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Plant-based vaccines for oral delivery of type 1 diabetes-related autoantigens: Evaluating oral tolerance mechanisms and disease prevention in NOD mice

View Article: PubMed Central - PubMed

ABSTRACT

Autoantigen-specific immunological tolerance represents a central objective for prevention of type 1 diabetes (T1D). Previous studies demonstrated mucosal antigen administration results in expansion of Foxp3+ and LAP+ regulatory T cells (Tregs), suggesting oral delivery of self-antigens might represent an effective means for modulating autoimmune disease. Early preclinical experiments using the non-obese diabetic (NOD) mouse model reported mucosal administration of T1D-related autoantigens [proinsulin or glutamic acid decarboxylase 65 (GAD)] delayed T1D onset, but published data are conflicting regarding dose, treatment duration, requirement for combinatorial agents, and extent of efficacy. Recently, dogma was challenged in a report demonstrating oral insulin does not prevent T1D in NOD mice, possibly due to antigen digestion prior to mucosal immune exposure. We used transplastomic plants expressing proinsulin and GAD to protect the autoantigens from degradation in an oral vaccine and tested the optimal combination, dose, and treatment duration for the prevention of T1D in NOD mice. Our data suggest oral autoantigen therapy alone does not effectively influence disease incidence or result in antigen-specific tolerance assessed by IL-10 measurement and Treg frequency. A more aggressive approach involving tolerogenic cytokine administration and/or lymphocyte depletion prior to oral antigen-specific immunotherapy will likely be required to impart durable therapeutic efficacy.

No MeSH data available.


At T1D onset or 32 weeks of age, (A–D) PLN and (E-H) MLN cells were stained for CD4, CD8, CD25, LAP, and Foxp3 for flow cytometric analysis. Live lymphocytes gated on CD4 were analyzed for the percent of CD25+Foxp3+ Tregs. There was no difference in Treg frequency in the (A) PLN or (E) MLN, P = 0.29 and P = 0.12, respectively. There was no significant difference in the percent of live lymphocytes that were (B,F) CD8+LAP+ T cells, P = 0.997 and P = 0.32, respectively; (C,G) CD4+LAP+ T cells, P = 0.58 and P = 0.23, respectively; and (D,H) CD4+CD25+Foxp3+LAP+ Tregs, P = 0.89 and P = 0.06, respectively (ANOVA). No Tx indicates untreated control NOD mice.
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f5: At T1D onset or 32 weeks of age, (A–D) PLN and (E-H) MLN cells were stained for CD4, CD8, CD25, LAP, and Foxp3 for flow cytometric analysis. Live lymphocytes gated on CD4 were analyzed for the percent of CD25+Foxp3+ Tregs. There was no difference in Treg frequency in the (A) PLN or (E) MLN, P = 0.29 and P = 0.12, respectively. There was no significant difference in the percent of live lymphocytes that were (B,F) CD8+LAP+ T cells, P = 0.997 and P = 0.32, respectively; (C,G) CD4+LAP+ T cells, P = 0.58 and P = 0.23, respectively; and (D,H) CD4+CD25+Foxp3+LAP+ Tregs, P = 0.89 and P = 0.06, respectively (ANOVA). No Tx indicates untreated control NOD mice.

Mentions: In phase 1, there were no observed changes in CD4+CD25+Foxp3+ Treg frequency in the spleens of mice treated with tobacco expressing 500 μg GAD, 250 μg CTB-hpINS, or WT tobacco (Fig. 4A). The inclusion of ATRA was similarly not associated with an increase in Treg frequency in the spleen (Fig. 4B). In phase 2, there were no differences in CD4+CD25+Foxp3+ Treg frequency within the spleen at 6, 10, and 14 weeks of age (Fig. 4C–E). However, splenic Treg frequencies were increased in mice treated with CTB-hpINS as well as CTB-hpINS plus GAD, relative to untreated animals at longitudinal endpoints (Fig. 4F). This effect was particularly evident among treated mice that did not progress to hyperglycemia (Fig. 4G). LAP+ T cells were not detectable in the spleen for any time point or treatment group. There were no changes in the frequency of CD4+CD25+Foxp3+ Tregs or LAP+ T cells in the PLN or MLN in any animals – progressors or not (Fig. 5 and S3).


Plant-based vaccines for oral delivery of type 1 diabetes-related autoantigens: Evaluating oral tolerance mechanisms and disease prevention in NOD mice
At T1D onset or 32 weeks of age, (A–D) PLN and (E-H) MLN cells were stained for CD4, CD8, CD25, LAP, and Foxp3 for flow cytometric analysis. Live lymphocytes gated on CD4 were analyzed for the percent of CD25+Foxp3+ Tregs. There was no difference in Treg frequency in the (A) PLN or (E) MLN, P = 0.29 and P = 0.12, respectively. There was no significant difference in the percent of live lymphocytes that were (B,F) CD8+LAP+ T cells, P = 0.997 and P = 0.32, respectively; (C,G) CD4+LAP+ T cells, P = 0.58 and P = 0.23, respectively; and (D,H) CD4+CD25+Foxp3+LAP+ Tregs, P = 0.89 and P = 0.06, respectively (ANOVA). No Tx indicates untreated control NOD mice.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5304332&req=5

f5: At T1D onset or 32 weeks of age, (A–D) PLN and (E-H) MLN cells were stained for CD4, CD8, CD25, LAP, and Foxp3 for flow cytometric analysis. Live lymphocytes gated on CD4 were analyzed for the percent of CD25+Foxp3+ Tregs. There was no difference in Treg frequency in the (A) PLN or (E) MLN, P = 0.29 and P = 0.12, respectively. There was no significant difference in the percent of live lymphocytes that were (B,F) CD8+LAP+ T cells, P = 0.997 and P = 0.32, respectively; (C,G) CD4+LAP+ T cells, P = 0.58 and P = 0.23, respectively; and (D,H) CD4+CD25+Foxp3+LAP+ Tregs, P = 0.89 and P = 0.06, respectively (ANOVA). No Tx indicates untreated control NOD mice.
Mentions: In phase 1, there were no observed changes in CD4+CD25+Foxp3+ Treg frequency in the spleens of mice treated with tobacco expressing 500 μg GAD, 250 μg CTB-hpINS, or WT tobacco (Fig. 4A). The inclusion of ATRA was similarly not associated with an increase in Treg frequency in the spleen (Fig. 4B). In phase 2, there were no differences in CD4+CD25+Foxp3+ Treg frequency within the spleen at 6, 10, and 14 weeks of age (Fig. 4C–E). However, splenic Treg frequencies were increased in mice treated with CTB-hpINS as well as CTB-hpINS plus GAD, relative to untreated animals at longitudinal endpoints (Fig. 4F). This effect was particularly evident among treated mice that did not progress to hyperglycemia (Fig. 4G). LAP+ T cells were not detectable in the spleen for any time point or treatment group. There were no changes in the frequency of CD4+CD25+Foxp3+ Tregs or LAP+ T cells in the PLN or MLN in any animals – progressors or not (Fig. 5 and S3).

View Article: PubMed Central - PubMed

ABSTRACT

Autoantigen-specific immunological tolerance represents a central objective for prevention of type 1 diabetes (T1D). Previous studies demonstrated mucosal antigen administration results in expansion of Foxp3+ and LAP+ regulatory T cells (Tregs), suggesting oral delivery of self-antigens might represent an effective means for modulating autoimmune disease. Early preclinical experiments using the non-obese diabetic (NOD) mouse model reported mucosal administration of T1D-related autoantigens [proinsulin or glutamic acid decarboxylase 65 (GAD)] delayed T1D onset, but published data are conflicting regarding dose, treatment duration, requirement for combinatorial agents, and extent of efficacy. Recently, dogma was challenged in a report demonstrating oral insulin does not prevent T1D in NOD mice, possibly due to antigen digestion prior to mucosal immune exposure. We used transplastomic plants expressing proinsulin and GAD to protect the autoantigens from degradation in an oral vaccine and tested the optimal combination, dose, and treatment duration for the prevention of T1D in NOD mice. Our data suggest oral autoantigen therapy alone does not effectively influence disease incidence or result in antigen-specific tolerance assessed by IL-10 measurement and Treg frequency. A more aggressive approach involving tolerogenic cytokine administration and/or lymphocyte depletion prior to oral antigen-specific immunotherapy will likely be required to impart durable therapeutic efficacy.

No MeSH data available.