A human dendritic cell-based method to identify CD[4.sup.+] T-cell epitopes in potential protein allergens - Mini-Monograph

Environmental Health Perspectives, Feb, 2003 by Marcia Stickler, Jeanette Mucha, Scott Power, Fiona Harding

We developed an assay to determine the location of immunodominant CD[4.sup.+] T-cell epitopes in any protein. The method uses CD[4.sup.+] T cells from community donors in conjunction with dendritic cells derived in vitro. Synthetic peptides constructed to describe the sequence of the protein of interest are cocultured with dendritic cells and CD[4.sup.+] T cells, and T-cell proliferation is measured. Data are compiled over a large replicate of human donors to pinpoint immunodominant, usually promiscuous epitope regions. We have applied this technique to a known food allergen, the Brazil nut 2S storage globulin protein, and to two potential food allergens, the Cry1Ab and Cry3Aa proteins. We show epitope data for these three proteins. This assay can be used as a tool to guide the selection and qualification of future potential food transgenes. Key word. e. dendritic cells, food allergens, human donors, T-cell epitopes. Environ Health Perspect 111:251-254 (2003). [Online 21 January 2003]

doi: 10.1289/ehp.5707 available via http://dx.doi.org/

For the immune system to respond to a protein with a finely tuned, high-affinity antibody response, antigen-specific CD[4.sup.+] helper T cells must be activated. Activation of CD[4.sup.+] T cells is a prerequisite for differentiation along either the Th1 or Th2 pathway. Activated Th2 cells are strongly associated with the advent of allergy (Kapsenberg et al. 1998; Mazzarella et al. 2000; O'Hehir et al. 1991; Van Neerven et al. 1996). Activation of T cells requires the recognition via their T-cell receptors of linear peptide antigens presented in the context of a cell-surface human leukoctye antigen (HLA) class II molecule. Any given protein immunogen will contain a discrete number of epitope regions capable of inducing activation for a particular HLA class II allele. Interestingly, the same T-cell epitopes are capable of inducing either Th1- or Th2-type responses (Van Neerven et al. 1994), depending on various environmental and antigen-presenting cell-specific factors (Constant and Bottomly 1997; Lanzavecchia and Sallusto 2001). There is considerable interest in the description of T-cell epitopes because the inclusion of helper epitopes improves the immune response to synthetic vaccine constructs (Alexander et al. 1994, 1998; del Guercio et al. 1997). Peptide epitopes have been used in the treatment of immunologic disorders such as allergy (Oldfield et al. 2001; Rolland et al. 2000) and cancer (Kobayashi et al. 2000; Slansky et al. 2000). Finally, manipulation of commonly promiscuous T-cell epitopes can be used to create reduced-immunogenicity proteins for use in a variety of applications (Warmerdam et al. 2002a, 2002b).

There are currently methods to determine peptide binding to some HLA class II-DR and -DQ molecules. Some of these methods measure the relative strength of the peptide-HLA interaction using isolated class II molecules and purified peptides. Other methods predict peptide binding to HLA using computer algorithms (Fleckenstein et al. 1999; Sturniolo et al. 1999; Yu et al. 2002). These predictive methods are more successful for class I molecules because of the more rigid requirements for peptide length and anchor residues. Unfortunately, peptide binding to HLA is not sufficient to predict the presence of a functional interaction with T cells. There are many examples in the literature of poorly binding T-cell epitopes and of tightly binding peptides that are not T-cell epitopes (Adorini et al. 1988; Fugger et al. 1996; Lo-Man et al. 1998; Ma et al. 1999; Velazquez et al. 2001). In addition, amino acids that flank the epitope core have been shown to have profound effects on T-cell activation in the absence of any effects on HLA binding (Godkin et al. 2001).

We have developed a functional assay that localizes T-cell proliferative responses to peptide epitopes using human community donor cells as the test material (Stickler et al. 2000). The localization of epitopes in our assay is based on a population approach, in that a large replicate of community donor responses is compiled and analyzed for the presence of an "immunodominant" peptide. Interestingly, many HLA class II alleles present similar epitopes ("promiscuous" T-cell epitopes), a property caused by shared binding pockets among the multitude of HLA class II alleles (Southwood et al. 1998). We have noted HLA-DR associations with particular peptide responses (Stickler et al. 2000) but assume that our assay largely identifies promiscuous HLA-DR supertype-associated epitopes.

Our assay was developed to predict functional T-cell epitopes in a population of individuals who have not been previously exposed to the protein under study. This is important for two reasons. First, many novel therapeutic and recombinant proteins are environmentally "new" in that human exposure to these proteins is not detected; therefore, methods are needed to predict a priori immunogenicity. Second, evidence from the literature suggests that as an immune response develops, T-cell epitope complexity increases. T-cell epitopes that "prime" the system can be completely discrete from secondary epitopes that arise as the response matures (Muraro et al. 2000). This is especially pronounced when studying T-cell clones, where an individual cloned line may have strict specificity for an epitope that is not an immunodominant epitope as defined by a pooled T-cell analysis. In human population-derived data where there is a known sensitization rate [e.g., the ~1% of the general population who are verified sensitive to peanuts (Sicherer and Sampson 2000)], our results likely expose both the immunodominant and the subdominant epitope regions in the protein of interest. Although not shown here, immunodominant and secondary epitopes could be distinguished in the described assay if the input CD[4.sup.+] T cells were separated into naive (CD[4.sup.+] CD45R[O.sup.-]) and memory (CD[4.sup.+] CD45R[O.sup.+]) populations.