scholarly journals Identifying Changes in Peripheral Lymphocyte Subpopulations in Adult Onset Type 1 Diabetes

2021 ◽  
Vol 12 ◽  
Author(s):  
Aina Teniente-Serra ◽  
Eduarda Pizarro ◽  
Bibiana Quirant-Sánchez ◽  
Marco A. Fernández ◽  
Marta Vives-Pi ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Type 1 Diabetes ◽  
B Cells ◽  
Peripheral Blood ◽  
Disease Onset ◽  
Lymphocyte Subpopulations ◽  
Effector Memory ◽  
Transitional B Cells

T- and B-lymphocytes play an important role in the pathogenesis of type 1 diabetes (T1D), a chronic disease caused by the autoimmune destruction of the insulin-producing cells in the pancreatic islets. Flow cytometry allows their characterization in peripheral blood, letting to investigate changes in cellular subpopulations that can provide insights in T1D pathophysiology. With this purpose, CD4+ and CD8+ T cells (including naïve, central memory, effector memory and terminally differentiated effector (TEMRA), Th17 and Tregs) and B cells subsets (naïve, unswitched memory, switched memory and transitional B cells) were analysed in peripheral blood of adult T1D patients at disease onset and after ≥2 years using multiparametric flow cytometry. Here we report changes in the percentage of early and late effector memory CD4+ and CD8+ T cells as well as of naïve subsets, regulatory T cells and transitional B cells in peripheral blood of adult patients at onset of T1D when compared with HD. After 2 years follow-up these changes were maintained. Also, we found a decrease in percentage of Th17 and numbers of T cells with baseline. In order to identify potential biomarkers of disease, ROC curves were performed being late EM CD4 T cell subset the most promising candidate. In conclusion, the observed changes in the percentage and/or absolute number of lymphocyte subpopulations of adult T1D patients support the hypothesis that effector cells migrate to the pancreas and this autoimmune process perseveres along the disease. Moreover, multiparametric flow allows to identify those subsets with potential to be considered biomarkers of disease.

scholarly journals Peripheral immune circadian variation, synchronisation and possible dysrhythmia in established type 1 diabetes

Diabetologia ◽  
2021 ◽  
Author(s):  
Craig A. Beam ◽  
Eleni Beli ◽  
Clive H. Wasserfall ◽  
Stephanie E. Woerner ◽  
Megan T. Legge ◽  
...  
Keyword(s):  
T Cells ◽  
Type 1 Diabetes ◽  
B Cells ◽  
Regulatory T Cells ◽  
Cd8 T Cells ◽  
Immune Cell ◽  
Circadian Variation ◽  
Time Of Day ◽  
Effector Memory

Abstract Aims/hypothesis The circadian clock influences both diabetes and immunity. Our goal in this study was to characterise more thoroughly the circadian patterns of immune cell populations and cytokines that are particularly relevant to the immune pathology of type 1 diabetes and thus fill in a current gap in our understanding of this disease. Methods Ten individuals with established type 1 diabetes (mean disease duration 11 years, age 18–40 years, six female) participated in a circadian sampling protocol, each providing six blood samples over a 24 h period. Results Daily ranges of population frequencies were sometimes large and possibly clinically significant. Several immune populations, such as dendritic cells, CD4 and CD8 T cells and their effector memory subpopulations, CD4 regulatory T cells, B cells and cytokine IL-6, exhibited statistically significant circadian rhythmicity. In a comparison with historical healthy control individuals, but using shipped samples, we observed that participants with type 1 diabetes had statistically significant phase shifts occurring in the time of peak occurrence of B cells (+4.8 h), CD4 and CD8 T cells (~ +5 h) and their naive and effector memory subsets (~ +3.3 to +4.5 h), and regulatory T cells (+4.1 h). An independent streptozotocin murine experiment confirmed the phase shifting of CD8 T cells and suggests that circadian dysrhythmia in type 1 diabetes might be an effect and not a cause of the disease. Conclusions/interpretation Future efforts investigating this newly described aspect of type 1 diabetes in human participants are warranted. Peripheral immune populations should be measured near the same time of day in order to reduce circadian-related variation. Graphical abstract

Low Frequency of Regulatory T Cells in the Peripheral Blood of Children with Type 1 Diabetes Diagnosed under the Age of Five

2012 ◽  
Vol 60 (4) ◽  
pp. 307-313 ◽  
Author(s):  
Agnieszka Szypowska ◽  
Anna Stelmaszczyk-Emmel ◽  
Urszula Demkow ◽  
Włodzimierz Łuczyński
Keyword(s):  
T Cells ◽  
Type 1 Diabetes ◽  
Regulatory T Cells ◽  
Peripheral Blood ◽  
Low Frequency

scholarly journals Position β57 of I-Ag7 controls early anti-insulin responses in NOD mice, linking an MHC susceptibility allele to type 1 diabetes onset

2019 ◽  
Vol 4 (38) ◽  
pp. eaaw6329 ◽  
Author(s):  
Louis Gioia ◽  
Marie Holt ◽  
Anne Costanzo ◽  
Siddhartha Sharma ◽  
Brian Abe ◽  
...  
Keyword(s):  
T Cells ◽  
Type 1 Diabetes ◽  
Disease Onset ◽  
Insulin Response ◽  
Nod Mice ◽  
Class Ii ◽  
Single Amino Acid ◽  
Specific Gene ◽  
Single Amino Acid Change

The class II region of the major histocompatibility complex (MHC) locus is the main contributor to the genetic susceptibility to type 1 diabetes (T1D). The loss of an aspartic acid at position 57 of diabetogenic HLA-DQβ chains supports this association; this single amino acid change influences how TCRs recognize peptides in the context of HLA-DQ8 and I-Ag7 using a mechanism termed the P9 switch. Here, we built register-specific insulin peptide MHC tetramers to examine CD4+ T cell responses to Ins12–20 and Ins13–21 peptides during the early prediabetic phase of disease in nonobese diabetic (NOD) mice. A single-cell analysis of anti-insulin CD4+ T cells performed in 6- and 12-week-old NOD mice revealed tissue-specific gene expression signatures. TCR signaling and clonal expansion were found only in the islets of Langerhans and produced either classical TH1 differentiation or an unusual Treg phenotype, independent of TCR usage. The early phase of the anti-insulin response was dominated by T cells specific for Ins12–20, the register that supports a P9 switch mode of recognition. The presence of the P9 switch was demonstrated by TCR sequencing, reexpression, mutagenesis, and functional testing of TCRαβ pairs in vitro. Genetic correction of the I-Aβ57 mutation in NOD mice resulted in the disappearance of D/E residues in the CDR3β of anti-Ins12–20 T cells. These results provide a mechanistic molecular explanation that links the characteristic MHC class II polymorphism of T1D with the recognition of islet autoantigens and disease onset.

scholarly journals Single-Cell RNA Sequencing Reveals Expanded Clones of Islet Antigen-Reactive CD4+ T Cells in Peripheral Blood of Subjects with Type 1 Diabetes

2017 ◽  
Vol 199 (1) ◽  
pp. 323-335 ◽  
Author(s):  
Karen Cerosaletti ◽  
Fariba Barahmand-pour-Whitman ◽  
Junbao Yang ◽  
Hannah A. DeBerg ◽  
Matthew J. Dufort ◽  
...  
Keyword(s):  
T Cells ◽  
Type 1 Diabetes ◽  
Single Cell ◽  
Rna Sequencing ◽  
Peripheral Blood ◽  
Cd4 T Cells ◽  
Single Cell Rna Sequencing ◽  
Islet Antigen

scholarly journals Activated but functionally impaired memory Tregs are expanded in slow progressors to type 1 diabetes

Diabetologia ◽  
2021 ◽  
Author(s):  
Joanne Boldison ◽  
Anna E. Long ◽  
Rachel J. Aitken ◽  
Isabel V. Wilson ◽  
Clare Megson ◽  
...  
Keyword(s):  
T Cells ◽  
Type 1 Diabetes ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Unsupervised Clustering ◽  
Autoimmune Response ◽  
Peripheral Blood Mononuclear ◽  
Slow Progressors ◽  
Healthy Donors

Abstract Aims/hypothesis Slow progressors to type 1 diabetes are individuals positive for multiple pancreatic islet autoantibodies who have remained diabetes-free for at least 10 years; regulation of the autoimmune response is understudied in this group. Here, we profile CD4+ regulatory T cells (Tregs) in a small but well-characterised cohort of extreme slow progressors with a median age 43 (range 31–72 years), followed up for 18–32 years. Methods Peripheral blood samples were obtained from slow progressors (n = 8), age- and sex-matched to healthy donors. One participant in this study was identified with a raised HbA1c at the time of assessment and subsequently diagnosed with diabetes; this donor was individually evaluated in the analysis of the data. Peripheral blood mononuclear cells (PBMCs) were isolated, and to assess frequency, phenotype and function of Tregs in donors, multi-parameter flow cytometry and T cell suppression assays were performed. Unsupervised clustering analysis, using FlowSOM and CITRUS (cluster identification, characterization, and regression), was used to evaluate Treg phenotypes. Results Unsupervised clustering on memory CD4+ T cells from slow progressors showed an increased frequency of activated memory CD4+ Tregs, associated with increased expression of glucocorticoid-induced TNFR-related protein (GITR), compared with matched healthy donors. One participant with a raised HbA1c at the time of assessment had a different Treg profile compared with both slow progressors and matched controls. Functional assays demonstrated that Treg-mediated suppression of CD4+ effector T cells from slow progressors was significantly impaired, compared with healthy donors. However, effector CD4+ T cells from slow progressors were more responsive to Treg suppression compared with healthy donors, demonstrated by increased suppression of CD25 and CD134 expression on effector CD4+ T cells. Conclusions/interpretations We conclude that activated memory CD4+ Tregs from slow progressors are expanded and enriched for GITR expression, highlighting the need for further study of Treg heterogeneity in individuals at risk of developing type 1 diabetes. Graphical abstract

scholarly journals Intralymphatic GAD-alum Injection Modulates B Cell Response and Induces Follicular Helper T Cells and PD-1+ CD8+ T Cells in Patients With Recent-Onset Type 1 Diabetes

2022 ◽  
Vol 12 ◽  
Author(s):  
Hugo Barcenilla ◽  
Mikael Pihl ◽  
Jeanette Wahlberg ◽  
Johnny Ludvigsson ◽  
Rosaura Casas
Keyword(s):  
T Cells ◽  
Type 1 Diabetes ◽  
B Cells ◽  
Lymph Nodes ◽  
Cd8 T Cells ◽  
Helper T Cells ◽  
Follicular Helper T Cells ◽  
Recent Onset ◽  
Follicular Helper

Antigen-specific immunotherapy is an appealing strategy to preserve beta-cell function in type 1 diabetes, although the approach has yet to meet its therapeutic endpoint. Direct administration of autoantigen into lymph nodes has emerged as an alternative administration route that can improve the efficacy of the treatment. In the first open-label clinical trial in humans, injection of aluminum-formulated glutamic acid decarboxylase (GAD-alum) into an inguinal lymph node led to the promising preservation of C-peptide in patients with recent-onset type 1 diabetes. The treatment induced a distinct immunomodulatory effect, but the response at the cell level has not been fully characterized. Here we used mass cytometry to profile the immune landscape in peripheral blood mononuclear cells from 12 participants of the study before and after 15 months of treatment. The immunomodulatory effect of the therapy included reduction of naïve and unswitched memory B cells, increase in follicular helper T cells and expansion of PD-1+ CD69+ cells in both CD8+ and double negative T cells. In vitro stimulation with GAD65 only affected effector CD8+ T cells in samples collected before the treatment. However, the recall response to antigen after 15 months included induction of CXCR3+ and CD11c+Tbet+ B cells, PD-1+ follicular helper T cells and exhausted-like CD8+ T cells. This study provides a deeper insight into the immunological changes associated with GAD-alum administration directly into the lymph nodes.

Characterization of T Cells Reactive to Hybrid Insulin Peptides in the Peripheral Blood of in Type 1 Diabetes Patients

Diabetes ◽  
2018 ◽  
Vol 67 (Supplement 1) ◽  
pp. 98-OR
Author(s):  
ROCKY L. BAKER ◽  
THOMAS DELONG ◽  
PETER GOTTLIEB ◽  
KATHRYN M. HASKINS
Keyword(s):  
T Cells ◽  
Type 1 Diabetes ◽  
Peripheral Blood ◽  
Diabetes Patients

Circulating immune markers predict the therapeutic effect in primary lung cancer.

2021 ◽  
Vol 39 (15_suppl) ◽  
pp. e21203-e21203
Author(s):  
Liangliang Xu ◽  
Jitian Zhang ◽  
Li Yang ◽  
Guangqiang Shao ◽  
Taiyang Liuru ◽  
...  
Keyword(s):  
Lung Cancer ◽  
T Cells ◽  
B Cells ◽  
Nk Cells ◽  
Cd8 T Cells ◽  
Peripheral Blood ◽  
Cd4 T Cells ◽  
Blood Lymphocyte ◽  
Lymphocyte Subpopulations ◽  
Significant Difference

e21203 Background: Radiotherapy (RT), surgical resection (SR), and immunotherapy (IT) as main therapies in lung cancer have either suppressive or stimulatory effects on the immune system. It’s still unclear the mechanism involved in the systemic changes of immune cells in the blood. Peripheral blood lymphocyte subpopulations were useful markers for evaluating immune response in tumor patients. Hence, we aimed to systematically investigate the alteration of lymphocyte subpopulations during the local therapies to evaluate antitumor treatment effects. Methods: Blood samples were obtained EDTA coated tubes and then centrifuged gently for white blood cell separation. The white blood cells in 10% DMSO and 90% FBS were frozen slowly in -80°C refrigerator. The following fluorochrome-conjugated surface and nuclear antibodies were used in the lymphocyte subtyping: CD11b, CD45, CD19, CD3, CD56, CD4, CD8a, CD25,CD127 and FOXP3. The staining cells were detected in the BD FACS machine and data were analyzed by the paired T-test. The percentage of Lymphocytes, Myeloid cells, B cells, T cells, Treg, CD8+ T cells, CD4+ T cells, NK cells, and NKT were examined. Results: Between July 2019 and January 2020, a total of 176 patients eligible, including 135 RT patients and 29 SR patients,12 IT patients, with both blood collection with both Pre, During and End therapies. Before local therapies, the percentage of total T cells in the RT group was significantly higher than SR (RT v.s SR mean:64.1 v.s 55.3, P = 0.02) while CD8+ T cells (RT v.s SR mean:28.2 v.s 34.5, P = 0.04)and Tregs (RT v.s SR mean:0.0 v.s 0.1, P = 0.055) were lower. The baseline level of T cells and their subtypes showed a significant difference in these two group patients. After local therapies, myeloid cells, lymphocytes, CD4+ T cells, CD8+ T cells, NK cells were significant different. There is no significant difference due to the smaller number of IT patients. In the RT group, lymphocytes (Pre-RT v.s End-RT mean:75.2 v.s 54.3, P = 0.004) and B cells (Pre-RT v.s End-RT mean:12.6 v.s 8.0, P = 0.03) were significantly decreased while other subpopulations didn’t show any significant difference after RT. Interestingly, in the SR group, there was a significant increase in CD4+ T cells (mean:59.0 v.s 62.1, p = 0.02) a trend of reduction in CD8+ T cells (mean:34.5 v.s 32.0, p = 0.055) after SR. In addition, there was an increased trend of Tregs after IT. Conclusions: There are some different patterns of distribution in subtypes of leukocytes in operable and inoperable patients and between different therapies. All RT, SR and IT changed the distribution of peripheral blood lymphocyte subpopulations. Further validation study is warranted to validate our findings particularly in circulating lymphocytes and B cells as a marker to evaluate immune status after RT, CD4+ T cells and CD8+ T cells after SR, Tregs after IT, as well as their relationship with tumor microenvironment and implication for personalized care.

Massive, Clinical Grade Expansion Of Polyclonal T Cells Using Blinatumomab For Adoptive Autologous Cellular Therapy Of CLL Patients

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3272-3272 ◽  
Author(s):  
Josée Golay ◽  
Anna D’amico ◽  
Gianmaria Borleri ◽  
Maria Chiara Finazzi ◽  
Giulia Quaresmini ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
B Cells ◽  
Peripheral Blood ◽  
T Cell Subsets ◽  
Th2 Cells ◽  
Adoptive Therapy ◽  
Specific Patient

Abstract Background The combined use of chemotherapy and monoclonal antibodies has proved highly effective for the treatment of CLL but often results in severe life threatening immunosuppression. The development of adoptive therapy with autologous T cells could be clinically relevant to overcome these problems. Methods We have devised a novel, simple and efficient method for ex vivo expansion of normal autologous T cells from the peripheral blood of CLL patients for adoptive therapy, using blinatumomab (CD3xCD19) and rhIL-2 in serum-free medium. The complete phenotype of in vitro expanded T cells was analyzed by flow cytometry and their cytotoxic activity by calcein release assays. Results We performed 18 expansions of T cells, starting from a very small volume of peripheral blood from untreated CLL patients (mean 10.3 ml, range 2-30 ml) that contained a mean of 9.2x106 T cells (range 0.4 to 51x106)(Fig.1). This method allowed reproducible expansion in about 21 days of a mean 410x106 CD3+ T cells (range 71 to 2184x106). The mean fold expansion of T cells in about 3 weeks of in vitro culture was 224 (range 4.4-1326). The only significant contaminant in final Blinatumomab Expanded T cell cultures (BET) were NK cells (mean 18.5%). Indeed addition of blinatumomab and rhIL-2 to the cultures led to a rapid decrease in CLL B cells, which took place from days 7 to 14 onwards and resulted in their complete depletion within 3 weeks (mean 0.2% CLL B cells at days 18-25). Only in one case, a significant percentage of CLL B cells could be observed at the end of culture, but this was due to the particularly high percentage neoplastic cells in the starting population in this patient (98%), resulting in relatively late depletion of these cells, which took place between days 14 and 21, and therefore remained detectable in this case at day 24 (3.8% CLL B cells at day 24). Despite the very low percentage of starting T cells in this specific patient (1.2%), 152x106 T cells could be obtained, equivalent to a 42 fold expansion. In the 18 expansions performed, the resulting BET cells contained both CD4+ and CD8+ cells in varying proportions (median 46.2% and 44.4% respectively). Only in two cases the final product was composed predominantly of CD4+ cells (95%). Expanded T cells were polyclonal, as shown by TCR Vβ expression which was within the normal range by flow cytometry. Indeed CMV specific clones, detected by CMV peptide (pp65495-503)-loaded HLA-A*0201 tetramer, were expanded using this method and detected in equivalent proportion before and after expansion. Final T cells were composed predominantly of the effector and central memory subsets. Th1 were slightly prevalent over Th2 cells (means 20% and 10%, respectively), whereas Th17 and Treg were less than 1%. Since CLL derived T cells have been shown previously to have enhanced expression of the synapse regulators CD272 and CD279 compared to normal T cells, leading to impaired immunological synapse formation, we have analyzed these markers in both starting and BET cells from 4 patients. We observed that CD272 and CD279 diminished in BET compared to the starting CLL T populations (from 73% to 19% and 61% to 18%, respectively). These data suggest that stimulation and expansion with blinatumomab and rhIL-2 has normalized expression of these regulators on CLL T cells. Indeed BET were highly cytotoxic against CD19+ targets cell lines or primary CLL cells, with 70-90% lysis at a 3:1 effector target ratio in presence of blinatumomab. Finally BET were compared to Xcellerated cells expanded using anti-CD3/CD28 Dynabeads and rhIL-2. The expansion protocols using either blinatumomab or anti-CD3/CD28 Dynabeads showed equivalent efficiency and comparable cell composition at the end of culture. Further comparison of the T cell subsets present in BET or CD3/CD28 cultures is in progress. Conclusions These data altogether suggest that the use of blinatumomab and rhIL-2 provides a reproducible, simple and GMP-compliant protocol, allowing expansion of large numbers of autologous polyclonal T cells depleted of CLL cells, from relatively small volumes of peripheral blood from CLL patients. This approach is an attractive option for adoptive therapy in these patients after immunosuppressive treatments. Disclosures: No relevant conflicts of interest to declare.

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