Helena Reijonen

Recurrence of type 1 diabetes after simultaneous pancreas-kidney transplantation, despite immunosuppression, is associated with autoantibodies and pathogenic autoreactive CD4 T-cells

OBJECTIVE To investigate if recurrent autoimmunity explained hyperglycemia and C-peptide loss in three immunosuppressed simultaneous pancreas-kidney (SPK) transplant recipients. RESEARCH DESIGN AND METHODS We monitored autoantibodies and autoreactive T-cells (using tetramers) and performed biopsy. The function of autoreactive T-cells was studied with in vitro and in vivo assays. RESULTS Autoantibodies were present pretransplant and persisted on follow-up in one patient. They appeared years after transplantation but before the development of hyperglycemia in the remaining patients. Pancreas transplant biopsies were taken within ∼1 year from hyperglycemia recurrence and revealed β-cell loss and insulitis. We studied autoreactive T-cells from the time of biopsy and repeatedly demonstrated their presence on further follow-up, together with autoantibodies. Treatment with T-cell–directed therapies (thymoglobulin and daclizumab, all patients), alone or with the addition of B-cell–directed therapy (rituximab, two patients), nonspecifically depleted T-cells and was associated with C-peptide secretion for >1 year. Autoreactive T-cells with the same autoantigen specificity and conserved T-cell receptor later reappeared with further C-peptide loss over the next 2 years. Purified autoreactive CD4 T-cells from two patients were cotransplanted with HLA-mismatched human islets into immunodeficient mice. Grafts showed β-cell loss in mice receiving autoreactive T-cells but not control T-cells. CONCLUSIONS We demonstrate the cardinal features of recurrent autoimmunity in three such patients, including the reappearance of CD4 T-cells capable of mediating β-cell destruction. Markers of autoimmunity can help diagnose this underappreciated cause of graft loss. Immune monitoring during therapy showed that autoimmunity was not resolved by the immunosuppressive agents used.

Ultra-sensitive detection of rare T cell clones

Advances in high-throughput sequencing have enabled technologies that probe the adaptive immune system with unprecedented depth. We have developed a multiplex PCR method to sequence tens of millions of T cell receptors (TCRs) from a single sample in a few days. A method is presented to test the precision, accuracy, and sensitivity of this assay. T cell clones, each with one fixed productive TCR rearrangement, are doped into complex blood cell samples. TCRs from a total of eleven samples are sequenced, with the doped T cell clones ranging from 10% of the total sample to 0.001% (one cell in 100,000). The assay is able to detect even the rarest clones. The precision of the assay is demonstrated across five orders of magnitude. The accuracy for each clone is within an overall factor of three across the 100,000 fold dynamic range. Additionally, the assay is shown to be highly repeatable.

Self-reactive human CD4 T cell clones form unusual immunological synapses.

Compared with influenza-specific T cells, self-reactive T cells from patients with multiple sclerosis or type 1 diabetes fail to slow down and do not form normal immunological synapses upon encounter with cognate self-peptide presented by MHC.

Th1 cytokines promote T-cell binding to antigen-presenting cells via enhanced hyaluronan production and accumulation at the immune synapse

Hyaluronan (HA) production by dendritic cells (DCs) is known to promote antigen presentation and to augment T-cell activation and proliferation. We hypothesized that pericellular HA can function as intercellular ‘glue’ directly mediating T cell–DC binding. Using primary human cells, we observed HA-dependent binding between T cells and DCs, which was abrogated upon pre-treatment of the DCs with 4-methylumbelliferone (4-MU), an agent which blocks HA synthesis. Furthermore, T cells regulate HA production by DCs via T cell-derived cytokines in a T helper (Th) subset-specific manner, as demonstrated by the observation that cell-culture supernatants from Th1 but not Th2 clones promote HA production. Similar effects were seen upon the addition of exogenous Th1 cytokines, IL-2, interferon γ (IFN-γ) and tumor necrosis factor α (TNF-α). The critical factors which determined the extent of DC–T cell binding in this system were the nature of the pre-treatment the DCs received and their capacity to synthesize HA, as T-cell clones which were pre-treated with monensin, added to block cytokine secretion, bound equivalently irrespective of their Th subset. These data support the existence of a feedforward loop wherein T-cell cytokines influence DC production of HA, which in turn affects the extent of DC–T cell binding. We also document the presence of focal deposits of HA at the immune synapse between T-cells and APC and on dendritic processes thought to be important in antigen presentation. These data point to a pivotal role for HA in DC–T cell interactions at the IS.

Risk Factors for Type 1 Diabetes Recurrence in Immunosuppressed Recipients of Simultaneous Pancreas–Kidney Transplants

Patients with type 1 diabetes (T1D) who are recipients of pancreas transplants are believed to rarely develop T1D recurrence in the allograft if effectively immunosuppressed. We evaluated a cohort of 223 recipients of simultaneous pancreas–kidney allografts for T1D recurrence and its risk factors. With long‐term follow‐up, recurrence was observed in approximately 7% of patients. Comparing the therapeutic regimens employed in this cohort over time, lack of induction therapy was associated with recurrence, but this occurs even with the current regimen, which includes induction; there was no influence of maintenance regimens. Longitudinal testing for T1D‐associated autoantibodies identified autoantibody positivity, number of autoantibodies, and autoantibody conversion after transplantation as critical risk factors. Autoantibodies to the zinc transporter 8 had the strongest and closest temporal association with recurrence, which was not explained by genetically encoded amino acid sequence donor–recipient mismatches for this autoantigen. Genetic risk factors included the presence of the T1D‐predisposing HLA‐DR3/DR4 genotype in the recipient and donor–recipient sharing of HLA‐DR alleles, especially HLA‐DR3. Thus, T1D recurrence is not uncommon and is developing in patients treated with current immunosuppression. The risk factors identified in this study can be assessed in the transplant clinic to identify recurrent T1D and may lead to therapeutic advances.

Development of diabetes mellitus in living pancreas donors and recipients

Previously, recurrence of insulin-dependent diabetes mellitus after pancreas transplants was only sporadically reported. Newer data, however, indicate recurrence rates as high as 5%. After identical-twin pancreas transplants, diabetes recurs in the absence of immunosuppressive therapy – strong evidence that it is an autoimmune disease. After deceased donor pancreas transplants, immunologic markers (autoantibodies, autoreactive T cells) herald recurrence. Selective destruction of β cells, still relatively uncommon, is not restricted to MHC compatibility. The development of diabetes in living pancreas donors is rare; it can be largely avoided by meticulous metabolic evaluation before donation and prevention of obesity after donation.