Comparison of cryopreservation methods on T‐cell responses to islet and control antigens from type 1 diabetic patients and controls

Brooks-Worrell, Barbara; Tree, Timothy; Mannering, Stuart I.; Durinovic‐Belló, Ivana; James, Eddie A.; Gottlieb, Peter A.; Wong, Samuel Yeung Shan; Zhou, Zhiguang; Yang, Lin; Cilio, Corrado; Reichow, Jessica; Menart, Barbara; Rütter, R; Schreiner, Robert S.; Pham, Minh N.; Marquesini, L; Lou, Olivia; Scotto, Matthieu; Mallone, Roberto; Schloot, Nanette C.

doi:10.1002/dmrr.1245

Cited by 21 publications

(9 citation statements)

References 26 publications

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“…Treg subsets with different suppressive ability can be distinguished based on CD39, CD45RA, FOXP3, and CD25 expression (23,44). As previous studies have shown, cryopreservation can alter the expression of various cell surface markers (20,29,45). In line with Zhang et al (46), our results indicate that the expression of CD39 on CD4 + CD25 + CD127 2 cells, with or without FOXP3, was not affected.…”

Section: Discussionsupporting

confidence: 87%

Subsets of CD4+, CD8+, and CD25hi Lymphocytes Are in General Not Influenced by Isolation and Long-Term Cryopreservation

Tompa

Nilsson-Bowers

Faresjö

2018

The Journal of Immunology

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Several key factors can affect the outcome of immunological studies; isolation/cryopreservation can possibly alter T, B, NK, and T-regulatory (Treg) cell marker expression patterns. Blood samples from 50 blood donors supplemented with Na-heparin or KEDTA were handled within 4 and 24 h after blood sampling. PBMC were isolated with different density gradients. Flow cytometric analysis of intracellular and extracellular CD markers was performed on blood samples freshly isolated PBMC, and PBMC was thawed 6 and 12 mo post-cryopreservation for the purpose of identifying B, NK, Th, T-cytotoxic, and Treg cells. No differences were observed in the percentages for CD3, CD3CD4, CD3CD8, CD19, or CD56CD16 cells within 24 h of sampling regardless of which supplement or isolation techniques were used. Differentiated (diff) CD4 cells were in general less affected by isolation and cryopreservation than diff CD8 cells. Terminally diff effector CD4 and CD8 cells were not affected by either isolation of lymphocytes or cryopreservation. In contrast, naive and early-diff effector memory CD4 and CD8 cells were affected by isolation and cryopreservation. The percentages of Treg cells defined as CD4CD25 expressing CD101 or CD129, CD4CD25CD127, and CD4CD25CD127FOXP3, respectively, remained stable after isolation and cryopreservation. Subsets expressing CD127, with or without FOXP3, were not affected by isolation/cryopreservation. Subsets expressing CD39, contrary to CD45RA, on CD4CD25CD127 cells with or without FOXP3 were not affected by either isolation or cryopreservation. In conclusion, subsets of CD4, CD8, and CD25 lymphocytes are in general not influenced by isolation and long-term cryopreservation.

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Section: Discussionsupporting

confidence: 87%

Subsets of CD4+, CD8+, and CD25hi Lymphocytes Are in General Not Influenced by Isolation and Long-Term Cryopreservation

Tompa

Nilsson-Bowers

Faresjö

2018

The Journal of Immunology

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“…In most cases this expansion yielded .100 million cloned CD4 + T cells. Aliquots of five million expanded T cells were cryopreserved in 10% DMSO/ FCS, as described previously (23), and stored over liquid nitrogen until required.…”

Section: Islet Isolationmentioning

confidence: 99%

Proinsulin-Specific, HLA-DQ8, and HLA-DQ8-Transdimer–Restricted CD4+ T Cells Infiltrate Islets in Type 1 Diabetes

et al. 2014

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Type 1 diabetes (T1D) develops when insulin-secreting b-cells, found in the pancreatic islets of Langerhans, are destroyed by infiltrating T cells. How human T cells recognize b-cell-derived antigens remains unclear. Genetic studies have shown that HLA and insulin alleles are the most strongly associated with risk of T1D. These longstanding observations implicate CD4 + T-cell responses against (pro)insulin in the pathogenesis of T1D. To dissect the autoimmune T-cell response against human b-cells, we isolated and characterized 53 CD4 + T-cell clones from within the residual pancreatic islets of a deceased organ donor who had T1D. These 53 clones expressed 47 unique clonotypes, 8 of which encoded proinsulin-specific T-cell receptors. On an individual clone basis, 14 of 53 CD4 + T-cell clones (26%) recognized 6 distinct but overlapping epitopes in the C-peptide of proinsulin. These clones recognized C-peptide epitopes presented by HLA-DQ8 and, notably, HLA-DQ8 transdimers that form in HLA-DQ2/-DQ8 heterozygous individuals. Responses to these epitopes were detected in the peripheral blood mononuclear cells of some people with recent-onset T1D but not in HLAmatched control subjects. Hence, proinsulin-specific, HLA-DQ8, and HLA-DQ8-transdimer-restricted CD4 + T cells are strongly implicated in the autoimmune pathogenesis of human T1D.Type 1 diabetes (T1D) is an autoimmune disease caused by the CD4 + and CD8 + T-cell-mediated destruction of pancreatic insulin-producing b-cells (1). b-Cell destruction leads to primary insulin deficiency, which is treated by exogenous insulin therapy, and currently there is no cure. The pathogenesis of T1D has been well characterized using the NOD mouse model, but the immune basis of T1D in humans is less clear.Genetic association studies have provided powerful insights into the etiology of human T1D (2,3). The HLA class II region has the strongest impact on risk of T1D. Some HLA alleles-DQB1*06:02 for example-dominantly protect against T1D (4). In contrast, of all alleles, HLA-DQ2

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“…Another study conducted by the T-Cell Workshop has underlined the difficulties in working with frozen samples, as none of the T-cell assays employed gave satisfactory results in terms of reproducibility when compared with fresh peripheral blood mononuclear cells [49]. Development of suitable freeze-thawing protocol and of dependable T-cell assays capable of interrogating this material is a key challenge for the next years, as it would greatly facilitate multicenter studies and immune monitoring of clinical trials.…”

Section: Tracking β-Cell Autoimmunity By T-cell Assays In Humansmentioning

confidence: 97%

Pathogenic and Regulatory T Cells in Type 1 Diabetes: Losing Self-Control, Restoring It, and How to Take the Temperature

Čulina

Mallone

2011

Curr Diab Rep

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The central role of T cells in type 1 diabetes pathogenesis is well established, but these cells continue to pose numerous challenges in understanding their dynamics and in following their modifications. Important progress has been recently made in pinpointing some novel antigens targeted by pathogenic T cells and the epitope sequences recognized. Studies on the interplay between effector T cells, their regulatory counterparts, and cells of the innate immune system have unraveled novel pathways and may inspire new therapeutic approaches. At the same time, the appreciation of the plasticity of regulatory T cells has raised important caveats on their use for cell-based therapies. Continuous development of T-cell assays exploring both pathogenic and regulatory players will be critical to "take the temperature" of undergoing disease progression and reversal.

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Comparison of cryopreservation methods on T‐cell responses to islet and control antigens from type 1 diabetic patients and controls

Cited by 21 publications

References 26 publications

Subsets of CD4+, CD8+, and CD25hi Lymphocytes Are in General Not Influenced by Isolation and Long-Term Cryopreservation

Subsets of CD4+, CD8+, and CD25hi Lymphocytes Are in General Not Influenced by Isolation and Long-Term Cryopreservation

Proinsulin-Specific, HLA-DQ8, and HLA-DQ8-Transdimer–Restricted CD4+ T Cells Infiltrate Islets in Type 1 Diabetes

Pathogenic and Regulatory T Cells in Type 1 Diabetes: Losing Self-Control, Restoring It, and How to Take the Temperature

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