Activated gut-homing CD8⁺ T cells for coeliac disease diagnosis on a gluten-free diet

Coeliac disease (CD) is a chronic immune-mediated systemic disease, which is triggered by gluten ingestion in genetically susceptible individuals. Clinical, serological, and histopathological data are used for CD diagnosis. However, these parameters generally normalized in patients following a gluten-free diet (GFD). In recent years, many patients come to the visit having started a GFD before a definitive CD diagnosis is established. In these patients, the diagnosis of CD can be problematic since a prolonged gluten challenge in a symptom-free patient on a GFD is always difficult. In recent years, low-invasive novel procedures have been proposed as good approaches for CD diagnosis in individuals following a GFD [1‐9]. This would represent an important step in clinical practice, since it would make it easier to diagnose individuals with self-prescribed GFD and those needing review of the initial diagnosis due to incomplete original testing, discrepant results, or slow or non-responsiveness to the GFD.

Determination of IL-2 has been described as the earliest potential marker for CD diagnosis after a short gluten challenge [7]. Even a whole blood IL-2 and IFN-γ release assay has been very recently described for diagnosis with no challenge requirement [8, 9]. Additionally, a 3-day gluten challenge mobilizes gut-homing memory T cells that can be detected in peripheral blood using different technologies [1, 3, 5, 7]. IFN-γ ELISPOT assays and HLA-DQ2-gliadin tetramer staining have been the most extensively described approaches, showing high sensitivity and specificity. Cytometry by time-of-flight mass spectrometry (CyTOF) enables to assay numerous parameters (>40) simultaneously and provides a deep phenotypic characterization of the mobilized cells. However, all of them show cost and technological limitations for translation into clinical practice and their sensitivity decreases in non-HLA-DQ2.5 subjects [7, 10]. Flow cytometry constitutes a more realistic alternative. This technique has become a routine tool in clinical laboratories, with increasing use in disease diagnosis [11]. We previously demonstrated that CD patients could be diagnosed in individuals on a GFD after a 3-day gluten challenge by analyzing only four markers in peripheral blood by flow cytometry [3]. The selected markers allow us to identify activated (CD38) gut-homing (CD103 and β7) CD8 T cells. In the current study, we aimed to further explore this diagnostic approach analyzing additional patients and controls and giving measures of diagnostic accuracy. As a secondary aim, we performed a pilot study to compare this test with the production of IFN-γ measured by ELISPOT in a subgroup of HLA-DQ2.5 patients.

In this work, CD8⁺ and TCRγδ⁺ T cell responses were evaluated in blood samples from subjects who underwent a 3-day gluten challenge. Activated gut-homing CD8⁺ T cells (CD8⁺ CD103⁺ β7^hi CD38⁺) were observed in the peripheral blood of CD patients, providing a diagnostic tool reaching 95% of specificity and 97% of sensitivity. Even a sensitivity of 100% can be considered if the patient with a very low anti-TG2 titer (below the manufacturer’s recommended cut-off) is excluded. Therefore, similar values to the previously reported for IFN-γ ELISPOT, blood cytokine release assays, or HLA-DQ2-gliadin tetramers are obtained [2, 4], but avoiding the technical constraints.

This finding constitutes an important advance into clinical practice. Gluten challenge is proposed for CD diagnosis of individuals with self-prescribed GFD and it is cumbersome for most of them. Our study shows that a blood test based on the analysis of four markers (CD8, CD103, β7, and CD38) by flow cytometry in individuals exposed to a 3-day gluten challenge seems as accurate in patients on a GFD as it is anti-TG2 serology in patients on a normal diet.

Gluten challenge is also recommended in doubtful cases: individuals with a Marsh 1 lesion or subjects with low-risk or non-permissive HLA genetics. The test here proposed shows high accuracy to diagnose Marsh 1 CD, which could allow distinguishing this disease from false positive serological results associated with either an unspecific mucosal lesion or other pathologies [21, 22]. The HLA influence could not be extensively investigated due to the very low number of non-HLA-DQ2.5 studied CD patients. However, it is particularly striking the CD8⁺ T cell response to gluten challenge in one CD patient lacking any HLA risk allele. Previously, this cell response had been detected in one HLA-DQ2.2 and one HLA-DQ8 patient [3, 23]. This could represent a notable advantage of the CD8⁺ T cell-based blood test when compared to ELISPOT, HLA-DQ2-gliadin tetramers technologies, and the recent proposed cytokine release assays [1, 5, 9], which require the use of selected gliadin peptides. CD patients respond to distinct gluten peptides, which imposes limits when testing the immunological response to specific gluten peptides. HLA genetic risk lies on the capacity of gluten peptides to form kinetically stable complex with the encoded HLA-DQ receptors, which is needed to elicit the T cell immune response leading to CD. It is beyond the scope of this work to speculate how CD can take place with the non-permissive HLA genetics, but once produced, CD is probably developed following the characteristic immunological cascade. The haplotype HLA-DQA1*03-DQB1*03:03 (HLA-DQ9.3), present in the studied CD subject lacking HLA risk, can reach considerable frequency in China, where it has been described as a CD susceptibility factor [24]. HLA-DQ9.3-restricted gluten-reactive T cells were detected in the small intestine of a CD patient [25]. Now, we show that they can also be detected in the blood.

Our work also solves an outstanding issue in the clinical practice, the amount and duration of dietary gluten necessary to elicit a measurable response. A 3-day challenge with 10 g of gluten allows accurate diagnosis. Some works have suggested that lower amounts of gluten can elicit changes in CD patients, thereby reducing possible discomfort and risk. However, a lower percentage of patients showing gut-homing activated CD8⁺ T cells was recently observed in patients enrolled in a 3-g vs. 10-g 3-day gluten challenge (17% vs. 83%) [7], although PBMCs instead of whole blood were tested, which could decrease sensitivity [26, 27].

Our results warn clinicians to be aware that symptoms developed after gluten challenge are unreliable indicators of the presence of CD since flatulence, abdominal distention, or altered bowel habits were also frequently reported by both healthy and disease controls, which is concordant with the literature [28]. It must be noted that most of our R-GFD disease controls probably show non-coeliac gluten sensitivity, but the diagnosis of this condition is complex, and our patients did not undergo a double-blind, placebo-controlled gluten challenge as suggested to establish a firm diagnosis [29]. Nevertheless, one limitation of our work is the use of white bread for the challenge, which can make that non-gluten dietary components such as fermentable carbohydrates (FODMAPs) confound clinical symptoms.

The CD8⁺ T cell response did not correlate with the intensity of the clinical symptoms triggered by the gluten challenge. This may seem to contrast with the correlation reported between symptom severity and cytokine levels, mainly IL-2, elicited after gluten challenge [6, 10]. However, that correlation was particularly evident for nausea and vomiting, two symptoms present in only two CD patients of our sample. This could be again related to the non-specificity of symptoms caused by the use of bread for the challenge. In any case, it is noteworthy that the present diagnostic approach is valid for subjects with an asymptomatic response to the gluten challenge.

The lack of correlation between the CD8⁺ T cell response and the duration of the GFD needs to be interpreted cautiously because the GFD compliance may differ among patients. With a good adherence to the GFD, it is expected a decline in antigen-specific memory T cells as the time from the initial immunological response increases. This probably justifies the lack of the T cell gluten-response in the patient who had been following a GFD for 25 years, which is concordant with previous observations [5]. Based on these findings, a negative result could not be considered valid when testing patients who follow a strict GFD for such a long time. Therefore, we excluded that patient to calculate the accuracy of the proposed diagnostic test.

We also investigated the subset of TCRγδ⁺ T cells. They are also mobilized after gluten challenge and do so at a similar magnitude than CD8⁺ T cells. However, TCRγδ⁺ T cells appear at low frequency in blood and the absolute numbers of the CD103⁺ β7^hi CD38⁺ TCRγδ⁺ T cells are in some CD patients as low as 2. Moreover, sensitivity and specificity of the proposed CD8⁺ T cell-based test are not increased by including TCRγδ⁺ as a marker. Therefore, this cell subset is not recommended for CD diagnosis, although our work adds evidence of its active role in CD pathogenesis [30, 31]. Remarkably, gut-homing TCRγδ⁺ and CD8⁺ T cells detected in the blood of CD patients after gluten challenge have been described as displaying a T cell repertoire partially overlapping with intraepithelial lymphocytes in the gut [32].

Leonard et al. observed quite similar sensitivity when evaluating the CD8⁺ T cell response and the production of IFN-γ measured by ELISPOT [7]. This is supported by our study, whose results warrant a formal comparison between the two diagnostic tests in HLA-DQ2.5⁺ patients at clinical practice. However, to be implemented into clinical practice, the analysis of gut-homing CD8⁺ T cells by flow cytometry shows numerous advantages over IFN-γ ELISPOT, being the most notorious: much faster processing and result delivery; easier to learn, teach, and use; need of devices (flow cytometer) routinely used in clinical settings; and non-requirement of gliadin peptides, probably allowing accurate diagnosis for subjects with different HLA-DQ receptors.

Besides the clear impact in CD diagnosis and clinical practice of our findings, our work corroborates the critical role of immunological memory in autoimmunity. The longevity and active functionality of memory T cells upon antigen rechallenge perpetuate histological damage and make it a chronic disease. Long-lived memory T cells carry TCRs that recognize antigens more effectively. On antigen re-exposures, they are expanded and mount a highly efficient quicker and stronger immune response. Therefore, these observations must be also considered when investigating new therapeutic targets.

Activated gut-homing CD8⁺ T cell measurement is a highly accurate blood test for CD diagnosis in patients on a GFD, being of easy implementation in daily clinical practice. Further multicenter studies on a larger patient sample are warranted to confirm the test diagnostic accuracy, mainly in non-HLA-DQ2.5 subjects.