In type 1 diabetes (T1D) there is a destruction of insulin producing cells as a consequence of an autoimmune response in which lymphocyte subpopulations are crucial. Antigen-specific CD8⁺ T lymphocytes play an important role in the autoimmune response in T1D at the pancreas (1, 2). Due to its inaccessibility, few studies have been performed analysing islet insulitis in the target tissue. Even so, it has been proved that at the onset of the disease resident memory CD8⁺ T cells are infiltrating the gland (3, 4). Moreover, an increase of Th17 cells and a decrease in number and functionality of Tregs has been found in pancreatic lymph nodes, supporting their role in the pathogenesis of T1D (5).

A large number of existing studies have examined alterations in peripheral lymphocyte subpopulations, although most of them have been performed in paediatric patients with T1D (6–11). In general, it has been reported an increase of Th1 and Th17 cells in peripheral blood, whereas Tregs cells are impaired, supporting their involvement in the pathogenesis of T1D (12–15).

In contrast to paediatric studies, the research of lymphocyte subpopulations in adulthood T1D remains limited. It was reported in the onset of the disease a predominance of an IFNγ-producing response (16). Additionally, higher amounts of Th17 cells were found in adult newly diagnosed patients (17). To our knowledge, only Apostolopoulou et al. performed a longitudinal study regarding the correlation of peripheral leukocyte subpopulations and their correlation with metabolic parameters (18).

Recent advances in the development of multiparametric flow cytometry have made possible an exhaustive and detailed characterisation of lymphocyte subsets in whole blood or isolated peripheral blood mononuclear cells (PBMC) of healthy donors and patients (19–22) and has been shown as a powerful tool for immunomonitoring of autoimmune diseases.

In order to properly address this question, we performed an exhaustive flow cytometric analysis in whole blood. We therefore analysed what are lymphocyte subpopulations like when found in peripheral blood in adulthood at onset of T1D and investigated whether these changes were maintained at established disease. The aim of this work is to define which changes detected in peripheral lymphocytes using flow cytometry at the onset of T1D could be potential biomarkers of disease in order to identify those individuals at risk to develop the autoimmune response.

In order to define changes that could be putative biomarkers of disease we performed an extensive whole blood multi-parametric flow cytometry analysis of T and B cell compartments in patients with adult-onset T1D and at established disease. We found an increase of naïve CD4⁺ and CD8⁺, of DP T cells and IgM-only B cells and a decrease of Treg and transitional B cells at onset of the disease. At established disease there is a decrease of Th17 cells and immature B cells, whereas IgM-only B cells increase when compared to onset. Percentage and absolute counts of late EM CD4 T cells are the best candidates to be studied as potential biomarkers of disease.

Previous studies described an increase of Th1 and Th17 cells (mainly at disease onset) and a decrease of Treg subsets (5, 13). Our results are in line with these findings, showing that at the onset of the disease there is an increase of total CD8⁺ T cells and a decrease of total CD4⁺ T cells in peripheral blood, and a bias towards a decrease of memory and effector T cells, which could be infiltrating the pancreatic tissue and perpetuating the autoimmune response. In fact, the presence of tissue resident memory CD8 T cells in insulitic lesions has been demonstrated by Kuric et al. in biopsies of adult patients at onset of the disease (3). We can also confirm that there is a decrease on the percentage of memory Treg cells in peripheral blood at the onset of disease. Disfunction of Treg cells has been associated to autoimmunity in a broad spectrum of autoimmune diseases. In T1D it has been described a decrease of this subset in peripheral blood although there is some controversy in the literature published (10, 26, 27), probably due to variability in the methodology used and in the characteristics of the patients included. Moreover, Ferraro et al. described a decrease in the frequency and functionality of Treg in pancreatic-draining lymph nodes (5). In the same study, authors found an expansion of Th17 cells in pancreatic-draining lymph nodes. In this context, although we didn’t find an increase in the percentage of Th17 cells at the onset of the disease when compared to HD, we showed that in the long-term disease this subset decreased when compared with disease onset. These results enforce the role of Th17 in the early stages of the disease, concurring with the moment with highest level of tissue damage. Moreover, a decrease in the percentage of Th17 in the long-term disease has also been reported in the second year of progression of the disease in pediatric patients (8).

The role of B cells in the initiation and progression of the disease is crucial (28, 29). Here we found a decrease in transitional B cells. Previous published results regarding this subpopulation were not conclusive: whereas Habib et al. (30) found an increase in peripheral blood, Thompson et al. (7) did not show differences. Interestingly, transitional B cells phenotype [defined as CD19⁺CD27^-CD24^hiCD38^hi (31)] can be included in the IL-10 producing regulatory B subset CD24^hiCD38^hi described by Blair et al. (32). Previous studies found a decrease of this regulatory B subset in T1D patients (33–35) suggesting that the lower frequencies of transitional B cells found in the present study could be related to an impairment in their regulatory properties. Moreover, here we show that patients at onset of the disease have higher percentages of naïve and IgM-only memory B cells, supporting that B cells can play a role in the pathogenesis of the disease, although more studies focusing on it are lacking.

When comparing our results to those in children at onset of T1D, a similar pattern in T cell subsets was found (8): a decrease of memory Tregs and increase of activated Tregs, decrease of Th17 at established disease and a decrease in CD4 and CD8 effector subsets. Interestingly, regarding B cells, no changes either in transitional or naïve and IgM-only memory B cells were observed in children at onset of disease.

It has been postulated that DP cells arise as a result of the failure of maturation of DP thymocytes that do not undergo negative selection and constitute a population of recent thymic emigrants (RTEs) in periphery. Alternatively, DP T cells may result from the expansion in the periphery of mature cells that re-express either CD4 or CD8 (36–38). Last years, our group has been working in elucidate if there is an increase of RTEs in peripheral blood of patients with organ-specific autoimmune diseases. Previous results in patients with Graves’ disease showed an increase of DP T cells in peripheral blood whereas in the thyroid gland an increase of TREC levels was found, suggesting an influx of T cells recently egressed from the thymus into the thyroid (25). However, these results were not confirmed in a second study (24). These discrepancies could be explained by differences in the duration of the disease between both groups of patients, being longer in those with expanded DP T cells. Interestingly, in the present study in adult-onset T1D there was an increase in peripheral blood of DP T cells with a naïve phenotype whereas DP T cells with an early EM phenotype are decreased, when compared with HD. These findings highly suggest the role of a failure in central tolerance mechanisms in the pathogenesis of T1D. However, it should be taken in account that inflammatory and metabolic alterations in T1D can influence the increase of DP T cells at the onset of the disease. For example, it has been described that episodes of hyperglycemia impair phagocytic function (39). So, it is reasonable to speculate that inflammation and hyperglycemia can alter thymic function in diabetes, as reported in ageing (40) and type 2 diabetes (41).

In a subgroup of patients analysed at onset of the disease and again at established disease (more than 2 years after the diagnosis of T1D), a decrease of Th17 cells was found, suggesting that this subset plays a role in the early stages of the disease. Furthermore, immature B cells decrease, and IgM-only memory B cells increase at established disease enforcing the role of B cells in the pathogenesis of the disease.

Until now, autoantibodies against pancreatic islet are the only immunological biomarkers used to define the risk to develop T1D (42). Interestingly, previous studies with antigen-specific cells and in miRNAs in CD4 subsets pointed T cell subsets potential markers of early autoimmune response (43). From all these studies and with the aim to find putative biomarkers of the lymphocyte subpopulations analysed we performed ROC curves. Here we found that, of the 33 subsets analysed, late EM CD4 T cells are the best candidate to be used as biomarkers of T1D. Future research should assess the potential of these subsets as putative biomarkers of developing T1D in those people at risk.

One of the limitations of the study is that this is a descriptive study, and it is difficult to interpret if some of the changes in cell subpopulations are biologically relevant. Functional studies would be necessary to address this. Even though, the presented results identify some changes that may be relevant as putative surrogate markers in the immunomonitoring of the disease.

In conclusion, multiparametric flow cytometry in peripheral blood adult-onset T1D pointed to an important role of minor lymphocyte subpopulations of effector T cells, but also B cells, in the pathogenesis of the disease being impaired those subsets with regulatory characteristics as Tregs and transitional B cells. Most of these changes are maintained at established disease. More studies focusing on late EM CD4 T cells as a potential tool as putative biomarker of disease should be performed.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The studies involving human participants were reviewed and approved by Comitè d’ètica de la investigació amb medicaments, Hospital Universitari Germans Trias i Pujol. The patients/participants provided their written informed consent to participate in this study.

AT-S designed the research, performed most of the experiments, analyzed, and interpreted the results, and drafted the manuscript. EP participated in the design of the study, recruitment of patients, interpretation of data and helped in writing the manuscript. BQ-S participated in the interpretation of data and helped in writing the manuscript. MF participated in the design and analysis of flow cytometry experiments. MV-P revised the manuscript. EM-C conceptualized and designed the study, supervised the research and revised the manuscript. All authors contributed to the article and approved the submitted version.

The authors are members of a consolidated group (2017SGR103) as recognized by the Agency for Management of University and Research Grants (AGAUR) of the Generalitat of Catalonia. This work has been partially funded by PI20/01313 integrated in the Plan Nacional de I+D+I and co-supported by the ISCIII-Subdirección General de Evaluación and the Fondo Europeo de Desarrollo Regional (FEDER).

MV-P holds a patent that relate to immunotherapy for T1D and is co-founder of Ahead Therapeutics S.L.

The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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