Peptide‐based immunotherapy of autoimmunity: a path of puzzles, paradoxes and possibilities (2001)

Our understanding of the basic mechanisms controlling the generation of productive immunity continues to evolve at a great pace. Models of how the immune response may be diverted from tackling invading pathogens into mounting an inappropriate response towards self tissue antigens also continue to develop. Armed with this knowledge, however, we are yet to develop effective and broadly applicable strategies to prevent or treat autoimmune disorders. This review assesses the prospects for developing antigen-specific therapies through the use of synthetic peptide antigens, and specifically altered peptide ligands (APL), to target pathogenic T-cell autoreactivity. Identification of T-cell epitopes within autoantigens has allowed the development of a minimalist approach to certain experimental models. Thus single peptides can replace purified autoantigen or homogenized tissue as the agents that elicit T-cell autoreactivity and therefore pathology. If we are using peptides to provoke disease, we should also be able to use peptides to prevent disease, and many reports over the last 15 years have confirmed this. But is this applicable to human autoimmune disorders and what is the best approach to use? By using analogue peptides with defined substitutions at individual residues, we can determine residues that interact either with the T-cell receptor (TCR) [analogues do not stimulate antigen-specific T cells but retain the ability to bind major histocompatibility complex (MHC)] or with the MHC (loss of both T-cell stimulation and MHC binding). The term ‘altered peptide ligand’ was first coined a decade ago by Evavold et al. to define peptides with alterations at TCR binding residues.1 The definition of APL has since become broadened to include analogue peptides containing one or more substitution(s) at any residue. APL can be divided based on their ability to stimulate antigen-specific T cells (Fig. 1). The native peptide or APL that give equivalent response patterns are referred to as agonists. APL requiring increased doses to induce qualitatively normal responses are weak or subagonists, whilst those able to stimulate at greatly reduced doses are superagonists. The original work by Allen and colleagues identified APL with ‘partial agonist’ activity, being able to stimulate effector function (cytokine production, target cell lysis) in T-cell clones in the absence of the concomitant proliferation provoked by agonist peptides.2, 3 Soon after, Sette's group reported APL with alterations at TCR contact residues that did not overtly stimulate T-cell clones, but inhibited activation induced by agonist when presented on the same antigen-presenting cell (APC).4, 5 This inhibition was not simply due to MHC blockade and was antigen-specific (i.e. an APL of antigen x would not inhibit activation of T cells specific for antigen y). APL with these inhibitory properties are termed TCR antagonists. APL have proved useful tools in the dissection of early signalling events proximal to TCR ligation. Debate continues over the precise mechanisms underlying partial agonist and TCR antagonist effects and is documented elsewhere.6, 7 Altered peptide ligands: subtypes and activities. APL can be divided according to their effects on antigen-specific T-cell populations in vitro. Pioneering work in the field of peptide-induced modulation of autoimmunity used murine experimental autoimmune encephalomyelitis (EAE)8 which serves as a model for multiple sclerosis (MS). EAE is induced by immunization with complete Freund's adjuvant (CFA) mixed with antigens of central nervous system (CNS) myelin, notably myelin basic protein (MBP), proteolipid protein (PLP) and myelin oligodendrocyte glycoprotein (MOG). Central to the disease is activation of myelin-reactive CD4+ T helper type 1 (Th1) cells which infiltrate the CNS and establish an inflammatory lesion. As there are several well-defined T-cell epitopes in these myelin antigens9-12, the disease can be induced using single peptides. For this reason it is the EAE model that has seen the most extensive investigations of peptide therapy. Three immunodominant regions have been of particular interest: PLP(139–151) and two regions within MBP, the acetylated N-terminal peptide (Ac1–9) and peptides contained within 80–105. The first epitope to be identified,9 and consequently tested as a therapy,13 was MBP Ac1–9. The molecular basis for the interaction of Ac1–9 with the Au MHC class II molecule and TCR has been characterized extensively13-15 and is summarized in Fig. 2. The residues responsible for binding to Au are 4Lys and 5Arg.13 However, the natural peptide binds extremely poorly,9, 16,17 due to 4Lys interacting unfavourably with a hydrophobic pocket within the Au peptide binding cleft, as highlighted by mutational studies and predictive computational analysis.18, 19 Hence we can produce APL with greatly increased binding affinities for Au by substitution of residue 4Lys, most notably with Ala, Val and Tyr13, 15,16 (Fig. 2). As we would predict, increased binding for class II translates into increased capacity to stimulate Ac1–9-specific T cells in vitro,13 such that femtomolar doses of the 4Tyr APL will stimulate responses that require nanomolar concentrations of wild-type Ac1–9.20 Thus these position 4 APL behave as superagonists. How the Ac1–9 peptide interacts with TCR and MHC class II. The Ac1–9 peptide of MBP binds to the Au class II molecule through interactions involving residues 4Lys and 5Arg. Residue 4 has a dominant effect on binding, with the wild-type Lys residue interacting unfavourably with the Au peptide-binding cleft. APL with alterations at position 4 can greatly increase binding to Au. These APL consequently act as superagonists in vitro, stimulating T cells raised against wild-type Ac1–9 at correspondingly low concentrations. Residues 3 and 6 interact with TCR. This scheme represents interactions with the Tg4 TCR for which residues 6 and 3 act as primary and secondary TCR contact residues, respectively. This preference in TCR contacts is reversed on the analysis of polyclonal Ac1–9-specific populations. This figure summarizes data from refs 13-15 and 20. The initial peptide therapy experiments of Wraith et al. focused on the 4Ala APL and uncovered a conundrum.13 Whilst the Ac1–9 4Ala peptide was a superagonist in vitro, it induced little or no EAE in vivo. Moreover, mice that were co-immunized with a mixture of 4Ala and wild-type 4Lys also showed little EAE. Preimmunization with 4Ala in incomplete Freund's adjuvant (IFA) also prevented EAE development on immunization with 4Lys in CFA.21 This was not due to the expansion of a regulatory population as T cells from 4Ala-primed mice could not transfer protection from EAE to syngeneic recipients. The basis for these paradoxical in vitro and in vivo effects has remained unclear for a decade but our recent data explain these findings (see below). Was this phenomenon peculiar to the Ac1–9 model? Later studies from van Eden's laboratory using EAE and adjuvant arthritis in rats suggested that it may be generally applicable.22 APL of MBP(72–85) and the arthritis-related peptide 180–188 of mycobacterial heat-shock protein 65 (hsp 65) were generated that showed increased binding affinities for the RT1B1 rat class II molecule. In co-immunization experiments it was found that the MBP APL specifically inhibited EAE but not arthritis, indicating direct effects on antigen-specific T cells. These early experiments therefore pointed to applications for APL in antigen-specific therapy of autoimmune disorders. The idea of TCR antagonist peptides as therapeutics was first applied to the EAE model induced with the immunodominant PLP[139–151] epitope in SJL mice.23 Residue 144Trp was identified as the dominant TCR contact for this epitope. Position 144 APL were identified that inhibited the in vitro activation of encephalitogenic 139–151-specific T-cell clones. When pools of these antagonist APL were added in equimolar amounts to the wild-type 139–151 prior to immunization in CFA, they were found to reduce significantly the incidence and severity of resulting EAE. Subsequently the approach was modified to design a single APL with substitutions at both 144 and 147.24 This L144/R147 APL inhibited in vitro activation of a panel of encephalitogenic T-cell clones showing distinct TCR gene usage. The analogue prevented EAE when co-administered with native 139–151 and, furthermore, could limit progression of EAE if given early the of T cells can also be by T-cell to a antigen has been early of type 1 APL based on this antigen were found to act as TCR when presented with wild-type T cells from were found to to two epitopes within the human receptor APL of these two or a APL both in a single showed effective antagonist on T-cell responses to wild-type These findings were in an experimental model of in which APL prevented the development of studies have also reported modulation of T cells from and specific for of is a however, with the use of TCR antagonist APL as Whilst are of early signalling these do not to have effects on T-cell partial agonist APL may T-cell clones in peptides inhibit when presented on the same as the agonist peptide when presented in How are we to effective with should have no effect on T cells in the absence of the native self antigen and therefore would not be a for As for disease it is not that antagonist and self antigen could be presented on the same This would most in however, and not in the pathogenic T cells would be a more approach therefore is to design APL that T cells to a or the for of self Central to the of most experimental autoimmune is the activation of CD4+ T cells of the such as and The development of antigen-specific responses can be by that the immune response towards the Whilst to an response by of cells have this approach has proved if the population is the of the analysis of the effects of the L144/R147 APL more TCR of T cells was not prevented by co-immunization with T-cell clones specific for the APL with native 139–151 but These clones were able to EAE when to syngeneic mice and to inhibit encephalitogenic of 139–151-specific T cells in vitro prior to transfer to Moreover, with L144/R147 EAE on immunization with epitopes from and This is a for of autoimmunity as should of the dominant TCR contact of a peptide to expansion of cells when using an immunization that clones from mice with either wild-type 139–151 (Th1) or a APL that cells residues 144 and clones residues and immunization with APL at residues as would cells. have been several reports that the use of APL the in vitro These of doses of agonist peptide have been reported to effect The however, a and the of a T-cell population being by for this should be in this system a use of the ability to a pathogenic response to a response has also been reported in EAE induced with APL from the in both mice and In these of T-cell to APL induced a in with in by in This of MBP is of particular it is also in the of human being the major MBP epitope by T cells from using human T-cell clones specific for this identified APL that as TCR of proliferation and the of and but specifically induced of These APL were therefore of both and in preference for studies T cells from APL that inhibited production, expansion of 1 as or is a of and with effects in several EAE is an for the of the progression of and of of the effects of have the expansion of populations in both EAE and indicating immune as a of has also been to against experimental and a inhibited activation of human T-cell clones specific for type II a autoantigen in their precise of to be may therefore an approach to more these studies APL can be used to experimental autoimmune this may be due to TCR immune or both and of TCR and immune can be in vitro using human T-cell populations. The findings would for the use of APL as TCR or immune in human autoimmune disorders. on our however, it is to a of have the for TCR using the model of The pathogenic T-cell is cells by the This model therefore to TCR effects may be to For these studies we used T cells from the Tg4 that a Ac1–9-specific As with tested Ac1–9-specific Tg4 cells residues and of Ac1–9 as primary and secondary TCR contact residues, respectively. Thus no substitution was at position and at position 3 could be of confirmed position 6 as the primary TCR contact we identified position 6 APL with TCR antagonist on Tg4 cells in vitro. tested these antagonist APL for encephalitogenic in vivo (i.e. immunization with individual APL in the absence of wild-type As Tg4 mice EAE immunization with wild-type but not immunization with the antagonist however, these APL were of EAE in normal Thus we the paradoxical findings that APL that inhibited encephalitogenic T cells in vivo induced disease in vivo. The reason for this when we the of polyclonal T-cell populations from normal These cells residues 3 and 6 as primary and secondary TCR the of Tg4 T cells. these polyclonal populations were by the position 6 APL that induced EAE in This T-cell against Ac1–9 APL pointed to in the T-cell in to of an in TCR usage. This represents a major to the development of EAE antagonist However, we identified a position 3 APL that inhibited activation of not Tg4 but also polyclonal T-cell This APL a substitution at the dominant TCR contact residue for the polyclonal was an effective of EAE when co-immunized with wild-type Ac1–9. position 3 APL that as an antagonist for Tg4 cells was a effective EAE in vivo. These the of the T-cell and that inhibition of the population is for effective of Our findings in the Ac1–9 EAE model on the of antagonist APL as therapies in TCR we to T-cell clones for in vitro This in the of T cells to the of but that are not of the in vivo The Ac1–9 system is to this as the Tg4 TCR was from one of a panel of clones showing TCR our antagonist APL on such clones can give a of the most peptide to this may be the with APL that induce immune Thus an APL that a to produce or vitro may have the effect on a distinct T-cell when given in vivo. This to against the use of antagonist or immune APL in human autoimmune an approach in an human population reduce pathology. on two II in include that this may be the The effects of APL based on were In one there was no or effect of two of with APL The APL for T cells that with the wild-type MBP and that could be from the The reported the of responses the of as by in vitro with either APL or wild-type Whilst no in was there was a of a in development of CNS in the of This of APL did however, induce immunity in those This was due to in a of an response to therapy for TCR and immune is therefore the of the T-cell the of the effects of and to the is that therapy will not be based on a single APL and may require for a of immune to of the T-cell epitope. If the use of antagonist or APL is is there a do we to do to the T cells the the is to is that CD4+ T are to cell doses of antigen can to of antigen-specific T cells in 65 This cell has been as a major that the immune by of T If the is by the of signalling through the can a of wild-type antigen be by using doses of superagonist The model to this due to the of APL with well-defined properties in vitro. can that T cells against wild-type Ac1–9 to the for Au and of a productive with Ac1–9 these T cells to induce EAE. however, superagonists to for EAE the in vivo of the T cells by Tg4 T cells into prior to immunization with either wild-type Ac1–9 or the 4Tyr 4Tyr immunization more of Tg4 cells showed of with in response to wild-type Ac1–9. cells therefore cell on in vivo to superagonist This an for the early that co-immunization of Ac1–9 with the 4Ala superagonist to induce T cells would be on 4Ala they a to an inflammatory in the also the to transfer protection using T cells from 4Ala-primed in response to superagonist stimulation has also been in vitro using an Ac1–9-specific TCR in vivo findings have been reported using a superagonist APL in the EAE cell has also been using low doses of an of The development of may therefore a for human would be that superagonists generally induce a prior to a to a is to therefore that APL have been that are of in T cells specific for but the of seen in with induced by native Our studies of Ac1–9 superagonists a T-cell populations generated by immunization with Ac1–9 or any of the APL showed to the antigen they were against responses first at T cells with class II a of TCR for that Au we that the T cells that immunization with superagonist distinct to the pathogenic T cells by wild-type Ac1–9. Thus T-cell generated against Ac1–9 were populations containing cells and T cells generated against the 4Tyr in used This low T cells are to with the same to superagonist as cells to Ac1–9. These to an model for immune responses to antigen in Fig. This model that due to the of TCR for the immune will a population of T cells of antigen with from low to T cells will not a and will of the of the T cells that are to will cell T cells for the antigen within a defined will therefore be allowed to expansion and the productive This would explain we and antigen-specific T cells with this for the effects of are of not the basis for our of and our is that T cells are also in the have the of T cells into the productive as use this to the effects on the This is distinct from at the single cell which of of the cell signalling model for The T-cell will a population of T-cell These will have a of for the T cells with therefore low for antigen will by to antigen or of self antigen T cells with will become but are from the by T cells with will be for and can and the productive immune This serves to T-cell for the antigen within a as yet the best approach is to induce T-cell using native has been for years that the in which antigen is has a dominant on the to a response or in or mixed with will provoke an antigen a of antigen-specific T-cell and have given peptides the or This approach can inhibit T-cell activation in response to antigen in Moreover, it has proved effective in the of disease in as as induced of and In of peptides from or 65 can inhibit the development of in the The underlying with peptides an of and has been to the of or a regulatory in antigen-specific T may in of these with cells being or diverted to regulatory function the The of of the T-cell epitopes that are to an to the use of peptide-induced in human autoimmune disorders. For certain the autoantigens are such as the in and and in autoimmune For autoantigens have been but we their in disease or the of as yet using mice have that the immunodominant which is in can elicit epitopes have yet to be in this is epitope a by which autoreactivity to epitopes due to tissue as disease This not the of epitopes but of the in of autoimmune the have The is how can we T cells against and epitopes in The may in the of By this we the of responses to multiple epitopes of a single epitope. This phenomenon has been using the as the model of the immunodominant epitope of in to this epitope and epitopes immunization with the tested the ability of encephalitogenic peptides to induce effects in Ac1–9 and of MBP and of Ac1–9 responses to both Ac1–9 and on immunization with myelin, but did not responses to The epitope proved an more effective responses to and both MBP could be induced at the of T-cell in the of a single peptide did not T-cell but could EAE immunization with a Thus PLP(139–151) EAE induced with or either of the two MBP Ac1–9 disease induced with of the MBP peptides but not the 4 a model for that can function in either the the of immunization with myelin or in the the CNS immunization with The increased of the epitope over the MBP epitope may a of in mice due to a of of this of a of cells could a low of cells. low of cell may not however, a of cells. for either in the or of autoimmune of with peptide to the expansion of an T regulatory cell immunization with antigen will to of this epitope in to pathogenic epitopes to which has not been induced in the This to of pathogenic T cells of However, T cells are also and activation of the pathogenic T cells either or of with the epitope against which the immune has not been will to T therefore the pathogenic T cells to be These cells to the target this the and an inflammatory lesion. tissue the epitope by the T cells which are to the target the target they can their has also been reported in studies of of peptides in These with those in the and the L144/R147 APL of a that the of using peptides will be the most effective The precise of the effects is an of In the peptides have been reported to the immune response towards a T cells as a of have been reported to were to a towards or immunity in normal in Tg4 however, of and production, but of peptide the of a to EAE in Tg4 The properties of have been in of in T cells that or have also been to have effects in of inflammatory of to the of peptides from a recent of in EAE EAE was induced with either PLP(139–151) or and when the same peptides were given in disease not when given This may a response with the of EAE. in our studies of the peptide of EAE induced with myelin to a in incidence and severity of of This of disease induced with myelin may not a to with peptide may induce a significantly T-cell many of which may no in due to of the peptide in a not generated by of native have that immunization with a population of cells of which a to and it is these cells that transfer EAE in the peptide-induced may be the of of cells not in This may not therefore be to the human in which T cells will be by of self antigen by immunization with a of synthetic is that there are in which we be able to the of peptide therapy by to the antigen Hence APL may be an the of a peptide for MHC a is at to the position 4 superagonists with increased for class II. Our experiments showing therefore used the 4Tyr APL to induce In our studies of the effects of MBP and peptides we that the peptide to prevent EAE induced with analysis complex T-cell responses to this peptide in to be epitopes within this peptide by distinct T-cell populations. of these epitopes are generated from antigen of native MBP the epitope is it is not generated containing the two epitopes are of the epitope is This a for T cells to epitopes from native MBP in the T-cell from mice of the that this is the dominant by the class Thus the encephalitogenic T-cell population a of the T-cell of to T cells the dominant but not those the This would for the to prevent EAE when with this to this peptide to EAE would be to APL with substitutions that the of the II The epitopes should become dominant and of the to the pathogenic T cells. an approach using APL would the of due to expansion of of T cells to epitopes as For in the system we are APL that stimulate those T cells that are to and induce EAE. These APL would be by cells that are not to it may be that these cells would be as they useful due to of antigens not to for the use of APL T-cell of antigens that have been antigens may be in the target but not in the the from central of T cells that specifically the modified of the Thus arthritis has been to T-cell in dominant T-cell responses to in disease have been to require the of residues to the of tissue alterations in antigen may have in the activation of T cells. The use of APL may therefore not the of but also our to the of cells. The use of APL over the decade has a central in our to the molecular for T-cell Identification of APL that act either as TCR or immune their use in of These however, have also highlighted the and of the T-cell in what first to be These will be in the human population and require when therapies as highlighted by recent more approach therefore is the use of natural peptide to the immune by expansion of T regulatory cells of this approach may to be however, for the of APL to the on those cells to the of to epitopes within the and of the of modified self antigens in autoimmune in the laboratory is by from the the and the and is an