Early induction of GAD(65)-reactive Th2 response in type 1 diabetic children treated with alum-formulated GAD(65).
BACKGROUND
We have previously shown that two injections of 20 µg alum-formulated glutamic acid decarboxylase 65 (GAD(65)) (GAD-alum; Diamyd(®)) in children with recent-onset type 1 diabetes lead to preservation of residual insulin secretion. In vitro cytokine production at the 15 months' follow-up indicated immunomodulation. In the present study, we took advantage of peripheral blood mononuclear cells, cryopreserved during early follow-ups, to investigate whether the immunomodulatory effect of GAD-alum was apparent earlier after treatment, preceding the changes previously reported at 15 months.


METHODS
Peripheral blood mononuclear cells from 70 type 1 diabetic children, randomly assigned GAD-alum (n = 35) or placebo (n = 35), that had been frozen at baseline (n = 27) and after 1 (n = 58), 3 (n = 67) and 9 (n = 66) months, were stimulated in vitro with GAD(65), tyrosine phosphatase-like protein IA-2 peptide, insulin peptide, GAD-alum, alum formulation or phytohaemagglutinin. Interleukin (IL)-5, -6, -10, -12, -13, -17, tumour necrosis factor and interferon-γ were measured in cell supernatants and serum samples using Luminex. Expression of FOXP3 and transforming growth factor-β was determined by real-time reverse transcription polymerase chain reaction.


RESULTS
Already 1 month after the first injection, GAD(65)-induced IL-5 and IL-13 together with FOXP3 were enhanced in GAD-alum-treated patients compared to those with placebo. The in vitro response at 3 and 9 months was characterized by a broader range of cytokines in the treated group. Notably, only the T-helper 2-associated cytokines IL-5 and IL-13 together with FOXP3 increased continuously over time.


CONCLUSIONS
Treatment with GAD-alum in type 1 diabetic children induced an early T-helper 2 immune enhanced response to GAD(65), followed by a wider spectrum of cytokines at 3 and 9 months.
Axelsson
Stina
S
Division of Paediatrics and Diabetes Research Centre, Department of Clinical and Experimental Medicine, Faculty of Health Sciences, Linköping University, Linköping, Sweden. stina.axelsson@liu.se
Hjorth
Maria
M
Akerman
Linda
L
Ludvigsson
Johnny
J
Casas
Rosaura
R
D000293:Adolescent; D000534:Alum Compounds; D001323:Autoantibodies / Q000097:blood / Q000276:immunology; D002648:Child; D003922:Diabetes Mellitus, Type 1 / Q000188:drug therapy* / Q000276:immunology; D005500:Follow-Up Studies; D051858:Forkhead Transcription Factors / Q000032:analysis / Q000276:immunology; D005968:Glutamate Decarboxylase / Q000378:metabolism* / Q000627:therapeutic use*; D006801:Humans; D007155:Immunologic Factors / Q000627:therapeutic use*; D007328:Insulin / Q000276:immunology / Q000378:metabolism; D007371:Interferon-gamma / Q000032:analysis / Q000276:immunology; D007378:Interleukins / Q000032:analysis / Q000276:immunology; D009000:Monocytes / Q000276:immunology; D015337:Multicenter Studies as Topic; D017027:Protein Tyrosine Phosphatases / Q000276:immunology; D016032:Randomized Controlled Trials as Topic; D018418:Th2 Cells / Q000201:enzymology / Q000276:immunology*; D016212:Transforming Growth Factor beta / Q000032:analysis / Q000276:immunology; D014409:Tumor Necrosis Factor-alpha / Q000032:analysis / Q000276:immunology
D000534:Alum Compounds; D001323:Autoantibodies; C418974:FOXP3 protein, human; D051858:Forkhead Transcription Factors; D007155:Immunologic Factors; D007328:Insulin; D007378:Interleukins; D016212:Transforming Growth Factor beta; D014409:Tumor Necrosis Factor-alpha; C041524:aluminum sulfate; D007371:Interferon-gamma; D017027:Protein Tyrosine Phosphatases; D005968:Glutamate Decarboxylase; C401141:glutamate decarboxylase 2