P-241

PANCREATIC ISLET TRANSPLANT IS A (RE)SOURCE OF ? CELLS ANTIGEN SPECIFIC B CELLS

E. Dugnani¹, V. Lampasona¹, V. Sordi¹, M. Powel², L. Piemonti^1
1 San Raffaele Scientific Institute, San Raffaele Diabetes Research Institute, Milan, Italy; ² RSR LTD, Cardiff, U.K.

Objective: Islet transplant (Tx) in T1D patients can trigger a rapid recurrence of antibodies to ? cells antigens, similarly to a recall Ab response. In models of secondary immune response, antigen specific B cells (asB) are mobilized as well. We therefore aim at evaluating frequency and features of asB to islet ags, essentially unknown in T1D, using post islet Tx samples as a source.

Methods: To identify asB in whole blood and in other tissues after lymphocyte isolation, we are applying a multicolor flow-cytometry strategy, based on the use of ?-CD19, ?-CD27, ?-CD38 and recombinant biotinylated(bi)-GAD or bi-IA-2 ags plus APC-streptavidin for staining of their cognate B cell receptor.

Results: Efficiency and specificity of the flow-cytometry strategy was confirmed by staining with bi-GAD and bi-IA2 of specific hybridomas. We then tested sequential blood samples after islet infusion in allo Tx T1D (n=7) and auto Tx oncological patients (n=6) as control. In allo Tx, GAD+ B cells appeared above background staining levels at day 2 post Tx (median 0.27.5% of CD19+; range 0.8-1.03%; 4 of 7 above LOD), increased until day 3-4 (0.31%; 0.22-0,64%; 5 of 7) and after day 6 decreased below detectable levels. No similar pattern was observed for IA2+ B cells apart from a slight and transient appearance of IA-2+ asB at day 4 (0.26 %; 0.18-0.59%). No association between asB frequencies and their corresponding autoantibody levels was observed. In vitro preliminary data suggest that a minority of GAD+ asB was able to secrete GAD autoantibodies after sorting and stimulation

Conclusions: Our strategy seems able to identify bona fide asB but its consistency remains to be validated through sorting, cloning and expression of antigen-specific Ig genes as recombinant mAbs. If successful the study of asBTx might be applied to the phenotyping of autoimmunity recurrence in islet Tx.

P-241

PANCREATIC ISLET TRANSPLANT IS A (RE)SOURCE OF ? CELLS ANTIGEN SPECIFIC B CELLS

E. Dugnani¹, V. Lampasona¹, V. Sordi¹, M. Powel², L. Piemonti^1
1 San Raffaele Scientific Institute, San Raffaele Diabetes Research Institute, Milan, Italy; ² RSR LTD, Cardiff, U.K.

Objective: Islet transplant (Tx) in T1D patients can trigger a rapid recurrence of antibodies to ? cells antigens, similarly to a recall Ab response. In models of secondary immune response, antigen specific B cells (asB) are mobilized as well. We therefore aim at evaluating frequency and features of asB to islet ags, essentially unknown in T1D, using post islet Tx samples as a source.

Methods: To identify asB in whole blood and in other tissues after lymphocyte isolation, we are applying a multicolor flow-cytometry strategy, based on the use of ?-CD19, ?-CD27, ?-CD38 and recombinant biotinylated(bi)-GAD or bi-IA-2 ags plus APC-streptavidin for staining of their cognate B cell receptor.

Results: Efficiency and specificity of the flow-cytometry strategy was confirmed by staining with bi-GAD and bi-IA2 of specific hybridomas. We then tested sequential blood samples after islet infusion in allo Tx T1D (n=7) and auto Tx oncological patients (n=6) as control. In allo Tx, GAD+ B cells appeared above background staining levels at day 2 post Tx (median 0.27.5% of CD19+; range 0.8-1.03%; 4 of 7 above LOD), increased until day 3-4 (0.31%; 0.22-0,64%; 5 of 7) and after day 6 decreased below detectable levels. No similar pattern was observed for IA2+ B cells apart from a slight and transient appearance of IA-2+ asB at day 4 (0.26 %; 0.18-0.59%). No association between asB frequencies and their corresponding autoantibody levels was observed. In vitro preliminary data suggest that a minority of GAD+ asB was able to secrete GAD autoantibodies after sorting and stimulation

Conclusions: Our strategy seems able to identify bona fide asB but its consistency remains to be validated through sorting, cloning and expression of antigen-specific Ig genes as recombinant mAbs. If successful the study of asBTx might be applied to the phenotyping of autoimmunity recurrence in islet Tx.