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T-cell Epitope Mapping Strategies

Page Contents
Synthesis of Peptides
Choice of N- and C-terminal Peptide Endings for Mapping of T Cell Epitopes
Mapping of T Cell Epitopes with Clones
Helper T Cell Clones
Cytotoxic T Cell Clones
Epitope Mapping with Polyclonal T Cells
General Method for In Vitro Stimulations with PBMC
Strategy and Experimental Design
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The Multipin Peptide Synthesis System is a rapid and simple method for the simultaneous production of many short peptides. The application described here is the use of solution-phase peptides for the mapping of T cell epitopes. T cell epitopes are defined as peptide sequences which, in association with proteins on antigen-presenting cells (APC), are required for recognition by specific T cells. Through the use of overlapping peptide sets, the epitopes can be identified in a systematic and thorough manner, and the minimal sequence(s) required for recognition can be determined. Synthetic peptides are added to T cell culture systems and parameters of T cell activation are measured. The method is equally applicable to T cell clones, T cell lines, and unpurified T cells, such as lymph node cells and peripheral blood mononuclear cells (PBMC).


Synthesis of Peptides

The peptide sequences of choice may be purchased economically and ready to use from Mimotopes as a PepSet, or may be synthesized on SynPhase Gears as supplied in our Cleavable peptide kits. These kit peptides contain a diketopiperazine ending when cleaved [1,2,3,4,5,6,7].

PepSets are available with a choice of C-terminal endings, and in higher yield than from kits. Kits to make peptides with a variety of C- terminal endings are not currently offered because the post- synthesis handling of the peptides requires a greater degree of expertise, and special equipment not available in many microbiology/immunology laboratories.


Choice of N- and C-terminal Peptide Endings for Mapping of T Cell Epitopes

For CD4+ (helper) cells, N- and C-terminal blocked peptides can be more effective than peptides with unblocked ends when short peptides (up to 15mers) are being tested [3,5,8,unpublished observations]. Peptides of 15 or more residues, with blocked ends, can also be very effective for initial location of epitopes for CD8+ (cytotoxic) cells [Moss et al., personal communication], but for the detailed mapping of minimal epitope sequences, peptides with unblocked ends are mandatory [9,10].


Mapping of T Cell Epitopes with Clones

The procedure for identifying epitopes for clones is much easier than for polyclonal cell preparations. Provided the clone(s) is in an antigen-responsive state, i.e. has not recently been stimulated with antigen or with IL-2, simple addition of a peptide containing the epitope to APC plus T cells is usually sufficient to bring about T cell activation. This qualitative approach can begin with longer peptides (e.g. >12mers) and be followed by testing with progressively shorter peptides from the active sequence until the minimal epitope has been defined. Many other factors can be varied and will have an effect on the quantitative aspects of the responses seen. These include:

   1. The peptide length
   2. The peptide concentration
   3. Presence or absence of end groups on the peptide
   4. The method used to measure T cell activation, including the type and number of APC used, and the time allowed between exposure of the T cell clone to the APC/peptide complex and the measurement of an activation parameter
   5. The extent of peptide processing or breakdown which can occur in the assay system [11]
   6. Other factors, such as the possible presence of toxic contaminants in the peptide preparation, low viability of the T cells or APC, unsuitable media, variation in incubation conditions etc. This latter set of factors would be properly controlled for in a well-designed experiment.

In extreme cases, such as when the peptide concentration is too low or the peptide is too long, the ability to measure T cell activation may be compromised. As there are so many possible variations of assay methods, we will not attempt to cover them here, but rather give references to some examples, and a brief outline of a method for helper T cell clones and one for cytotoxic T cell clones.


Helper T Cell Clones

Clones are usually maintained by regular cycles of stimulation with antigen plus APC, or with growth factors such as recombinant IL-2 or supernatants of nonspecifically stimulated PBMC. For an epitope mapping experiment, the T cells should be "rested" without these stimuli, e.g. for 5-7 days in unsupplemented media [12]. For the epitope mapping experiment, a suitable type of APC must be chosen. The APC must be histocompatible with the T cell clone, must express MHC class II and are often unable to proliferate under the test conditions. For human T cell clones, typical examples of APC would be "autologous" EBV- transformed B cells or PHA-blasts which have been prevented from proliferating by lethal irradiation or treatment with mitomycin C.Peptide at one or more concentration levels (e.g. a range of concentrations from 10 micromolar to 0.01micromolar) is added to the APC. After an incubation period of 1h or more, the excess peptide may be washed away or the T cells may be added directly to the "sensitized" APC. After a further incubation period of 1-3 days, a parameter of T cell response is measured, e.g. incorporation of tritiated thymidine into cellular DNA or release of a lymphokine such as IL-2.


Cytotoxic T Cell Clones

The generation of CTL clones usually requires efficient processing/presentation of antigen in association with MHC class I molecules, a process which can be accomplished by infecting APC with viruses capable of expressing the antigen. Alternatively, the antigen can be supplied as a short peptide (if an appropriate sequence is known), but the addition of whole protein antigens may fail to stimulate the development of CTL due to inappropriate presentation [13,14].The choice of APC (target cells) for CTL killing assays is in principle much wider than the choice of APC for helper cells, as any histocompatible cell expressing MHC class I could be used. The sensitization of target cells with the peptide is similar to that described above for helper cells, but if the killing of target cells is to be measured, e.g. by release of 51Cr-chromate, actively growing targets can be used (in contrast to the APC in proliferation assays), and the assay may be carried out in hours rather than days. Simple addition of the peptide to the medium containing CTL and targets can give rise to T cell-T cell killing [15], so it is probably best to sensitize targets and wash away excess peptide before adding the T cells. Controls for peptide toxicity and spontaneous target cell lysis should always be included. Conventionally, titration of the effector:target ratio gives some indication of the "strength" of recognition. CTL response can also be measured by lymphokine release rather than cytotoxicity.As mentioned above, mapping CTL epitopes to their minimal length requires use of unblocked peptides of 8, 9 or 10 residues. Effective peptide doses may be three or more orders of magnitude lower for CTL target sensitization than for helper APC sensitization [9,16,17,F. Carbone, D. Moss: personal communications].


Epitope Mapping with Polyclonal T Cells

The requirements and limitations of epitope mapping studies in animals are different from those in man. In animal studies, immunization or infection can be used to ensure the presence of antigen-specific T cells, whereas in man one normally relies on therapeutic or natural exposure to immunogens. For animal studies, cells from lymphoid organs would be used (e.g. spleen, lymph nodes) whereas peripheral blood is the only readily available source of lymphocytes in man. It is important to note that murine T cells require the presence of 2-mercaptoethanol [18] in order to proliferate in vitro, unlike human T cells [19]. The entire set of epitopes for an antigen/animal strain combination can be mapped using polyclonal T cells and overlapping peptides [3].


General Method for In Vitro Stimulations with PBMC

At Mimotopes we have been using peptides for mapping T cell epitopes using human peripheral blood as the source of polyclonal T cells. If you are interested in designing experiments with peripheral blood mononuclear cells (PBMC), we include here a method we have found to be reliable.PBMC are isolated from heparinized or defibrinated venous blood by Ficoll/hypaque density interface centrifugation. We routinely use 200,000 PBMC per round-bottom microtitre well in a volume of 200 microlitre. The medium consists of RPMI#1640 with 2mM glutamine, antibiotics, 5mM HEPES buffer and 10% human serum, either screened, pooled serum or autologous serum from defibrinated blood. Cultures are incubated with added peptide for four to six days at 37°C in humidified 5% carbon dioxide/air, then trace-labeled with 0.25 microCuries of tritiated thymidine of high specific activity (>40Ci/mmol) for 6h before harvesting the DNA and scintillation counting. Because of the relatively low frequency of T cells specific for any given peptide epitope it is important to set up sufficient replicates of peptide-stimulated and control PBMC cultures to allow an estimate of the frequency of positive peptide-specific responses to be made. We have developed IBM-compatible software which assists with the design of these assays, and analyses the data from them. The software calculates a precursor frequency of responding T cells in each peptide treatment group.


Strategy and Experimental Design

The following strategy has been developed for studies on PBMC but aspects are applicable to T cell lines and clones also.

1. Synthesis of Overlapping Peptides

The strategy we have used up until now is to make all possible overlapping peptides, of a defined length (e.g. 15mers), homologous with the antigen of interest. Other strategies which are more economical on pin/peptide numbers can be used, e.g. making longer peptides, with their N-terminal residues offset along the sequence by 2, 3 or more residues. The disadvantage of not making every overlapping peptide is the loss of resolution and the risk that an epitope will be missed altogether. For instance, a minimal length epitope of ten amino acid residues will be present in four successive 13mer peptides each offset by one amino acid residue. The same decamer epitope will only occur in two successive 13mers offset by three residues, and will occur in only one peptide if successive 13mer peptides are offset by four residues.Further considerations in designing the synthesis are cost and overall size of the assays using the peptides. Decreasing the degree of overlap will reduce the total number of peptides synthesized for a given sequence, and thus the cost. However, this decreases the number of times a epitope will occur within sequential overlapping peptides as described above. Increasing the length of each peptide may help overcome this but in turn increases the cost. Thus the degree of overlap, the peptide length, and the expected epitope length all need to be considered when designing a synthesis.

2. Post-Synthesis Peptide Pooling

Pooling of peptides can reduce the "size" of a screening test with synthetic peptides. The "size" of the stimulation test for the initial screen, defined as the amount of blood or number of T cells required, is affected by the number of peptides in each pool, i.e. the size of the test reduces as the number of peptides in each pool increases. The size of the test could be kept small by making the pools very large; we do not know the upper practical limit to the number of peptides in a pool.Experiments with pools of different sizes suggest that pools of 20 or more peptides are not only feasible, but also desirable, because the sensitivity for detection of a "determinant region" is enhanced by the use of large pools of overlapping peptides, due to synergism between overlapping epitopes. The amount of blood required is also affected by the amount of further testing required to "decode" the results of tests with pooled peptides, i.e. to find the individual peptides responsible for proliferation incurred by pools. If individual peptides from a pool are to be screened by the same method as used for the pools themselves, the size of the "decoding" experiment can be larger than the original screening with the pooled peptides! The size of a total screening experiment, i.e. an experiment to screen every part of a protein sequence, is reduced each time a pool is found to be negative (nonstimulatory). Conversely, no benefit (in terms of reduced scale of the screening test) is gained if nearly all pools are positive (stimulatory)! If the pools are large to start with (e.g. 20 or more peptides), it may be more "economical" on scarce blood cells, peptide, and other resources to decode by testing a set of smaller pools prior to testing individual peptides.

3. Peptide Yield and Utilization

The expected yield of 13mer peptide from a pin in a synthesis kit is more than 100nmol (about 150 micrograms). More than 500 wells of PBMC can be stimulated with this amount of peptide when used at 1micromolar, a level we have found to be very effective in these tests. This allows the allocation of 24 wells per donor for each pool in the screen using pools, with the possibility that a further 24 wells per donor will be required for decoding a positive result with that pool. For a peptide which was positive with every individual tested, this allows 10 donors to be fully screened (10x24x2=480 wells). The number of individuals who can be screened with a particular pool increases by one for each individual whose cells give a negative screening result on that pool, because the decoding test does not need to be carried out. Thus it is probable that 15 individuals can be screened with high precision using 100nmol of peptide. Peptides ordered as sets from Mimotopes are made at the 500-1000nmol scale and will thus allow 5 to 10 times the amount of work to be done as can be done with peptide made with a kit.

4. Human Sera for Medium Formulation

The selection of sera for supplementation of the defined growth medium is a vital factor for success. We have recently found that autologous serum prepared from defibrinated blood (defibrinated with glass balls) gives more consistent support of proliferation over a panel of donors than a single batch of human serum made by an exhaustive process of screening and pooling of the "best" sera. Our experiments suggest that it may be an advantage, when testing whole antigens, not to heat- inactivate the autologous serum for medium supplementation, but we routinely do so for safety reasons and to reduce the risk of proteolytic breakdown of the added peptides. If not using autologous sera, one would ideally start with human AB sera and screen with a panel of donors' PBMC of known ability to respond to an antigen, to select those sera which give low "cells alone" backgrounds while also strongly supporting antigen- driven proliferation.



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