Peptide pools can cover long peptide sequences and even entire proteins. Alternatively, they can contain multiple non-contiguous peptides sequences that represent key immunodominant epitopes. Peptide Pools stimulate antigen-specific CD4+ and CD8+ T cells efficiently and cost-effectively. The activated T cells can then be detected, quantified or isolated for analysis based on released cytokines and upregulated activation markers.

Peptide pools were first used in immunological research in 1980 [1]. However, their full potential was not recognized until more than 40 years later. Today, peptide pools are employed in a wide range of immunological research fields. Examples are:

• antigen and epitope discovery [2]
• immune monitoring in research and clinical trials [3,4]
• in-vitro T-cell activation and expansion in adoptive cancer immunotherapies [5]
• development of diagnostic tests.

Peptides offer several benefits over proteins in T cell stimulation tests: They can be synthesized reproducibly under the strict requirements of ISO 9001:2015. Protein expression systems are not required, and at the same time sequences can be varied as needed and post-translational modifications can be introduced as required.

Protein-Spanning Peptide Pools: Peptide Length and Overlap

Peptide pools typically represent a complete protein by mapping its primary amino acid sequence into 15mer peptides, each overlapping by 11 amino acids. The 15-amino acid length is a good compromise, ensuring epitopes can bind to both MHC Class I and II binding grooves. Figure 1 illustrates the necessary properties for peptides to bind to the two different binding grooves. Although a length of 9-amino acids would be optimal for the MHC Class I binding groove, peptides with 15-amino acids can still effectively stimulate CD8+ T cell responses in ELISPOT experiments, either because they are still short enough or due to extracellular trimming. Because of the overlap of 11 amino acids, every possible epitope up to 12 amino acids in length is covered in at least one of the overlapping 15-amino acid peptides. Each 15-amino acid peptide is generally present at a concentration of 1 µg/ml, but the optimal concentration can vary based on the specific peptide sequence and the intended application.

Sequence Variants in Peptide Pools

Pathogenic viruses and tumors often exhibit sequence diversity due to frequent mutations, with viruses also experiencing recombination events. This variability can significantly complicate immune recognition. Therefore, it's crucial to consider this diversity when designing peptide pools for immune monitoring or vaccination, requiring complex peptide pools to encompass all potential sequence variations. Bioinformatics algorithms can be employed to calculate all possible peptides and assess their prevalence, thereby ensuring comprehensive sequence coverage of the peptide pool. This type of pool is useful for studying the presence or absence of T cell responses and monitoring longitudinal changes. For instance, it has been successfully utilized to monitor HIV infections, which are characterized by high mutation rates [6].

Pools Covering Key Immunodominant Epitopes

Given that the entire proteome of a pathogen is often too extensive to be covered by protein-spanning peptide pools, a peptide pool containing all identified T cell stimulating peptides of the pathogen can serve as an efficient alternative. Furthermore, combining key immunodominant epitopes from various pathogens in a single peptide pool is another viable approach.
In addition to utilizing already published peptide sequences, peptide-MHC binding predictions can be employed to design the peptide pool. While this method might overlook some potentially stimulating peptides, it provides an efficient and cost-effective means to epitomize all T cell stimulating components of a pathogen in a single stimulation.

Epitope Mapping with Matrix Pools

Matrix pools efficiently identify T cell epitopes, requiring minimal material. In this process, peptides are grouped in a matrix design: Each peptide is placed in a table field, with row and column peptides combined into mapping sub-pools. As a result, each peptide features in one row and one column sub-pool. A positive response to both a row and column sub-pool in a T cell assay indicates the shared peptide that stimulates the T-cells. This method significantly decreases the number of assays required for individual peptide testing. The identified stimulating peptide should be confirmed in a seperate test. For epitope mapping, a peptide length of 8-20 amino acids, overlapping by 1-4 amino acids, can be optimal.

Positive control pools: essential for successful immune monitoring

T cells and antigen-presenting cells (APCs) are sensitive and can be damaged throughout the entire experimental process: through shear forces during blood collection, delays in transport, exposure to extreme temperatures, cryopreservation and thawing processes. Positive controls are therefore essential for T cell immune monitoring. Antigen-specific T cell immunity can only be reliably assessed, if the functional fitness of the T cells and APCs is ensured.
The CEF Control Peptide Pool advanced is most popular for testing the functionality of antigen-specific T cells. It is a lyophilized mixture of 32 key immunodominant epitopes from three widespread viruses: Cytomegalovirus (CMV), Epstein-Barr virus (EBV) and Influenza A virus (Flu). The selected peptides are CD8+ T cell epitopes that match common HLA class I alleles. They reliably stimulate human CD8+ T cells to secrete granzyme B and cytokines such as IFN-γ and IL-2. By checking cytokine secretion, the CEF Control Peptide Pool advanced ensures the functionality of T cells and APCs after storage, freezing and thawing. It is used in methods that are central to vaccine development, tumor targeting and other immunological research areas:

• ELISPOT
• FluoroSpot
• CTL assay
• Analysis of intracellular cytokine
• Flow cytometry

The  is also frequently used as a positive control, as it typically triggers robust immune responses in most individuals. Given the high vaccination coverage for tetanus, the Tetanus Toxin Peptide Pool also serves well as a positive control, eliciting vigorous responses in most cases.

Peptide Pools as Negative Controls

In addition to false-negative signals, T cells can exhibit reactions not specific to a particular antigen. Such false-positive signals can also influence the outcomes of T cell assays. Researchers often employ media with an equivalent peptide solvent quantity as in the test series as a negative control. However, peptide-containing control pools can provide a more accurate assessment of the true background signal in a test. The HUMAN (Actin) Peptide Pool, spanning non-immunogenic human ß-actin, is a suitable option. Alternatively, virus-based peptide pools can be utilized, when testing material from previously unexposed individuals – such as the HIV control Peptide Pool for HIV-negative subjects. Nevertheless, virus-based negative control pools can cross-react with antigens to which an individual has had prior exposure, leading to T cell reactions that surpass the genuine biological background.

Ideal Size of a Peptide Pool

The number of peptides in a peptide pool can vary significantly. There are small peptide pools such as the HBV control peptide pool with a mere nine epitopes, while large peptide pools such as the tetanus toxin peptide pool encompass 326 overlapping peptides. In general, it's advisable to avoid overloading a pool with peptides, as they can interfere with each other and are more prone to precipitate in high concentrations. Competition for MHC binding sites can also pose an issue with extensive pools. However, the ideal size of a peptide pool hinges on the peptides' solubility and intended application.

Literature

1. Kazim, A L, and M Z Atassi. “A novel and comprehensive synthetic approach for the elucidation of protein antigenic structures. Determination of the full antigenic profile of the alpha-chain of human haemoglobin.” The Biochemical journal vol. 191,1 (1980): 261-4. doi:10.1042/bj1910261
2. Li Pira, Giuseppina et al. “High throughput T epitope mapping and vaccine development.” Journal of biomedicine & biotechnology vol. 2010 (2010): 325720. doi:10.1155/2010/325720
3. Dahlke, Christine et al. “Comprehensive Characterization of Cellular Immune Responses Following Ebola Virus Infection.”  The Journal of infectious diseases vol. 215,2 (2017): 287-292.doi:10.1093/infdis/jiw508
4. Nathan, Anusha et al. “Structure-guided T cell vaccine design for SARS-CoV-2 variants and sarbecoviruses.” Cell vol. 184,17 (2021): 4401-4413.e10. doi:10.1016/j.cell.2021.06.029
5. Tran, Eric et al. “'Final common pathway' of human cancer immunotherapy: targeting random somatic mutations.” Nature immunology vol. 18,3 (2017): 255-262. doi:10.1038/ni.3682
6. Al-Kolla, Rita et al. “Design and validation of HIV peptide pools for detection of HIV-specific CD4+ and CD8+ T cells.” PloS one vol. 17,8 e0268370. 16 Aug. 2022, doi:10.1371/journal.pone.0268370