To determine the specificities and frequencies of islet autoreactive CD8 T-cells in pancreas draining lymph nodes, control lymph nodes, spleen and/or peripheral blood from diabetic and non-diabetic organ donors, we used the Diab-Q-kit, a nanotechnology that allows the direct detection of circulating autoreactive CD8 T-cells against an array of islet-epitopes simultaneously, reducing the needs of patient resources. The detection of these T-cells is based on the T-cell receptor (TCR) specificity for its peptide-HLA ligand. We produced HLA-monomers for an array of islet epitopes presented in the context of HLA -A2, -A3 and -B7. This allowed expanding the number of patients that could be analyzed considerably. Moreover, to characterize the memory phenotype of the autoreactive T-cells, we validated and upgraded the Diab-Q-kit through the inclusion of cell surface markers to distinguish naïve, effector memory and central memory T-cells. From the nPOD repository we selected a total of 29 cases to be analysed for this study from which we could obtain cell suspensions from blood and pancreas draining lymph nodes, control lymph nodes and/ or spleen.
Upon thawing of the samples we unexpectedly observed that far fewer viable cells could be recovered from the majority of the samples than the number reported to be frozen on the vial. For some cases, we also counted the number of dead cells in each vial. We found that the percentage of dead cells was very high in some cases, but not all. As an internal control, we always included samples from healthy controls collected and cryopreserved in our laboratory, which showed very high viability after thawing (>90%), indicating that the thawing procedure was correctly performed.
Similar issues on viability and recovery have since been reported by other partners in nPOD. Importantly, the quality control to detect any issues was critical to identify these problems and potential causes. For instance, issues with viability and recovery were determined immediately after thawing, while FACS analyses performed hours after thawing of the samples would have suggested excellent viability outcome, even in cases that QC earlier on had identified poor recovery and viability.
The consequences of low viability and recovery may differ depending on the type of analysis per project (functional assays vs. mRNA) and incentive (cloning T-cells may still be possible, but may not be representative of the total tissue sample). Such issues have been evaluated in depth and at length between the central facility and participants of nPOD. Indeed, the nPOD central facility was indispensable in identifying important correlations between recovery, viability and donor characteristics that may differ between tissue source of the leukocytes and that need not be independent co-variates ( PBMC: demographics, cause of death [anoxia vs > CV/stroke], disease [T2D/Ab+>T1D], donor age [o>y]; splenocytes: donor age [y>o], BMI [low>hi], transmission time [short>long]), underscoring that the thawing procedure was not the primary parameter of poor outcome and implying that intrinsic features of the biosamples determines quality and reward.
Despite lower cell recovery than expected, we nevertheless proceeded performing the Diab-Q-kit analysis on all samples, to take maximal knowledge, benefit and reward from the precious nPOD material. Unfortunately, for more than half of the samples no reliable data could be obtained, due to the low number of CD8 T-cells present in each sample. This precluded reliable comparisons in frequencies and specificities of islet-autoreactive CD8 T-cells in the individual organs. The results from this study have been discussed at length with JDRF nPOD, and it has helped the nPOD consortium to decide on the feasibility of projects where viable cell suspensions are needed for analysis and functional assays. Measures have been undertaken to adjust allocations of samples on basis of this important new insight.