Intralymphatic Glutamic Acid Decarboxylase-Alum Administration Induced Th2-Like-Specific Immunomodulation in Responder Patients: A Pilot Clinical Trial in Type 1 Diabetes

Beatriz Tavira, Hugo Barcenilla, Jeannette Wahlberg, Peter Achenbach, Johnny Ludvigsson, Rosaura Casas, Beatriz Tavira, Hugo Barcenilla, Jeannette Wahlberg, Peter Achenbach, Johnny Ludvigsson, Rosaura Casas

Abstract

GAD-alum given into lymph nodes to type 1 diabetes patients participating in an open-label pilot trial resulted in preservation of C-peptide similar to promising results from other trials. Here, we compared the immunomodulatory effect of giving GAD-alum directly into lymph nodes versus that induced by subcutaneous administration. Samples from T1D patients (n = 6) who received 4 μg GAD-alum into lymph nodes (LNs), followed by two booster injections one month apart, and from patients (n = 6) who received two subcutaneous injections (SC) (20 μg) given one month apart were compared. GADA, IA-2A, GADA subclasses, IgE, GAD65-induced cytokines, PBMC proliferation, and T cell markers were analyzed. Lower doses of GAD-alum into LN induced higher GADA levels than SC injections and reduced proliferation and IgG1 GADA subclass, while enhancing IgG2, IgG3, and IgG4. The cytokine profile was dominated by the Th2-associated cytokine IL-13, and GAD65 stimulation induced activated CD4 T cells. Patients responding clinically best account for most of the immunological changes. In contrast, SC treatment resulted in predominant IgG1, predominant IFN-γ, higher proliferation, and activated CD4 and CD8 cells. Patients from the LN group with best metabolic outcome seemed to have common immune correlates related to the treatment. This trial is registered with DIAGNODE (NCT02352974, clinicaltrials.gov) and DIABGAD (NCT01785108, clinicaltrials.gov).

Figures

Figure 1
Figure 1
CONSORT flow diagram. A total of 12 patients were recruited in two different clinical studies (n = 6 each study) using different doses and administration of GAD-alum. None of the patients were excluded during these trials' follow-up.
Figure 2
Figure 2
GADA titers and GADA subclass distribution in GAD-alum lymph node and subcutaneous treatment. (a) Median values of GADA titers in the lymph node (LN, n = 6) or subcutaneous vaccination (SC, n = 6). (b) Change of the frequency (%) of IgG1, IgG2, IgG3, and IgG4 GADA subclasses. Frequencies were calculated with respect to the combined sum of the AUs of the 4 subclasses in each sample. The median percentage with respect to the total IgG is shown for each respective subclass. (c) GADA subclass relative contribution at baseline, 90, and 180 days for LN and SC groups.
Figure 3
Figure 3
GAD65-induced cytokine secretion upon in vitro PMBC stimulation. Relative contribution (%) of the cytokines in LN patients and SC group at 90 and 180 days.
Figure 4
Figure 4
T cell activation induced by GAD65 in lymph node (LN, n = 6, black circles) and subcutaneous (SC, n = 6, white circles) GAD-alum patients. (a) Percentage of GAD65-activated CD4+CD25+CD127+ T cells and (b) CD8+CD25+CD127+ T cells. (c) Mean percentage of CD4+CD25+CD127lo/−FOXP3+ (Treg) in resting samples (medium alone) and (d) induced by GAD65 stimulation. (e) Mean percentage of FOXP3loCD45RA− nonsuppressor regulatory T cells in resting samples (medium alone) and (f) GAD65-stimulated samples.
Figure 5
Figure 5
Heat map representing the immunological changes induced by GAD-alum treatment. Variations were calculated as the ratio of the values at 90 and 180 days with respect to the baseline. Patients received GAD-alum injections into the lymph nodes (LNs, n = 6) or subcutaneously (SC, n = 6), and they were stratified from left to right according to their clinical outcome at 180 days. Clinical variables are expressed as percentage of change from baseline (%). At 90 days, max. stimulated, and AUC C-peptide were not calculated, as meal tolerance tests were not performed, and are represented by “×”. The color scales illustrate the posttreatment increase (green) or decrease (red) of variables in relation to baseline values.

References

    1. Schloot N. C., Meierhoff G., Lengyel C., et al. Effect of heat shock protein peptide DiaPep277 on beta-cell function in paediatric and adult patients with recent-onset diabetes mellitus type 1: two prospective, randomized, double-blind phase II trials. Diabetes/Metabolism Research and Reviews. 2007;23(4):276–285. doi: 10.1002/dmrr.707.
    1. Keymeulen B., Vandemeulebroucke E., Ziegler A. G., et al. Insulin needs after CD3-antibody therapy in new-onset type 1 diabetes. New England Journal of Medicine. 2005;352(25):2598–2608. doi: 10.1056/NEJMoa043980.
    1. Herold K. C., Hagopian W., Auger J. A., et al. Anti-CD3 monoclonal antibody in new-onset type 1 diabetes mellitus. New England Journal of Medicine. 2002;346(22):1692–1698. doi: 10.1056/NEJMoa012864.
    1. Pescovitz M. D., Greenbaum C. J., Krause-Steinrauf H., et al. Rituximab, B-lymphocyte depletion, and preservation of beta-cell function. New England Journal of Medicine. 2009;361(22):2143–2152. doi: 10.1056/NEJMoa0904452.
    1. Orban T., Bundy B., Becker D. J., et al. Co-stimulation modulation with abatacept in patients with recent-onset type 1 diabetes: a randomised, double-blind, placebo-controlled trial. The Lancet. 2011;378(9789):412–419. doi: 10.1016/S0140-6736(11)60886-6.
    1. Pozzilli P., Pitocco D., Visalli N., et al. No effect of oral insulin on residual beta-cell function in recent-onset type I diabetes (the IMDIAB VII). IMDIAB group. Diabetologia. 2000;43(8):1000–1004. doi: 10.1007/s001250051482.
    1. Walter M., Philotheou A., Bonnici F., Ziegler A.-G., Jimenez R., on behalf of the NBI-6024 Study Group No effect of the altered peptide ligand NBI-6024 on beta-cell residual function and insulin needs in new-onset type 1 diabetes. Diabetes Care. 2009;32(11):2036–2040. doi: 10.2337/dc09-0449.
    1. Ludvigsson J., Faresjö M., Hjorth M., et al. GAD treatment and insulin secretion in recent-onset type 1 diabetes. New England Journal of Medicine. 2008;359(18):1909–1920. doi: 10.1056/NEJMoa0804328.
    1. Wherrett D. K., Bundy B., Becker D. J., et al. Antigen-based therapy with glutamic acid decarboxylase (GAD) vaccine in patients with recent-onset type 1 diabetes: a randomised double-blind trial. The Lancet. 2011;378(9788):319–327. doi: 10.1016/S0140-6736(11)60895-7.
    1. Ludvigsson J., Krisky D., Casas R., et al. GAD65 antigen therapy in recently diagnosed type 1 diabetes mellitus. New England Journal of Medicine. 2012;366(5):433–442. doi: 10.1056/NEJMoa1107096.
    1. Tavira B., Cheramy M., Axelsson S., Akerman L., Ludvigsson J., Casas R. Effect of simultaneous vaccination with H1N1 and GAD-alum on GAD65-induced immune response. Diabetologia. 2017;60(7):1276–1283. doi: 10.1007/s00125-017-4263-x.
    1. Beam C. A., the Type 1 Diabetes TrialNet Study Group, MacCallum C., et al. GAD vaccine reduces insulin loss in recently diagnosed type 1 diabetes: findings from a Bayesian meta-analysis. Diabetologia. 2017;60(1):43–49. doi: 10.1007/s00125-016-4122-1.
    1. Pfaar O., Bachert C., Bufe A., et al. Guideline on allergen-specific immunotherapy in IgE-mediated allergic diseases. Allergo Journal International. 2014;23(8):282–319. doi: 10.1007/s40629-014-0032-2.
    1. Dhami S., Nurmatov U., Pajno G. B., et al. Allergen immunotherapy for IgE-mediated food allergy: protocol for a systematic review. Clinical and Translational Allergy. 2016;6(1):p. 24. doi: 10.1186/s13601-016-0113-z.
    1. Tsabouri S., Mavroudi A., Feketea G., Guibas G. V. Subcutaneous and sublingual immunotherapy in allergic asthma in children. Frontiers in Pediatrics. 2017;5:p. 82. doi: 10.3389/fped.2017.00082.
    1. Chelladurai Y., Suarez-Cuervo C., Erekosima N., et al. Effectiveness of subcutaneous versus sublingual immunotherapy for the treatment of allergic rhinoconjunctivitis and asthma: a systematic review. The Journal of Allergy and Clinical Immunology: In Practice. 2013;1(4):361–369. doi: 10.1016/j.jaip.2013.04.005.
    1. Maloy K. J., Erdmann I., Basch V., et al. Intralymphatic immunization enhances DNA vaccination. Proceedings of the National Academy of Sciences. 2001;98(6):3299–3303. doi: 10.1073/pnas.051630798.
    1. Senti G., Prinz Vavricka B. M., Erdmann I., et al. Intralymphatic allergen administration renders specific immunotherapy faster and safer: a randomized controlled trial. Proceedings of the National Academy of Sciences. 2008;105(46):17908–17912. doi: 10.1073/pnas.0803725105.
    1. Patterson A. M., Bonny A. E., Shiels W. E., II, Erwin E. A. Three-injection intralymphatic immunotherapy in adolescents and young adults with grass pollen rhinoconjunctivitis. Annals of Allergy, Asthma & Immunology. 2016;116(2):168–170. doi: 10.1016/j.anai.2015.11.010.
    1. Hylander T., Larsson O., Petersson-Westin U., et al. Intralymphatic immunotherapy of pollen-induced rhinoconjunctivitis: a double-blind placebo-controlled trial. Respiratory Research. 2016;17(1):p. 10. doi: 10.1186/s12931-016-0324-9.
    1. Senti G., Crameri R., Kuster D., et al. Intralymphatic immunotherapy for cat allergy induces tolerance after only 3 injections. Journal of Allergy and Clinical Immunology. 2012;129(5):1290–1296. doi: 10.1016/j.jaci.2012.02.026.
    1. Mackensen A., Krause T., Blum U., Uhrmeister P., Mertelsmann R., Lindemann A. Homing of intravenously and intralymphatically injected human dendritic cells generated in vitro from CD34+ hematopoietic progenitor cells. Cancer Immunology, Immunotherapy. 1999;48(2-3):118–122. doi: 10.1007/s002620050555.
    1. Johansen P., Häffner A. . C., Koch F., et al. Direct intralymphatic injection of peptide vaccines enhances immunogenicity. European Journal of Immunology. 2005;35(2):568–574. doi: 10.1002/eji.200425599.
    1. Ludvigsson J., Wahlberg J., Casas R. Intralymphatic injection of autoantigen in type 1 diabetes. New England Journal of Medicine. 2017;376(7):697–699. doi: 10.1056/NEJMc1616343.
    1. Ludvigsson J., Tavira B., Casas R. More on intralymphatic injection of autoantigen in type 1 diabetes. New England Journal of Medicine. 2017;377(4):403–405. doi: 10.1056/NEJMc1703468.
    1. Cheramy M., Skoglund C., Johansson I., Ludvigsson J., Hampe C. S., Casas R. GAD-alum treatment in patients with type 1 diabetes and the subsequent effect on GADA IgG subclass distribution, GAD65 enzyme activity and humoral response. Clinical Immunology. 2010;137(1):31–40. doi: 10.1016/j.clim.2010.06.001.
    1. Bonifacio E., Scirpoli M., Kredel K., Füchtenbusch M., Ziegler A. G. Early autoantibody responses in prediabetes are IgG1 dominated and suggest antigen-specific regulation. Journal of Immunology. 1999;163(1):525–532.
    1. Axelsson S., Cheramy M., Akerman L., Pihl M., Ludvigsson J., Casas R. Cellular and humoral immune responses in type 1 diabetic patients participating in a phase III GAD-alum intervention trial. Diabetes Care. 2013;36(11):3418–3424. doi: 10.2337/dc12-2251.
    1. Axelsson S., Chéramy M., Hjorth M., et al. Long-lasting immune responses 4 years after GAD-alum treatment in children with type 1 diabetes. PLoS One. 2011;6(12, article e29008) doi: 10.1371/journal.pone.0029008.
    1. Harrison L. C., Honeyman M. C., Steele C. E., et al. Pancreatic beta-cell function and immune responses to insulin after administration of intranasal insulin to humans at risk for type 1 diabetes. Diabetes Care. 2004;27(10):2348–2355. doi: 10.2337/diacare.27.10.2348.
    1. Russell M. A., Cooper A. C., Dhayal S., Morgan N. G. Differential effects of interleukin-13 and interleukin-6 on Jak/STAT signaling and cell viability in pancreatic β-cells. Islets. 2013;5(2):95–105. doi: 10.4161/isl.24249.
    1. Usero L., Sánchez A., Pizarro E., et al. Interleukin-13 pathway alterations impair invariant natural killer T-cell-mediated regulation of effector T cells in type 1 diabetes. Diabetes. 2016;65(8):2356–2366. doi: 10.2337/db15-1350.
    1. Wood K. J., Sawitzki B. Interferon gamma: a crucial role in the function of induced regulatory T cells in vivo. Trends in Immunology. 2006;27(4):183–187. doi: 10.1016/j.it.2006.02.008.
    1. Fenimore J., Young H. A. Regulation of IFN-γ expression. Advances in Experimental Medicine and Biology. 2016;941:1–19. doi: 10.1007/978-94-024-0921-5_1.
    1. Pihl M., Barcenilla H., Axelsson S., et al. GAD-specific T cells are induced by GAD-alum treatment in type-1 diabetes patients. Clinical Immunology. 2017;176:114–121. doi: 10.1016/j.clim.2017.01.010.
    1. Hjorth M., Axelsson S., Ryden A., Faresjo M., Ludvigsson J., Casas R. GAD-alum treatment induces GAD65-specific CD4+CD25highFOXP3+ cells in type 1 diabetic patients. Clinical Immunology. 2011;138(1):117–126. doi: 10.1016/j.clim.2010.10.004.
    1. Pihl M., Åkerman L., Axelsson S., et al. Regulatory T cell phenotype and function 4 years after GAD-alum treatment in children with type 1 diabetes. Clinical & Experimental Immunology. 2013;172(3):394–402. doi: 10.1111/cei.12078.
    1. Schmid S., Molteni A., Fuchtenbusch M., Naserke H. E., Ziegler A. G., Bonifacio E. Reduced IL-4 associated antibody responses to vaccine in early pre-diabetes. Diabetologia. 2002;45(5):677–685. doi: 10.1007/s00125-002-0816-7.
    1. Martinez-Gomez J. M., Johansen P., Erdmann I., Senti G., Crameri R., Kundig T. M. Intralymphatic injections as a new administration route for allergen-specific immunotherapy. International Archives of Allergy and Immunology. 2009;150(1):59–65. doi: 10.1159/000210381.
    1. Brewer J. M., Conacher M., Hunter C. A., Mohrs M., Brombacher F., Alexander J. Aluminium hydroxide adjuvant initiates strong antigen-specific Th2 responses in the absence of IL-4- or IL-13-mediated signaling. Journal of Immunology. 1999;163(12):6448–6454.

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