Immune responses against islet allografts during tapering of immunosuppression--a pilot study in 5 subjects

Abstract

Transplantation of isolated islet of Langerhans cells has great potential as a cure for type 1 diabetes but continuous immune suppressive therapy often causes considerable side effects. Tapering of immunosuppression in successfully transplanted patients would lower patients' health risk. To identify immune biomarkers that may prove informative in monitoring tapering, we studied the effect of tapering on islet auto- and alloimmune reactivity in a pilot study in five transplant recipients in vitro. Cytokine responses to the graft were measured using Luminex technology. Avidity of alloreactive cytotoxic T Lymphocytes (CTL) was determined by CD8 blockade. The influence of immunosuppression was mimicked by in vitro replenishment of tacrolimus and MPA, the active metabolite of mycophenolate mofetil. Tapering of tacrolimus was generally followed by decreased C-peptide production. T-cell autoreactivity increased in four out of five patients during tapering. Overall alloreactive CTL precursor frequencies did not change, but their avidity to donor mismatches increased significantly after tapering (P = 0·035). In vitro addition of tacrolimus but not MPA strongly inhibited CTL alloreactivity during tapering and led to a significant shift to anti-inflammatory graft-specific cytokine production. Tapering of immunosuppression is characterized by diverse immune profiles that appear to relate inversely to plasma C-peptide levels. Highly avid allospecific CTLs that are known to associate with rejection increased during tapering, but could be countered by restoring immune suppression in vitro. Immune monitoring studies may help guiding tapering of immunosuppression after islet cell transplantation, even though we do not have formal prove yet that the observed changes reflect direct effects of immune suppression on immunity.

Fig. 1

Overview of clinical and immunological parameters of five islet cell transplant recipients during tapering of immunosuppression. Shown are immunosuppression levels: tacrolimus trough level (green dots), MMF dosage (blue open circles); Plasma C-peptide levels (black squares) and period of insulin use (gray bar); Cellular autoimmunity: stimulation index of autoreactive proliferative response to IA-2 (green squares) and GAD (blue open circles); Total cellular alloreactivity: CTLp frequency in response to 3–4 different graft specific stimulator cells; Highly avid cellular alloreactivity: CTLp frequency after addition of anti-CD8; Antibodies: serum levels of allo-antibodies (black squares) and auto-antibodies ICA (red inverse triangles), IAA (gray diamonds), GADA (blue circles), IA2A (green triangles).

Fig. 2

Alloreactive CTLp frequency before, during (median if multiple values) and after tapering. Data represent CTLp frequencies in response to different graft-specific stimulator cells without addition of anti-CD8 (a) and with addition of anti-CD8 (b) (only high avidity CTLs). % inhibition by anti-CD8 significantly decreases after tapering (c) (P = 0·0013 by repeated measures ANOVA; *P < 0·05, **P < 0·01 after Bonferonni's correction for multiple comparisons), indicating a relative increase in highly avid CTLs. Horizontal lines represent median values.

Fig. 3

Effect of addition of tacrolimus and/ or MPA on CD4+ T-cell proliferation (a), CD8+ CTLs (b) and highly avid CTLs (c) in high responsive patient and graft-specific stimulator combinations. Data represent stimulator-responder combinations of five patients before, during (median if multiple values) and after tapering of immunosuppression (mean+SEM) without addition of immuno-suppression (○), with tacrolimus (), with MPA (▴) or tacrolimus and MPA (▾). *P < 0·05, **P < 0·01, ***P < 0·001 by Friedman test. In all significant tests with P < 0·01, the difference between No IS and TAC/MPA remains significant after Dunn's correction for multiple comparison.

Fig. 4

Graft-specific production of cytokines after addition of tacrolimus and/or MPA. Data represent of IL2 (a), IFNγ (b), IL13 (c), IL10 (d) and TNFα (e) of five patients' PBMC in high responsive patient and graft-specific stimulator combinations before, during (median if multiple values) and after tapering of immunosuppression (mean+SEM) without addition of immunosuppression (○), with tacrolimus (), with MPA (▴) or tacrolimus and MPA (▾). *P < 0·05, **P < 0·01, ***P < 0·001 by Friedman test. For all comparisons significant differences remain after Dunn's correction for multiple comparison: for IL10, pre and post tapering No IS – tacrolimus and MPA – tacrolimus and during tapering MPA – tacrolimus/MPA; for IL2, pre tapering No IS – tacrolimus, during tapering No IS – tacrolimus and MPA – tacrolimus and post tapering MPA – tacrolimus/MPA remain significant. For TNFα during tapering No IS – tacrolimus.