gp100[209-217](2M).

When immunized with the modified protein, the T cells will still respond to native protein induced cytokine release.

“The gp100 specific CD8 T cells were not merely low affinity CTLs that recognized exogenously loaded peptides at nonphysiologically high concentrations, but they also recognized the wild type epitope naturally expressed by the murine melanoma cells.”

“… suggests that the stability of peptides associated with MHC, not simply the peptide levels was critical to induce reactivity against the weak immunogen, gp100.”

Strategic mutations in the class I major histocompatibility complex HLA-A2 independently affect both peptide binding and T cell receptor recognition.

Baxter TK, Gagnon SJ, Davis-Harrison RL, Beck JC, Binz AK, Turner RV, Biddison WE, Baker BM.J Biol Chem. 2004 Jul 9;279(28):29175-84. Epub 2004 May 6.

Exactly what the title says: they created a whole bunch of mutated peptides to try and determine which residues on the MHC complex are important in protein binding and stability in HLA-A2 (which is an MHC class I by the way). They characterize specific mutations on the HLA molecule correlated to binding assays.

Peptide dissociation over time fit a biexponential decay function.

“T cell activity does not correlate with peptide-MHC half life:o The observation that clones RS56 and 10B7 can still

lyse K66R targets indicates that although lysine at position 66 is required for slow peptide dissociation, neither the lysine nor a slow peptide dissociation rate is requisite for T cell recognition.

o These findings demonstrate that the K66A mutation does not diminish the ability of all TCRs to recognize Tax-HLA-A2 despite the increased peptide dissociation rate.”

Affinity testing: “The observation that the K66R mutant binds peptide more tightly than wild type despite a dramatic increase in peptide off rate indicates that the mutation results in either faster peptide association and / or a shift in the conformational equilibrium of the peptide free molecule toward a more peptide accessible state.”

Q: Everybody thinks that peptide-MHC dissociation rates / stability governs immunogenicity – what happened here?

A: “Immunological potency measured in vitro need not correlate with the half life of the peptide MHC complex as is often assumed.

o “The fast peptide dissociation rate resulting from the K66A and K66R mutations is still above some “minimum threshold” for efficient antigen presentation in an in vitro assay of cytotoxicity. Although data that correlate the peptide dissociation rate with in vivo immunogenicity exist, our findings indicate that this correlation does not necessarily hold for in vitro experiments.”

o “However, our data highlight an important difference between in vitro measurements of cytotoxicity, where exogenous peptide is present and can bind at the cell surface, and in vivo situations, where there is little or no exogenous peptide and biding occurs in the tightly regulated environment of the endoplasmic reticulum. Thus, the peptide dissociation rate rather than affinity may be expected to have a greater influence on cell surface presentation levels in vivo than in vitro and vice versa.”

Their claim that dissociation rates are irrelevant for T cell cytotoxicity assays seems to be simply a claim about the dissociation rates themselves. What they represent however, is how long the peptide remains bound to the MHC molecule which is important. Since in their in vitro system, there is always exogenous peptide around, the MHC complex is always loaded and is always set to prime the TCR.

o What it does show however, is that you do not require constant stimulation by the very same MHC-peptide complex to stimulate the TCR. Rather, even if the MHC swaps peptides every so often – cytotoxicity is still induced. This is important regarding the synapse and where the MHC-peptide complexes are found.

Antigenicity and immunogenicity of peptide analogues of a low affinity peptide of the human telomerase reverse transcriptase tumor antigen.

Hernandez J, Schoeder K, Blondelle SE, Pons FG, Lone YC, Simora A, Langlade-Demoyen P, Wilson DB, Zanetti M.Eur J Immunol. 2004 Aug;34(8):2331-41.

Seems like a great idea to try to immunize T cells against telomerase to try to kill cancer cells. “Not surprisingly, over 85% of all types of human tumors express high levels of telomerase activity that is virtually absent in normal tissues.”

“TCR recognition is somewhat degenerate and that a single TCR may recognize different peptide/MHC combinations even when the sequences of these peptides are highly divergent.”

“Our results demonstrate therefore, that peptides with a very low binding affinity for MHC class I can mediate lysis of target cells if present in solution at the time of TCR engagement.”

o This seems in accord with the last thought on the previous paper.

o They note that peptides with similar avidities may still induce different levels of immunogenicity:

“Factors other than binding to the MHC may contribute to their immunogenicity.”

They continue to quote (Mol Immunol. 1994 Aug;31(11):813-22) that states that immunogenicity is a function of MHC peptide dissociation, but perhaps they meant a different paper by the same author: The relationship between class I binding affinity and immunogenicity of potential cytotoxic T cell epitopes (J Immunol. 1994 Dec 15;153(12):5586-92).

The relationship between class I binding affinity and immunogenicity of potential cytotoxic T cell epitopes.

Sette A, Vitiello A, Reherman B, Fowler P, Nayersina R, Kast WM, Melief CJ, Oseroff C, Yuan L, Ruppert J, et al.J Immunol. 1994 Dec 15;153(12):5586-92.

They attempt to find an affinity threshold for peptide-MHC class I binding that elicits a CTL response. They also try to differentiate between two different theories of responder-nonresponder status:

Determinant selection: genetic control of T cell responses is the consequence of the capacity of certain antigenic determinants to bind specific MHC alleles and not others.

Holes in the T cell repertoire: as a reflection of the deletion and / or functional inactivation of potentially autoreactive T cells, or of structural constraints on the generation of appropriate TCR molecules, certain TCR specificities are simply not present in the functional T cell repertoire.

The difference seems to be that according to the first theory the TCRs for these peptides exist, they just are not elicited due to poor MHC presentation whereas according to the second theory, even if the MHC could present the peptide properly, there are no T cells equipped to handle such a peptide.

“Only peptides with a relatively high binding affinity for MHC are immunogenic.”

Categorize three levels of binding affinity:o Less than 50nM: all are immunogenico 50-500nM: only 60% are immunogenico Greater than 500nM: none are immunogenic

They seem to assume that binding rates in vivo and ex vivo are the same since they try to compare using transgenic mice T cells and CTLs from hepatitis B patients (testing recall).

o Need to look up the in vitro binding protocol to see what temperature it is performed at.

Speculations on their part:o Q: How could it be that some peptides exist that have a low

binding affinity but are still immunogenic?o A: “It is possible that self derived antigenic peptides may be

characterized by relatively low MHC binding affinity because of selective elimination by thymic education and / or T cell tolerance of T cells reactive against high affinity MHC binding peptides.”

o Alternatively, it is important to see the details of how these studies were done. Restifo (I think) showed that if you have excess exogenous peptide in the field, the low dissociation rate is overcome. Is the same true for low affinities?

o Mechanisms for peptide-class I interactions: “An affinity threshold might be dictated by some basic

feature intrinsic to peptide-class I interactions and be independent of the particular protocol used and experimental situation examined.”

“Peptides with much lower affinities (i.e., lower than 500nM) might form complexes that are very unstable. It is tempting to speculate that the instability of such complexes might be responsible for their lack of immunogenicity, even in situations in which supplying the potential epitope in the form of an excess synthetic peptide might have been expected to overcome he hurdles of intracellular competition with other HLA binding peptides.”

Restifo, as mentioned previously, has shown that this is indeed not the case.

o “The type of APC presenting the peptide MHC/complexes may have dramatic effects on immunogenicity and antigenicity:

“MHC affinity does ultimately control the number of complexes expressed on the APC surface, whereas the type of APC, the lymphokine environment, and the presence or absence of costimulatory signals may ultimately dictate what number of such complexes are necessary for T cell activation. Previous studies demonstrated that, at least in the class II system, this is indeed the case.”

o The whole discussion is somewhat fuzzy since it is all speculation.

Immunogenicity of peptides bound to MHC class I molecules depends on the MHC-peptide complex stability.

van der Burg SH, Visseren MJ, Brandt RM, Kast WM, Melief CJ.J Immunol. 1996 May 1;156(9):3308-14.

They compare the correlations between affinity and immunogenicity vs. dissociation rate and immunogenicity.

“The dissociate rate of peptides bound to MHC class I is influenced neither by the presence of competing peptides nor by the concentration of competing peptides.”

Measure dissociation of peptide stabilized complexes by the loss of a conformation specific anti-HLA-A2 Ab. The loss of Ab represents the dissociation of the peptide from the class I molecule to which the peptide is bound. The stability is then presented by the time required for 50% of the molecules to decay (DT50).

“All high affinity binding peptides form stable MHC peptide complexes and are immunogenic, whereas the group of peptides of intermediate affinity contains either peptides that are immunogenic and form stable MHC peptide complexes or are non-immunogenic and do not from stable MHC peptide complexes as shown by their high dissociation rates.”

o In the intermediate affinity-binding group, the determination of immunogenicity is dependent upon dissociation rates.

o The talk about “forming stable MHC peptide complexes” is merely their overextending the function of dissociation rates.

Data: “A significant correlation exists between the immunogenicity of a peptide and the dissociation rate:”

o Immunogenic: 21 of 23 had a DT50 of > 3hr.o Non-immunogenic: none had a DT50 of > 3hr.o “This correlation (p=0.0000003) is closer than that

between peptide binding affinity and immunogenicity (p=0.0005).”

o Intermediate affinity: dissociation correlation (p=0.00007) is closer than affinity correlation (p=0.04).

o The data seems only relevant for the intermediate binders; peptides with really high affinities are less likely to dissociate quickly and in general probably form more stable complexes.

Dissociate rates “predict whether a peptide can persist at the cell surface for a time sufficient to allow the induction of a CTL response. Unfortunately, it does not predict whether a peptide will be endogenously processed and presented in the context of MHC class I.”

o This is still relevant for vaccine production since the peptides are introduced exogenously.

“There might also be a relation between the amount of peptides created and immunogenicity; it is conceivable that due to a limited amount of protein in the cytoplasm, not enough peptides are presented at the cell surface to induce a CTL response.”

This paper is 2 years later than the previous paper and so can amend their results. It seems that dissociation rates become important only when the affinity cannot provide a stable complex. However, there are always some peptides (here, 2 out of 23) that despite their high affinities, have low dissociation rates; nonetheless, these peptides induced immunogenicity. Are these exceptions that prove the rule or that require amending the rule?

Increased immunogenicity of an anchor-modified tumor-associated antigen is due to the enhanced stability of the peptide/MHC complex: implications for vaccine design.

Borbulevych OY, Baxter TK, Yu Z, Restifo NP, Baker BM.J Immunol. 2005 Apr 15;174(8):4812-20.

This paper attempts to show the structural basis for the differences between the gp100 native vs. modified peptides.

“The T2M modified peptide, which is more immunogenic in vitro and in vivo, binds HLA-A2 with a 9 fold greater affinity and has a 7 fold slower dissociation rate at physiological temperature.”

Three theories:o A: The native peptide stimulates a discrete population of T

cells that can crossreact with the native peptide.o B: Native TAA function as partial agonists or antagonists,

stimulating anergy in responding cells, whereas APL trigger T cells to signal in a more productive fashion.

o C: Peptide affinities and dissociation rates with the MHC class I molecule.

Crystallography: structures presented to the T cell are identical. “T cells from vaccinated [melanoma] patients [with the T2M

peptide] are unable to distinguish between the native and T2M modified peptides.

o These patients were vaccinated so that their T cells could attack their own native gp100 in the hopes of defeating their tumors.

o “The number of cells activated by the native peptide is highly correlated with the number of cells activated by the T2M modified peptide.”

o “There is little, if any, trend indicating the existence of T cells that can discriminate between the two peptides.”

o This makes sense if structurally the T cells see the same thing in both instances.

Thermodynamically, the modified peptide has a harder time dissociating.

These results contradict two previous papers that showed a structural difference between the two peptides:

o “Cloids” of T cells that were capable of recognizing the T2M modified but not the native gp100 peptide:

Assume he is dealing with T cells with weak affinity in general for the native peptide.

T cells with weak affinity to the native peptide do not respond well because of the fast dissociation rate.

The T2M peptide induces a much higher density of peptide-MHC complexes that can overcome T cells with weak TCR affinity for the peptide.

o Antibodies can bind the modified but not the native peptide:

??? Implications for vaccine design: if you can understand the

crystallography stuff, you can make better peptides.

Dissociation of the peptide-MHC class I complex limits the binding rate of exogenous peptide.

Ojcius DM, Abastado JP, Casrouge A, Mottez E, Cabanie L, Kourilsky P.J Immunol. 1993 Dec 1;151(11):6020-6.

“The rate of exogenous peptide binding is limited by the dissociation rate of the previously bound peptides.”

Measured dissociation by fluorescently labeling a peptide which would bind to MHC and then watch as the fluorescence decreases (binding of DAN-PbCS to Kd results in fluorescence enhancement).

Addition of Kd restricted, unlabeled, exogenous peptide “results in a sudden increase in the extent and rate of MHC-peptide dissociation.

o This is not true for peptides that are not allele specific.o This effect is independent of the concentration of

exogenous peptide used. This would seem to indicate that the rate limiting

step in peptide binding is the dissociation rate of the previously bound peptide.

o “The complex undergoes partial dissociation at early time points, and the remainder attains a plateau that was essentially stable for several days. The dissociation could be accelerated by an excess of allele restricted peptides but not by an excess of peptides that are presented by other MHC alleles.”

Recombinant HLA-DR1 produced by insect cells has been shown to bind faster than the native molecule.

o Association rate of exogenous peptide was similar in the recombinant system as well as a preloaded system.

o “The results imply that the binding kinetics of exogenous peptide are limited primarily by the affinity of the peptide previously occupying the Kd binding site.”

o They then claim to have found endogenous peptides in the previously-thought empty MHC II molecules from the insect system indicating that their exogenous peptide associate profile should be similar to the other systems tested.

Speculation:o “These results suggest that there is always a small

proportion of receptive class I molecules on the cell surface, and the size of this population may depend on the MHC allele and cell type used. The fact that most of the class I molecules on living cells cannot be loaded with

labeled peptides suggest that most surface class I molecules already harbor high affinity peptides. The extremely slow dissociation of these peptide-MHC complexes would preclude subsequent binding of exogenous peptides.”

o This makes sense that the dissociation constant is important in line with the previous Restifo and Meleif (1996) paper above.

However, they do not explain why the addition of exogenous, allele specific peptide should increase the dissociation rate. They provide a reference however…

Extensive peptide ligand exchange by surface class I major histocompatibility complex molecules independent of exogenous beta 2-microglobulin.

Smith JD, Lie WR, Gorka J, Myers NB, Hansen TH.Proc Natl Acad Sci U S A. 1992 Aug 15;89(16):7767-71.

This paper is referenced by Ojcius to try to show that exogenous, competitive peptides increase dissociation rates.

They first try to determine the appropriate temperatures for optimum binding and use H-2k (MHC I) with an Ld allele known for its ability to be loaded with exogenous peptide.

They are looking at peptide stability from two angles:o What is the relationship between peptide binding on MHC

stability, surface expression and turnover?o What is the relationship of bound peptide on the capacity

to bind exogenous peptide? “Peptide occupancy prolongs cell surface turnover” of the MHC

molecule.o “Ld turnover of cells pulsed with peptide is significantly

shorter [2hrs.] than that previously observed on cells treated with continuous peptide [6hrs.].”

“This difference implies that a single Ld molecule can rebind the fed peptide when available continuously in the culture medium.”

This assumes that the MHC I does not get internalized or shed while still attached to the peptide. This explanation assumes that the MHC first sheds its peptide and then becomes unstable until it binds another peptide. They do NOT prove this.

Binding of exogenous peptide to the MHC molecule is dependent on the dissociation rate of previously bound peptide to the MHC.

o “Pre-coating led to a striking enhancement in the amount of I(125)-CMV binding [the exogenous peptide].”

o “The off-rate of the precoated peptide determined how much secondary binding was observed regardless of which peptide was used to measure subsequent peptide binding” as per the previous paper.

Conclusions:o “The majority of peptide binding to live cells occurs at the

cell surface.”o “The amount of peptide exchange is governed by the

number of Ld molecules and most importantly, the dissociation rate of the bound peptide.”

Theories: Dissociated labeled peptide competes for

rebinding with unlabeled peptide. Competing unlabeled peptide may facilitate the

dissociation of bound peptide.

Free ligand-induced dissociation of MHC-antigen complexes.

Pedrazzini T, Sette A, Albertson M, Grey HM.J Immunol. 1991 May 15;146(10):3496-501.

“The addition of high affinity IEd binding peptides accelerated the dissociation rate of preformed MHC-Ag complexes. These data suggest a more complicated mechanism of dissociation than classical first order kinetics of dissociation of other ligand-receptor systems.”

Functionally measuring the capacity of T cell hybridoma cells to produce IL-2 after stimulation with different amounts of the peptide on MHC II complexes.

“Dissociation of the complexes was measured as a function of the time required for the APC to lose sufficient complexes so that their stimulatory capacity was decreased to a level equivalent to that obtained when APC were incubated with one half that amount of Ag.”

o They assume that dissociation over time should effect T cell activity. While it makes sense that IL-2 production should correlate with the dissociation rate – the dissociation rate can only be given as a relative value. The fact that a T cell produces IL-2 for 6 hrs. following Ag pulse says nothing about the absolute dissociation kinetics other than it is larger or smaller than some other condition.

Competitive, allele restricted peptides increase the dissociation rate of bound peptide.

o Two theories: Inhibitor peptide prevented further association

between free Ag from within the cell and MHC – a somewhat allosteric effect.

If this is true though, they do not understand why it should be MHC allele restricted since the allosteric effect is presumably on the invariant portion of the MHC molecule.

Some affect on the dissociation of previously formed MHC-Ag complexes.

They try to prove that the second theory is more correct since they saw the same dissociation effect with fixed cells and they think that it is unlikely that fixed cells actively present peptide on MHC intracellularly.

“Recent crystallographic analysis of class I MHC proteins, in which discrete pockets of the MHC-Ag binding groove have been delineated that might represent subsites of binding between antigen and MHC in the class I MHC … dissociation of Ag from one subsite, followed by binding of free competitor to that subsite, would result in the more rapid dissociation of the peptide from the MHC molecule, once the Ag dissociated from the second subsite. Thus, in the absence of inhibitor it would be anticipated that dissociation from the first subsite would be followed by reassociation to that subsite prior to dissociation from the second subsite. If this reassociation was inhibited because the competitor peptide bound to that subsite, then accelerated dissociation would be observed.”

Serial triggering of many T-cell receptors by a few peptide-MHC complexes.

Valitutti S, Muller S, Cella M, Padovan E, Lanzavecchia A.Nature. 1995 May 11;375(6527):148-51.

“A small number of peptide MHC complexes can achieve a high TCR occupancy, because a single complex can serially engage and trigger up to 200 TCRs.”

“T cells conjugated with peptide pulsed APCs undergo a profound and time dependent downregulation of the TCR/CD3 complex.”

o This downregulation is specific to those TCRs that have been engaged.

o What they really show is that if you put two TCRs of different specificities on a cell, only the form that is engaged becomes downregulated. This does not mean that each TCR that binds MHC is downregulated, but that all TCRs that are specific for the peptide are downregulated. Therefore, their claim that “bystander TCRs being essentially unaffected” only applies to their model system, but cannot be applied to the in vivo situation.

“APCs pulsed with high peptide concentrations (20uM) display 7,500 complexes and induce downregulation of 93% of TCRs. Strikingly, APCs pulsed with a low peptide concentration (50nM) display only 100 peptide-DR complexes, yet they downregulate 62% of TCRs.”

o “Each peptide-DR complex must engage a large number of TCRs in successive rounds.”

o They do not prove this, but rather that triggering induces downregulation of TCRs specific for that trigger.

“Production of IFNg by T cells correlates with the number of TCRs downregulated.”

“The high sensitivity low affinity paradox of T cell antigen recognition can be explained by our finding that a small number of peptide MHC complexes serially trigger a much larger number of TCRs leading to an amplified and sustained signal. The high off rate of TCR can be instrumental to this mechanism, because it allows a single peptide MHC complex to engage many TCRs in successive rounds of ligation, triggering and dissociation.”

Serial triggering of TCRs: a basis for the sensitivity and specificity of antigen recognition.

Valitutti S, Lanzavecchia A.Immunol Today. 1997 Jun;18(6):299-304. PMID: 9190117

“The TCR MHC interaction is characterized by a very high off rate, with half lives ranging from four seconds to a few minutes.”o However, “T cells interact with APCs for a prolonged

period.”o “Sustained signaling in an antigen driven system is

required for T cell activation.”o “TCRs that are activated at any given time are not able to

sustain the overall signaling process, suggesting that a

sequential recruitment and triggering of TCRs is necessary to maintain signaling.”

“A few peptide MHC complexes must serially engage and trigger many TCRs. The number of triggered TCRs is a function of the logarithm of the number of complexes offered.”

“An appropriate off rate allows a single peptide MHC complex to dissociate from the engaged TCR following triggering making it possible to engage and trigger several TCRs serially.”

o “Implies that a TCR-CD3 complex can be triggered by monovalent ligation for a minimum time – probably a few seconds.”

o “Thus the TCR would function like a simple switch that needs to be pressed for enough time to release the signal.”

The number of TCRs that can be triggered in a given time period by a single peptide MHC complex varies as a function of:

o The time of ligation required for triggeringo The stability of the complex

This model assumes that the serial triggering process will take place in a limited area of T cell APC contact and will consequently be dependent on three factors:

o T cell adhesion that facilitates the TCR peptide MHC interaction

o A supply of signal transduction components to the triggered TCRs

o A supply of new TCRs to replace those removed following downregulation

Gabrielle tells me that in fact the nanoclusters seem to follow this model pretty well. Will look into…

Altered ligands presumably induce partial T cell activation or anergy depending on their kinetics in relation to the kinetics of effector peptides.

o Those ligands that promote shorter dissociation times will only partially activate the T cells.

Correlation between TCR downregulation and T cell response.o T cells can ‘count’ the number of triggered TCRs and

respond above a given ‘threshold.’ The exquisite sensitivity of cells is explained by the fact that a

few agonists with optimal kinetics of binding can trigger many TCRs, thus achieving the high level of TCR occupancy required to induce T cell activation.

o Antibodies are selected for increasing affinity, whereas T cells are selected for optimal kinetics.

Quantitative contribution of CD4 and CD8 to T cell antigen receptor serial triggering.

Viola A, Salio M, Tuosto L, Linkert S, Acuto O, Lanzavecchia A.J Exp Med. 1997 Nov 17;186(10):1775-9. PMID: 9362538

During T cell-APC interaction, triggered TCRs and coreceptors are downregulated and degraded with identical kinetics and with fixed stoichiometry.

o This was an attempt to show the purpose of the coreceptors even though they are sometimes unnecessary.

Coreceptors:o Stabilize the TCR peptide MHC interaction.o Carry Lck in contact with the TCR to initiate

phosphorylation events. Approximately 2 CD4 and 4 CD8 molecules were downregulated

per each downregulated TCR.o The downregulation was not necessarily a result of MHC

engagement since bacterial superantigens and anti-CD3 antibodies caused the same effect.

o Downregulation of the coreceptor was due to an intracellular association with triggered TCRs.

o This process occurs after TCR triggering, although not necessarily by MHC interaction.

Coreceptor contribution:o Ligands with higher off rates may require the extracellular

interaction of the coreceptor with the cognate molecule to increase the stability of the complex and , consequently, the rate of triggering.

o The sensitivity to inhibition by coreceptor antibodies is inversely correlated to the efficiency of the TCR ligand interaction.

o The inhibition of T cell response by anti-CD4 antibodies precisely correlated with a reduced level of TCR downregulation both when strong and weak agonists were used.

However, in spite of a decreased efficiency of TCR triggering, the threshold of T cell activation and the type of cytokines produced were not affected by anti-CD4.

The binding of a coreceptor to cognate MHC molecules becomes critical in the case of low affinity ligands because in this case the coreceptor can initially stabilize the TCR ligand interaction, thus

increasing the probability that an engagement event will result in triggering.

This could be very relevant to the gp100 peptide. It might be wise to look at CD8 as well during stable and unstable synapse formation.

Interaction between the CD8 coreceptor and MHC class I stabilizes TCR-antigen complexes at the cell surface.

Wooldridge L, van den Berg HA, Glick M, Gostick E, Laugel B, Hutchinson SL, Milicic A, Brenchley JM, Douek DC, Price DA, Sewell AK.J Biol Chem. 2005 Apr 18; [Epub ahead of print]

Try to quantify the stabilization that CD8 binding provides to the pMHCI/TCR interaction by using MHC mutants with varying affinity for CD8.

“Simultaneous binding of the TCR and CD8 by pMHCI allows the potential for CD8 induced stabilization of the TCR/pMHCI interaction.”

Using MHCI tetramers and NOT monomers:o “The analysis of interaction kinetics between pMHCI and

cell surface TCR has been hindered by the extremely short half lives of such interactions (1-12 seconds at 25 deg). Tetrameric pMHCI molecules have considerably longer interaction times and allow a quantitative assessment of dissociation.”

o “The use of soluble pMHCI proteins enables TCR/pMHCI interactions and kinetics at the cell surface to be studied without interference from other adhesion or costimulatory molecules.”

“The decay of pMHCI tetramers from the cell surface of 868 CTLs clearly correlates with the affinity of the pMHCI/CD8 interaction.”

o “A decreased pMHCI/CD8 interaction leads to more rapid tetramer dissociation and that an increased pMHCI/CD8 interaction results in slower tetramer dissociation.”

o “The maximum effect of the pMHCI/CD8 interaction is a prolongation by a factor of 2.16 of the mean TCR/pMHCI binding time.”

“The dependency on the pMHCI/CD8 interaction for stable tetramer binding correlates with the functional avidity of the CTL and is thought to reflect the intrinsic affinity of the TCR for pMHCI ligand amongst other factors such as cell surface organization and density of the TCR.”

o “Tetramers derive their high avidity from the large probability that a monovalently bound tetramer will bind bivalently before the single bound site dissociates.”

“If a ligand is already optimal in the absence of CD8, it becomes 17% less effective in the saturating presence of CD8. Hence if we rank the strong agonists for a given T cell by potency, we conclude that the CD8 stabilization effect can alter the order of that ranking, allowing CTL to focus their functional avidity on a ligand by adjusting CD8 expression levels.”

The immunological synapse balances T cell receptor signaling and degradation.

Lee KH, Dinner AR, Tu C, Campi G, Raychaudhuri S, Varma R, Sims TN, Burack WR, Wu H, Wang J, Kanagawa O, Markiewicz M, Allen PM, Dustin ML, Chakraborty AK, Shaw AS.Science. 2003 Nov 14;302(5648):1218-22. Epub 2003 Sep 25.

C-SMAC is not involved in signaling.o Rather, it is a site for both strong receptor triggering and

increased TCR degradation.o CD2AP deficiency profoundly altered these morphological

features of the two rings. CD2AP deficiency:

o Enhanced proliferation.o Delayed and prolonged tyrosine phosphorylation of the

TCRz.o No C-SMAC formation.o C-SMAC does not potentiate TCR signaling.

TCR-pMHC binding:o When the C-SMAC does not form, the median time for a

pMHC to rebind a TCR after dissociation is 20.3e6 MC steps. In contrast, the corresponding time is only 2.7e6 MC steps when TCRs cluster in the C-SMAC.

o Receptor coupling in the C-SMAC should therefore directly enhance the frequency of TCR-pMHC complex formation without any change in the TCR-pMHC half life.

TCR degradation:o Only fully phosphorylated receptors are subject to

degradation.o Enhanced triggering in the C-SMAC also serves to limit

sustained tyrosine kinase activity in the C-SMAC over long times.

o The only way to sustain TCR signaling over longer periods would be to have TCR replenishment from new synthesis.

o Attenuation of phosphotryrosine levels in the C-SMACis due to enhanced receptor triggering, which results in increased receptor degradation.

The immunological synapse of CTL contains a secretory domain and membrane bridges.

Stinchcombe JC, Bossi G, Booth S, Griffiths GM.Immunity. 2001 Nov;15(5):751-61.

Although target cell death occurs within 5 minutes of CTL-target cell contact, an immunological synapse similar to that seen in CD4 cells rapidly forms in CTL, with a ring of adhesion proteins surrounding an inner signaling molecule domain.

o Kill in a short time and can serially kill up to 6 target cells per hour!

o Seems like the machinery is all ready before they even get there.

CTL polarize their lytic granules and trigger target cell apoptosis rapidly after cell contact.

o An indentation appears in the target cell membrane after about 90s, which becomes the location for secretion of lytic granules.

o Labeled granules still appeared inside the CTL even after target cell death indicating that CTL do not need to exocytose all of their granules for a target cell to be killed.

Moreover, this allows for rapid killing without necessitating continual protein synthesis.

o Granule polarization is preceded by reorientation of the MTOC followed by the Golgi complex to the point of contact between the two cells.

o Talin accumulation also precedes granule polarization, but occurs after MTOC polarization.

Immunological synapse:o CD11a-CD18 (LFA-1 complex) like talin, becomes

concentrated at the contact site with a target cell, forming an outer ring resembling the pSMAC described upon CD4 interaction with APCs.

o High levels of Lck and PKC-th become concentrated at the contact site [c-SMAC] upon target cell recognition.

Low levels of PKC-th in the pSMAC.o CD45 (phosphatase for Lck) is largely excluded.

o Asymmetric!!! Always leaves a gap between the signaling

molecules and the adhesion molecules only on one side.

CTL Acquire target ell membrane protein and form membrane bridges with the targets.

o They go through a detailed analysis of how this happens.o They propose that the more a CTL kills, it obtains more an

more foreign membrane protein.o This in turn, produces fratricide, and is a self limiting

control of CTL killing.

Cytotoxic T lymphocytes form an antigen-independent ring junction.

Somersalo K, Anikeeva N, Sims TN, Thomas VK, Strong RK, Spies T, Lebedeva T, Sykulev Y, Dustin ML.J Clin Invest. 2004 Jan;113(1):49-57.

Used bilayers with 40 molecs/um2 MHC class I and 200 molecs/um2 ICAM-1 but could not visualize the TCR nor the MHC.

IS formation with CD8 cells was completed within 0.5-4 minutes – cSMAC with a pSMAC.

CTLs form an antigen independent ring junction:o Even in the absence of agonist MHCp complexes, the LFA-

1-ICAM-1 complexes formed a right junction.

o These junctions were transient, with an average duration of 15 minutes.

o The number of LFA-1-ICAM-1 interactions per pSMAC was 6,000-7,000, representing about 10% of the total LFA-1 molecules on the cell.

o CD8 is not required, since the CD8- CTL clone 115ix formed ring junctions with a 15 minute lag time.

o Ring junctions were formed above an ICAM-1 density of 160 molecs/um2, while significant adhesion and ICAM-1 accumulation was observed at 40 molecs/um2.

o MICA, a ligand for NKG2D significantly enhanced ring formation at low ICAM-1 densities.

o The formation of ring junctions is responsive to at least two inflammatory/transformation frlated signals:

ICAM-1 expression level MICA

CD8 accumulated in nearly 90% of cell contacts in the presence of agonist MHCp complexes.

o In many ISs, a central hole was detected in the CD8 pattern.

o CD3 and CD8 were present in the central region of the ring junction on ICAM-1 alone, but they were only enriched in 30% of the contacts, and the degree of enrichment was lower than in the IS.

LFA”D: it seems difficult to ignore the MHCp [complexed with TCR presumably] in the cSMAC region. Although it is not necessary, when it is there, CD8 goes there too and CD3 is enriched. The fact that when you are able to remove its effect, there is still a ring junction, is interesting, but not necessarily relevant in anyway. This may have no physiological validity.

Differences between CD8 and CD4 ISs:o CD8 have no nascent synapses with TCR engagement in

the periphery.o Many cSMACs had a central hole, lacking CD8.

Were they lacking MHC also? “It is likely that increasing ICAM-1 expression facilitates the

effectiveness of T cells’ hunting for targets … possibly an adaptation for CTL function.”

o “The ring junction causes the CTL to pause and form a central domain with CD3, CD8 and lipid rafts, an ideal signal reception surface, but does not mobilize the secretory apparatus. We would suggest that the cell is on “high alert” but its weapons are not “armed” yet.”

o Assume that the ICAM-1 layer functions as an adhesion gasket, sort of what John Loike showed with neutrophils and CD11b/c-CD18.

This should help limit the cytotoxic potential of released granules to the cleft between cells only.

Two pathways for IS formation:o Antigen independent through ring junctions – “presynapse”

– sets the stage for CTL killing.o Antigen dependent through a nascent IS.

The whole thing is sort of weird since when antigen is there, the whole apparatus is more effective

LFA”D: Restifo showed that immunizing with the modified peptide primes CTLs to kill cells expressing the native peptide, but immunizing with the native peptide does not do this. This means that the CTLs form “synapses” with target cells presenting the

native peptide and effectively kill them. Something seems wrong though when the CTL interact with an APC presenting the native peptide – perhaps this will be reflected in the IS as well. It will be interesting to see how the cells react towards the bilayers – will they view them as APCs or targets?

T-cell receptor triggering is critically dependent on the dimensions of its peptide-MHC ligand.

Choudhuri K, Wiseman D, Brown MH, Gould K, van der Merwe PA.Nature. 2005 Jul 28;436(7050):578-82.

Increasing the dimensions of the TCR-pMHC interaction by elongating the pMHC ectodomain greatly reduces TCR triggering without affecting TCR-pMHC ligation.

Assume that TCR triggering is dependent upon proper dimensions in the cSMAC area.

o This should exclude large molecules, such as CD45 which you don’t want there anyway.

Abrogation of TCR triggering seen with elongated forms of pMHC is not the result of decreased TCR engagement.

o Increases the intermembrane distance and allows CD45 greater access to engaged TCR-CD3 complexes at the interface.

o CD45 depleted early but then enriched by 5 minutes at the interface – limiting signaling.

Kinetic Segregation model of TCR signaling:o Signaling is the result of tethering of the TCR-CD3 complex

within close contact zones in which tyrosine phosphorylation is favored because of size dependent exclusion of tyrosine phosphatases such as CD45.

The role of the secretory immunological synapse in killing by CD8+ CTL.

Stinchcombe JC, Griffiths GM.Semin Immunol. 2003 Dec;15(6):301-5.

The formation of an immunological synapse may be important in mediating the functional responses resulting from target cell

recognition in effector cells with a variety of differing roles within the immune system.

o In activated CTL, the signaling function of the immunological synapse may be less important, but that synapse formation is vital for specific, controlled and directed killing.

Ring of ICAM-1 adhesion molecules with associated talin.o Cytotoxic enzymes are presynthesized and are secreted in

a specific area, into a cleft in the target cell membrane. Secretory lysosomes first cluster around the MTOC, then move

round the Golgi complex one by one to align and dock with the plasma membrane opposite the target cell.

o Involves the small G protein, Rab27a. Downregulation of the immune response as in their previous

paper via fratricide.

T cell killing does not require the formation of a stable mature immunological synapse.

Purbhoo MA, Irvine DJ, Huppa JB, Davis MM.Nat Immunol. 2004 May;5(5):524-30. Epub 2004 Mar 28. Erratum in: Nat Immunol. 2004 Jun;5(6):658.

Only 3 pMHC complexes were required for killing, showing that stable synapse formation and complete signaling are not required for cytotoxicity.

o CD4 cells require engagement with as few as ten pMHC ligands.

o In CD8 cells, even a single pMHC complex induced a detectable calcium signal, and additional ligands resulted in greater calcium increase, with maximum responses first noted with approximately ten pMHC complexes.

o Suggests that the number of pMHC complexes at the immunological synapse remains constant after initial CTL activation and that it is unlikely that pMHC complexes continue to be recruited to the immunological synapse.

o Need 10 pMHCs for a stable synapse, even though 1 complex is sufficient to extend conjugate stability.

The earliest sign of cell death was usually apparent shortly (5-15 min) after CTL-APC engagement, indicating that the decision to proceed with the cytotoxic response is dependent on the events during the early stage of CTL-APC contact.

CD8 clustering preceded and did not extensively coincide with clustering of its MHC ligand or the TCR. Instead, CD8 was rapidly

internalized from the center of the IS, with only remnant surface accumulation persisting in association but not colocalization with MHC clusters.

o The lack of CD8 engagement with pMHC greatly reduced the response to any number of pMHC complexes.

o Suggests that CD8 acts to initiate signal transduction and is distinct from the purpose of MHC and TCR clustering as the synapse matures.

o TCR clustering only began as the synapse matured, indicating a function for antigen receptor clustering during the effector phase of CTL activation.

Direct observation of ligand recognition by T cells.

Irvine DJ, Purbhoo MA, Krogsgaard M, Davis MM. Nature. 2002 Oct 24;419(6909):845-9.

T cells expressing the CD4 antigen respond with transient calcium singalling to even a single agonist peptide-MHC ligand, and that the organization of molecules in the contact zone of the T cell and APC takes on the characteristics of an immunological synapse when only about ten agonists are present.

Single ligands:o Calcium increase decayed rapidly and was only typically

sustained with 10 or more peptide ligands. T cell responses to a few agonist peptides were not rare events,

as indicated by the frequency of responses.o T cells stopped and formed close contacts with APCs in

95% of frontal contacts (that is, the leading edge of the T cell contacting peptide) with 1-10 peptides.

o A stable, central accumulation of IEk forms when ten or more agonist peptides are present.

This accumulation corresponds to a very large excess of endogenous MHC molecules relative to the number of specific peptides.

It would be expected that many endogenous peptide-MHCs would not be able to bind a given TCR owing to interference with the bound peptide.

There is a marked dependence on CD4 for the recognition of low numbers of specific peptides. But these effects can be overcome with a much higher density of ligand, and T cell stimulation then seems to proceed normally.

o This is unexpected, since even high conce3ntrations of monomeric peptide-MHC in solution do not trigger CD4

positive T cell activation. Instead this required peptide-MHC trimers or dimers.

o CD4 binds the MHC at almost a 90 degree angle, which makes it unlikely that a CD4 molecule that is closely associated with a TCR (as many are in activated T cells) can bind to the same peptide-MHC ligand that the TCR is contacting.

o Pseudo-dimers: a weak interaction between the TCR and the peptide-MHC complex could be stabilized by the weak binding of CD4 to the MHC.

The c-SMAC: sorting it all out (or in).

Lin J, Miller MJ, Shaw AS.J Cell Biol. 2005 Jul 18;170(2):177-82. Epub 2005 Jul 11.

Only talin and LFA-1 are known to reside in the p-SMAC. Cytochalasin D, and actin polymerization inhibitor, can block c-

SMAC formation.o Because the formation of the immunological synapse

occurs concomitantly with MTOC polarization, microtubules and microtubule motors may also be important.

Unclear if specific c-SMAC and p-SMAC formation is necessary for signaling or only synapse formation.

Two models:o The c-SMAC might help to initiate T cell activation by

driving receptor aggregation. But, it is not necessary!

o Maybe it enhances T cell activation by concentrating the TCR and pMHC in one area of the plasma membrane and holding them there for long periods of time.

This would facilitate rebinding events due to the high concentration of TCR and ligand – serial triggering.

TCR downregulation:o The inability to downregulate activated receptors results in

sustained signaling, suggesting that the c-SMAC enhances TCR downregulation and signal attenuation.

Sustained signaling:o Nobody knows why the T cell sticks around for hours when

all that is needed is a couple of minutes.o It is interesting to speculate that endogenous self-peptides

are responsible for sustained TCR signaling events. In this model, antigenic peptides initiate c-SMAC formation, but the c-SMAC may allow the usually nonstimulatory self

peptides to induce a signal sufficient to sustain [albeit, not initiate] T cell activation.