Wheat genotypes containing minimally harmful gluten sequences

“Previous studies have documented that landraces and older wheat varieties contain more diverse gene combinations for prolamins (wheat proteins) in comparison to modern varieties (10, 11). Literature shows variations for specific gene sequences mainly in the epitopic regions of Glia-α9, Glia-α2, Glia-α20, and Glia-α in older landraces (9). In the last decade in the context of CD, the immunogenicity of T-cell specific epitopes has been bought to the forefront (9, 12). The immunogenic potential amongst different hexaploid wheat varieties is variable; hence it is possible that there are breeding-induced differences in the presence and expression of T-cell stimulatory epitopes in modern varieties of wheat (13, 14). This raises the question of, whether there is any specific variety of wheat which is less immunogenic and can be used in breeding programs for developing a wheat genotype completely safe for consumption by patients suffering from CD”.

Highlights:
• The identification of less/non-immunogenic wheat species is an important milestone that could help patients or even prevent CD.
• With the use of gluten-specific T-cells and PBMCs, wheat genotypes containing minimally harmful gluten sequences can be selected.

….omissis. “In the current pilot study, the immune toxicity of wheat antigen from four genotypes of old hexaploid wheats, C591, C273, 9D, and K78 and the current wheat variety PBW-621was assessed using in vitro assays on CD patient-derived gliadin reactive Tcells and PBMCs. These specific varieties were selected from our previous study, where based on in silico and gene sequencing analysis we reported these to carry a reduced load for T-cells stimulatory epitopes (15). Our findings show that a substantial immunogenic difference exists among these four varieties of wheat. The results predicted that total gliadin extract from four test varieties K78, 9D, C591, C273, and PBW621 manifested differential ability to stimulate celiac mucosal T-cell lines. This was specified by cell proliferation assays and IFN-γ and TNFα production. Gliadin extract from an old variety C273 released by the Department of Agriculture, Punjab, India in 1957 showed least immunogenicity amongst all test varieties analyzed. Rate of multiplication of patient derived T-cells and PBMCs was observed to be minimal in cultures stimulated with C273 gliadin. On the contrary an increased proliferation was shown with other varieties K78, C591, and PBW621 (control) (Figures 3A,B, Table 2). Similarly, secretion of IFN-γ and TNF-α were least in C273 activated cultures (Table 3, Figures 4, 5). These findings on comparison with other wheat genotypes taken (K78, C591, 9D, and PBW621), with controls and with unstimulated cultures were consistent with low immunogenic potential of C273. We feel that proliferation assay from both gluten reactive T-cell and PBMCs when combined with production of inflammatory cytokines, should detect the potential of gliadin peptides to activate celiac lesion T-cells.

The identification of less/non-immunogenic wheat species is an important milestone that could help patients or even prevent CD. One of the major restricting factors, however, is the lack of an operative animal model that could fully correlate with the disease pathogenesis; hence in vitro models have been developed. Staining with mAbs limits the revelation of all present T-cell epitopes due to its shorter epitope recognition site, and due to interference of some other sites that do not represent complete epitopes (9). Therefore, intestinal T-cell clones are considered as sensitive and accurate monitors to analyze complex protein digests from different wheat accessions (13, 23). A potential pitfall is that direct in vivo gluten exposure causes destruction of villous architecture along with inducing T-cell activation (24). This relationship cannot be formally established ex vivo but we believe that assessing gluten response of celiac derived T-cells is a prime in vitro marker to explore the toxicity of any wheat variety. To minimize this, polyclonal T-cells from four different treated CDpatients and two non-CD healthy controls for complimentary testing were taken.

A higher degree of polymorphism exists in gluten genes in individual wheat accessions, which may be suggestive of distinct toxicity profile of different wheat genotypes (12). This has prompted analysis of selected old hexaploid wheat genotypes to find allelic variants in modern bread wheats which are purported to be significant in CD. Previous studies have failed to identify non-toxic wheat. However, as the knowledge on Tcells stimulatory epitopes is increasing interesting observations are being reported (9, 25–28). Both the amount of gluten in the wheat genotypes along with the level of toxic T-cell stimulating epitopes contribute toward the noxious nature of wheat for CD patients (29, 30). In 2005, Molberg et al. defined highly immunodominant and immunostimulatory αG33 mer fragment encoded by genes located on Gli-2 locus of chromosome 6D (23). Experimental data from different Triticum species with AA, AABB and AABBDD genotypes reinforced the cytotoxicity of wheat varieties against intestinal epithelial cells, however; in contrast the studies conducted on CaCO-2/TC7 and K562(S) cells reported no cytotoxicity (28, 31). Another study documented emmer and durum wheats to exhibit lower reactivity than common wheat due to lack of D-genome (32). But even these species have been shown to express T-cell immunogenic αand γ-epitopes, their reactivity profile is not universal, therefore, safety of wheat species depends on an individual’s response to gluten (29, 33). Previous studies comparing European heritage and modern wheats, identified less toxic wheat landraces (9). Despite a seemingly high prevalence of CD in India (1.04%) (34), only one report from our group; has been published so far to evaluate Indian wheat cultivars (15). This trial is an important step in the direction where old Indian wheat varieties have been examined for their potential to elicit CD……….omissis C273 is a pre-green revolution variety which is medium tall and low yielding. Hence, it failed to become a popular choice among cultivators (39, 40). C273 along with other C series varieties, however, are reported to be high quality wheats especially for flat bread (chapatti) purposes. The increasing prevalence of CD worldwide is a concern for both the consumers and health professionals. There is no doubt that improved and convenient diagnostics and hygiene hypothesis have contributed to this increase. But owing to extensive breeding of wheat in past 50–100 years, genes coding for gluten proteins may have lost their heterogeneity (41, 42). Kaur et al. (15, 40) and the present study emphasize the testing of old wheat accessions. In conclusion, by considering the level of toxic T-cell epitopes our data indicate that with the use of gluten-specific T-cells and PBMCs, wheat genotypes containing minimally harmful gluten sequences can be selected. Our results based on in vitro analysis, exposing duodenal mucosal biopsy derived T-cells extracted from CD patients in remission to different wheat varieties reveal C273 genotype as a potential safer variety, but a larger study should be conducted to confirm the finding.” Variable Immunogenic Potential of Wheat: Prospective for Selection of Innocuous Varieties for Celiac Disease Patients via in vitro Approach. Jasmine Grover, Parveen Chhuneja , Vandana Midha et alt. Frontiers in Immunology | www.frontiersin.org ORIGINAL RESEARCH published: 04 February 2019