Art Seminario 25 - 08 - 23

HHS Public Access
Author manuscript
J Immunol. Author manuscript; available in PMC 2023 August 01.
Author Manuscript
Published in final edited form as:

J Immunol. 2022 August 01; 209(3): 465–475. doi:10.4049/jimmunol.2200208.
Tolerogenic Immune Modifying Nanoparticles Encapsulating

Multiple Recombinant Pancreatic β Cell Proteins Prevent Onset
and Progression of Type 1 Diabetes in NOD Mice
Joseph R. Podojil*,§, Samantha Genardi§, Ming-Yi Chiang*, Sandeep Kakade§, Tobias Neef*,
Tushar Murthy§, Michael T. Boyne II*,§, Adam Elhofy§, Stephen D Miller*,#,1
*Department of Microbiology-Immunology, Feinberg School of Medicine, Northwestern University,
Author Manuscript
Chicago, IL, USA;

#Department of Interdepartmental Immunobiology Center, Feinberg School of Medicine,
Northwestern University, Chicago, IL, USA;
§COUR Pharmaceutical Development Company, Inc., Northbrook, IL, USA
Abstract
Type 1 diabetes (T1D) is an autoimmune disease characterized by T and B cell responses to
proteins expressed by insulin-producing pancreatic β cells, inflammatory lesions within islets
(insulitis), and β cell loss. We previously showed that antigen (Ag)-specific tolerance targeting
single β cell protein epitopes is effective in preventing T1D induced by transfer of mono-specific
diabetogenic CD4 and CD8 transgenic T cells to NOD.scid mice. However, tolerance induction
Author Manuscript
to individual diabetogenic proteins, e.g., GAD65 or insulin, has failed to ameliorate T1D both in
wildtype NOD mice and in the clinic. Initiation and progression of T1D is likely due to activation
of T cells specific for multiple diabetogenic epitopes. To test this hypothesis, recombinant
insulin, GAD65, and chromogranin A proteins were encapsulated within poly(lactic-co-glycolic
acid) (PLGA) nanoparticles (COUR CNPs) to assess regulatory T cell induction, inhibition of
Ag-specific T cell responses, and blockade of T1D induction/progression in NOD mice. While
treatment of NOD mice with CNPs containing a single protein inhibited the corresponding
Ag-specific T cell response, inhibition of overt T1D development only occurred if all three
diabetogenic proteins were included within the CNPs (CNP-T1D). Blockade of T1D following
CNP-T1D tolerization was characterized by regulatory T cell induction and a significant decrease
in both peri-insulitis and immune cell infiltration into pancreatic islets. As we have recently
published that CNP treatment is both safe and induced Ag-specific tolerance in a Phase 1/2a
Author Manuscript
celiac disease clinical trial, Ag-specific tolerance induced by nanoparticles encapsulating multiple
diabetogenic proteins is a promising approach to T1D treatment.
1
Corresponding Author: Stephen D. Miller, Ph.D., Department of Microbiology-Immunology, Northwestern University Medical
School, Tarry 6-718, 303 E. Chicago Ave, Chicago, IL 60611, 312-503-7674, FAX: 312-503-1154, s-d-miller@northwestern.edu.
Author Contributions:
J.R.P., M.Y.C., T.N., T.M., A.E., and S.D.M. formed the hypotheses, designed experiments, performed experiments, analyzed results,
and prepared the manuscript. T.N., S.G., S.K., and M.T.B. helped design experiments, analyze results, and synthesized the CNP-T1D
nanoparticles. All authors read and edited the manuscript.
Podojil et al. Page 2
Keywords
Author Manuscript
immune tolerance; type 1 diabetes; PLGA nanoparticles; regulatory T cells; cell trafficking
Introduction
Type 1 diabetes (T1D) is an autoimmune disease affecting around 1.6 million people in the
US and 4.7 million people world-wide (1, 2). Presently, the standard of care for patients
with T1D is exogenous insulin replacement combined with diet and exercise modifications
(3). Evidence from both the non-obese diabetic (NOD) mouse model of spontaneous T1D
and T1D patients suggests that insulin is only one of several dominant measurable disease-
associated Ags recognized by autoreactive CD4+ T cells and CD8+ T cells driving disease
pathogenesis (4, 5). The disease initiating epitope or epitopes in T1D are not known,
Author Manuscript
and therefore not surprisingly, single protein or peptide tolerance approaches have been
unsuccessful to date in both NOD mice and human T1D patients. T1D is not a single antigen
(Ag)-mediated disease, rather once inflammation within the pancreas and tissue destruction
occurs the autoimmune response expands to include both T cell and B cell responses
to additional epitopes, a process known as epitope spreading (6–9). In support of a role
for epitope spreading in T1D, autoreactive responses to Ags other than insulin, including
glutamic acid decarboxylase 65 (GAD65), islet specific glucose 6 phosphate catalytic
subunit related protein (IGRP), Islet antigen-2 (IA-2), phogrin (IA-2β), Chromogranin A,
zinc transporter 8 (ZnT8), and vasostain-1 (5, 10–12), have been identified in both T1D
patients and NOD mice. Therefore, the failure of tolerance approaches targeting single Ags,
such as GAD65 or insulin, to inhibit the onset and eventual progression of disease in clinical
trials suggests that tolerance to multiple disease Ags may be necessary to provide efficient
Author Manuscript
disease control (6, 8, 9).
Currently, there are no available therapies that treat the root cause of T1D, i.e., activated
autoimmune T cells mediating continued pancreatic β cell destruction, either through CD8+
T cells, CD4+ T cell-mediated inflammation, or autoantibodies. Therapies in the pipeline are
alternatively targeting broad T cell suppression with anti-CD3 or transient suppression with
single Ag-specific CAR-Tregs. Our laboratory has studied the ability to induce Ag-specific
tolerance via the induction of Ag-specific Treg cells and the induction of IL-10, CTLA-4,
and PD-1 expression following treatment with Ag-coupled apoptotic splenocytes, Ag-
coupled nanoparticles, and nanoparticles that contain encapsulated Ag [reviewed (13, 14)].
We have shown that intravenous treatment with carboxylated poly(lactide-co-glycolide)
(PLGA) nanoparticles induces Ag-specific immune tolerance in a wide variety of mouse
disease models via CD4+ regulatory T cell (Treg)-dependent mechanisms (3, 15–17).
Author Manuscript
Biodegradable carboxylated PLGA nanoparticles containing encapsulated Ag [tolerance-

inducing microparticles (TIMPs); named CNPs] were shown to prevent and treat disease
in several mouse models of T1D through reprogramming of APCs and induction of Treg
cells. Nanoparticle size (400–800 nm) and negative charge results in phagocytic uptake
of the Ag-encapsulating nanoparticles by tolerogenic APCs via the MARCO scavenger
receptor in the splenic marginal zone and liver, resulting in presentation of the Ag in the
context of immune regulatory signals including PD-L1/2 and IL-10 (14). For example,

the treatment with PLGA nanoparticles encapsulating or coated with the chromogranin A
mimotope peptide, p31, following the transfer of diabetogenic transgenic CD4+ BDC-2.5 T
Author Manuscript
cells to NOD.scid mice, led to the sequestration of the autoreactive effector CD4+ T cells
within the spleen and increased CD4+FoxP3+ Treg cells in the spleen, pancreatic lymph
nodes and pancreas (16). The induction of the Treg cells was found to be required for
long-term tolerance as the removal of CD25+ T cells drastically reduced the time through
which tolerance was maintained. Additionally, in the BDC-2.5 CD4+ T cell adoptive transfer
model, PLGA nanoparticles coupled with a hybrid insulin peptide, 2.5HIP, also increased
the numbers of FoxP3+ Treg cells in the pancreas and reduced the trafficking of effector
CD4+ T cells (18). These findings suggest that a common regulatory mechanism is induced
at the cellular level across mouse strains, in that Ag-loaded PLGA nanoparticle treatment
induced various subsets of Ag-specific Treg cells capable of regulating autoimmune disease.
With regards to clinical translation, CNP nanoparticles have demonstrated efficacy to

Author Manuscript
induce tolerance in mouse models of EAE, T1D (adoptive transfer), celiac disease,
and Th2-induced allergic airway inflammation (16, 17, 19). In these models, CNP
treatment reduced pathogenic cytokine production (IFN-γ, IL-17, IL-4, and IL-5) (16,
17, 19), increasing anti-inflammatory IL-10 cytokine production (16), and increasing
CD25+Foxp3+ Treg cells at the site of disease (16). In pre-clinical studies to assess CNP
function in celiac disease, treatment with gliadin encapsulating PLGA nanoparticles (TIMP-
GLIA, TAK-101/CNP-101) significantly decreased gliadin-specific T cell proliferation,
inflammatory cytokine secretion, circulating gliadin-specific IgG/IgG2c, gliadin-specific
DTH response, gluten-dependent enteropathy, and weight loss (17). The functionality of
TAK-101/TIMP-GLIA treatment was also demonstrated in HLA-DQ8 transgenic mice, as
treatment increased the level of FoxP3 expression, and induced gene signatures associated
with tolerance induction (17). The CNP nanoparticle design was first introduced into
Author Manuscript
human clinical studies with TAK-101/TIMP-GLIA and demonstrated successful induction

of tolerance in celiac patients orally challenged with gluten (20). These studies assessed
safety, tolerability, pharmacokinetics, and efficacy of TAK-101/TIMP-GLIA treatment to
induce gliadin-specific T cell tolerance in patients with biopsy-confirmed celiac disease
(20).
In the present study we sought to address both a fundamental immunological question

regarding identification of disease-dependent Ag-specific T cell responses involved in the
onset of T1D in NOD mice and to determine if treatment of NOD mice with CNPs
containing recombinant diabetogenic proteins would inhibit the development of T1D. The
spontaneous NOD mouse model recapitulates some key disease characteristics of human
T1D, where multiple diabetogenic Ags drive disease progression. Insulin, GAD65, and
Author Manuscript
chromogranin A proteins known to be potential targets in TID were encapsulated within

TIMPs (CNP-T1D), enabling us to test the simultaneous tolerogenic targeting of multiple
diabetogenic CD4+ T cell and CD8+ T cell epitopes for efficacy in preventing T1D onset
(21). Treatment with CNP-T1D beginning at either 6 or 10 weeks of age prevented β cell
destruction and further epitope spreading, as well as inhibiting the secretion of IFN-γ upon
splenocyte ex vivo recall culture. Encapsulation of multiple disease epitopes in CNP-T1D
covering three major T1D autoantigens was necessary to prevent T1D onset as single
protein tolerance proved insufficient. CNP-T1D-induced tolerance was durable lasting for

13.5 weeks post third dose administration. Taken together, these data show CNP-T1D is a
Author Manuscript
promising therapeutic for treatment of T1D and provides an advantage over single protein
or single epitope targeting therapies by inducing tolerance to multiple encapsulated cognate
epitopes as well as spread epitopes.
Materials and Methods

Materials
Acid-terminated Poly(D, L-lactic-co-glycolic acid) (PLGA) polymer was purchased from
Lactel Absorbable Polymers (Evonik Corporation, AL). Recombinant Insulin was purchased
from Sigma Aldrich (St., Louis, MO). Recombinant GAD65 and Chromogranin proteins
were custom ordered from Biomatik, Inc. (Ontario, Canada). MOG92–106, p31, Pro-ins C19-
A3, and the linkered peptide p31-NRPA7-InsB9–23 were purchased from Genemed Synthesis
(Torrance, CA). 2.5-HIP was generously provided by the Kathryn Haskins laboratory at the
Author Manuscript
University of Colorado. See Supp. Table 1 for peptide sequences.
Manufacture of CNP nanoparticles (PLGA nanoparticles)

For CNPs encapsulating diabetic proteins (Insulin, Chromogranin A, or GAD65), PLGA
solution was mixed with each recombinant protein to generate a water-in-oil emulsion.
This was mixed with a proprietary blend of surfactants and organic solvents to form an
oil-in-water secondary emulsion. The solvent was removed by evaporation, yielding PLGA
nanoparticles encapsulating diabetic protein, which were washed, filtered, and concentrated
via tangential flow filtration. CNP-Insulin, CNP-Chromogranin A, and CNP-GAD65 were
supplied as a lyophilized powder containing approximately 100 mg of PLGA nanoparticles
per vial with average nanoparticle diameters 400–800 nm and zeta potentials of -30 to −60
mV. The protein content was ≥ 3 μg of recombinant protein per milligram of PLGA (Supp.
Author Manuscript
Figure 1). Nanoparticles used in initial attempts to inhibit T1D were created by slightly
different methods. PLGA(Insulin) was created by double-emulsion, solvent evaporation
as described previously (22). PLGA/MOG92–106, PLGA/Insulin, PLGA/Pro-insC19-A3,
PLGA/p31-NRPA7-InsB9–23 were synthesized by first coupling peptide to PLGA polymer
in dimethyl sulfoxide (DMSO) using ethylene carbodiimide (ECDI), followed by single
emulsion, solvent evaporation as previously described (23).
NOD mice, blood glucose monitoring, and treatment

Female NOD mice, 5 weeks of age, were purchased from Jackson Labs (Bar Harbor,
ME). All mice were housed under specific pathogen-free conditions in the Northwestern
University Center for Comparative Medicine and maintained according to protocols
Author Manuscript
approved by the Northwestern University Animal Care and Use Committee (Chicago, IL).
NOD mice were treated with CNPs (2.5 mg/dose in 200 μL sterile saline) on the specified
days. Blood glucose levels were measured in female NOD mice with UltraTRAK Pro
Blood Glucose Monitoring System weekly starting at the 6 weeks of age. Mice with two
consecutive readings at or above 250 mg/dL were determined to be diabetic.

Ex vivo recalls
Author Manuscript
On indicated days mice were euthanized and spleens were collected for isolation of single
cell suspensions. For ex vivo reactivation cultures, 1×106 total splenocytes were plated
in replicate wells on a 96-well flat-bottomed plates with complete RPMI medium plus
anti-CD3 (1 μg/mL), OVA323–339, Chromogranin A359–372, Insulin B9–23, Insulin A14–20,
GAD65286–300, GAD65515–524, IGRP206–214, HSP60437–460, or Znt8345–359 (20 μg/mL)
(Supp. Table 2). The culture supernatants were collected at 72 hours post culture and levels
of secreted cytokines were determined via Luminex using a Mouse Cytokine/Chemokine
Magnetic Bead Panel (Millipore).
Pathology Scoring of Pancreata

For histological analysis, the pancreas was removed and snap frozen in OCT. Multiple 10
μm sections were stained with hematoxylin and eosin and scored blindly for insulitis (Score:
Author Manuscript
no infiltrate; peri-insulitis present; <25%; and >25% of the islet is infiltrated). Average
insulitis percentages were determined from the total number of islets counted from each
treatment group.
Tissue processing and flow cytometry

The spleens and pancreatic lymph nodes were processed via physical disruption, and RBCs
were lysed using ammonium chloride prior to processing for flow cytometry. For the
pancreas, single cell suspensions were prepared by mincing the tumor tissue in 2 mL of
Accutase (MilliPore) plus 1 mg/mL collagenase, and the samples were incubated at 37°C for
30 min. Following the enzyme digestion, the pancreas samples were disrupted with a 100
μm cell strainer, the cell strainer washed 2x with 10 mL of HBSS+ 5% FCS, and the cells
pelleted. Cells were washed in PBS, stained with LIVE/DEAD® Fixable Aqua Dead Cell
Author Manuscript
Stain (Life Technologies; Grand Island, NY), blocked with anti-CD16/32 (ThermoFisher
Scientific), and then stained with the indicated antibodies. 106 viable cells were analyzed
per individual sample using a BD Celesta (BD Bioscience), and the data analyzed using
FloJo Version 9.5.2 software (Tree Star, Inc.; Ashland, OR). The specific antibodies used are
presented in Supp. Table 3.
Statistical analyses
Comparisons of T1D incidence were analyzed by X2 using Fisher’s exact probability.
Two-way ANOVA with a Bonferroni post-test was used to determine statistical differences
between treatment groups.
Results
Author Manuscript
Multiple diabetogenic peptide-specific T cell populations are present within the spleen of
NOD mice
Identification of the specific disease-inducing protein/peptide in spontaneous type 1 diabetes
(T1D) in NOD mice and human patients has been a topic of study for many years.
While the induction of tolerance to a single peptide is very reproducible after intravenous
infusion of peptide encapsulating PLGA nanoparticles (tolerance-inducing microparticles/

TIMP; named CNP), the specific disease-associated T cell epitopes need to be known for
Author Manuscript
this tolerance therapy to be effective. We previously showed that treatment of NOD.scid

mice PLGA nanoparticles encapsulating with the BDC2.5 mimetope 1040-31 (p31), the
NY8.3 mimetope NRPA7, and the linked p31-NRPA7-Insulin B9–23 peptide significantly
inhibit T1D-induced by the transfer of transgenic CD4+ BDC2.5 or CD8+ NY8.3 T
cells, respectively (16). However, individual treatment of wildtype NOD mice with PLGA
nanoparticles containing p31, p31-NRPA7-Insulin B9–23, pro-insulin C19-A3 or intact insulin
(Figure 1A) failed to inhibit development of T1D. Similarly, while treatment of NOD.scid
mice alone with PLGA-2.5HIP inhibits CD4+ BDC2.5 transgenic T cell-induced T1D (18),
this treatment is not able to delay the onset of T1D in NOD mice (Figure 1B). Thus,
targeting of individual diabetogenic peptide epitopes is incapable of inhibiting onset of T1D
supporting the hypothesis that tolerogenic targeting of multiple CD4+ T cell and CD8+
T cell epitopes using a combination of several recombinant diabetogenic proteins may be
Author Manuscript
required.
Based on previously published data and the above results, we asked if detectible T cell
responses to multiple diabetogenic peptides were present within the spleen of 6 and 9
week-old NOD mice. These two time points were chosen as this represents ages where
the number of immune cells within the pancreatic islets increases (24), there is detectible
circulating anti-insulin antibody (Ab) (25), and there is a detectible splenic T cell response
to various insulin peptides (26, 27). Splenocytes from 6 and 9 week-old NOD mice and
6 week-old C57BL/6 mice were cultured in the presence of 20 μg/mL of the following
peptides: OVA323–339, chromogranin A359–372, insulin B9–23, insulin A14–29, GAD65286–300,
GAD65515–524, IGRP206–214, HSP60437–460, or ZnT8345–359 (20). At 72 hours of culture,
supernatants were collected, and the levels of secreted IFN-γ measured. We found that
splenocytes from 6 week-old NOD, but not C57BL/6, mice, produced low, but significant,
Author Manuscript
levels of IFN-γ in response to all of the diabetogenic peptides, but not to the control
OVA323–339 peptide (Figure 1C). Additionally, there is a trend toward an age-dependent
increase in the level of IFN-γ secreted by splenocytes cultured in the presence of each of
the diabetogenic peptides, with the interesting exception of GAD65515–524. These findings
indicate that T cells of various specificities are present and activated within young NOD
mice, as early as 6 weeks of age, well prior to the onset of overt T1D.
CNP-T1D treatment inhibits the development of T1D in NOD mice

Based on the above data, we tested if the induction of tolerance to multiple diabetogenic
proteins would block the onset of T1D. NOD mice were randomized into two treatment
groups and treated with either 2.5 mg/dose of Unloaded CNP or CNPs containing
recombinant insulin, GAD65, and chromogranin A proteins. A 2.5 mg dose of CNP-
Author Manuscript
T1D consisted of 0.83 mg of CNP-Insulin, 0.83 mg of CNP-GAD65, 0.83 mg of

CNP-Chromogranin A equaling a total 2.5 mg/dose. We hypothesized that CNP-T1D
treatment would induce tolerance to chromogranin A359–372, insulin B9–23, insulin A14–29,
GAD65286–300, and GAD65515–524, and thereby allow for an enhanced regulatory response
within both the spleen and the pancreas. At 6 weeks of age NOD mice have anti-insulin
antibodies (26) which is associated with T1D progression and is similar to the Ab profile
for pre-diabetic stage 2 T1D patients (27). Studies in NOD mice (28) and humans (29, 30)

suggest that 40–85% of the β cell mass has been destroyed by onset of diabetes (stage 3 in
Author Manuscript
humans). Therefore, tolerogenic therapies administered prior to complete destruction of the

β cell mass may serve the greatest benefit for patients. Mice were treated with 2.5 mg/dose
of Unloaded CNP or CNP-T1D at 6, 7, and 11 weeks of age to replicate stage 2 disease in
T1D patients, and the level of blood glucose measured bi-weekly. Mice were considered to
have overt diabetes after two consecutive blood glucose readings of >250 mg/dL. As shown
in Figure 2A, treatment with CNP-T1D significantly decreased the number of NOD mice
that developed T1D (1/10) versus CNP alone (9/10) and the effect was durable for at least
13.5 weeks post the third dose. CNP-T1D treatment significantly decreased the percentage
of pancreatic islets that contained >25% immune cells (Figure 2B and C). Approximately
50% of the islets within the pancreas of CNP-T1D treated NOD mice had no detectible
infiltrating immune cells. Additionally, the greatest efficacy of CNP-T1D required 2 or 3
doses (Figure 2D).
Author Manuscript
To better simulate treatment closer to onset of dysglycemia, we next tested CNP-T1D

treatment of NOD mice at 10, 11, and 15 weeks of age. Similar to mice treated at 6, 7,
and 11 weeks of age, this delayed treatment regimen significantly decreased the incidence
of mice developing T1D (Figure 2E) as well as the percentage of pancreatic islets that
contained >25% immune cells (Figure 2F) and the effect was durable for at least 7 weeks
post third dose administration. Collectively these results show that tolerogenic treatment
with CNP-T1D containing multiple diabetogenic CD4+ T cell and CD8+ T cell epitopes
starting at either 6 weeks of age to mimic Stage 2 T1D (prior to onset of clinical symptoms)
or at 10 weeks of age to mimic late Stage 2/early Stage 3 T1D (prior to overt diabetes but
after dysglycemia) significantly prevented T1D development which developed in >90% of
control mice starting at 13 weeks of age.
Author Manuscript
Durable tolerance requires all 3 diabetogenic proteins in CNP-T1D and decreases IFN-γ
and increases IL-10 secretion
As further confirmation for the requirement of all three diabetogenic proteins to
be contained within the CNP-T1D dose, we compared tolerance with the individual
recombinant proteins to CNP-T1D. NOD mice were treated with Unloaded CNP (2.5 mg/
dose), CNP-Insulin (0.83 mg/dose + 1.67 mg/dose of Unloaded CNP), CNP-Chromogranin
A (0.83 mg/dose + 1.67 mg/dose of Unloaded CNP), CNP-GAD65 (0.83 mg/dose + 1.67
mg/dose of Unloaded CNP), or CNP-T1D (2.5 mg/dose) at 6, 7 and 11 weeks of age.
As such, all mice received the same dose of 2.5 mg CNPs, and the amount of each
respective recombinant diabetogenic protein within this dose was constant across respective
treatment groups. Strikingly, the results show that only treatment of NOD mice with CNP-
T1D containing the three diabetogenic proteins significantly inhibited the onset of T1D as
Author Manuscript
compared to nanoparticles encapsulating any one of the individual recombinant proteins

(Figure 3A). In the present experiment, NOD mice treated with CNP-Insulin and CNP-
Chromogranin A exhibited a significant increase in disease incidence as compared to the
unloaded CNP treatment group (Figure 3A). However, this finding varied from experiment
to experiment, as PLG(Insulin) and PLG/PLG did not increase in disease incidence as
presented in Figure 1A.

To confirm the specificity of the various tolerogenic treatments, IFN-γ and IL-10 production
Author Manuscript
were measured from splenocyte ex vivo recall cultures at 9 weeks of age, a time point
at which the mice had received two doses of the indicated CNPs. Total splenocytes from
individual mice were cultured in the presence of OVA323–339, chromogranin A359–372,
insulin A14–29, insulin B9–23, GAD65286–300, GAD65515–524, IGRP206–214, HSP60437–460,
or ZnT8345–359 (20 μg/mL) for 72 hours (Figure 3B–J). The treatment of NOD mice with
CNP-T1D significantly decreased the level of IFN-γ secreted upon splenocyte culture in the
presence of insulin A14–20, insulin B9–23, and IGRP437–460, while also increasing the level
of IL-10 secreted upon splenocyte culture in the presence of insulin A14–20, insulin B9–23,
GAD65286–300, GAD65515–524, and chromogranin A359–372 (Figure 3C–H). In contrast,
CNP-Insulin only decreased the level of IFN-γ secreted upon splenocyte culture in the
presence of insulin A14–20, insulin B9–23, and IGRP437–460. While CNP-GAD65 treatment
increased the level of IL-10 secreted upon splenocyte culture similar to CNP-T1D, CNP-
Author Manuscript
GAD65 treatment did not significantly modulate the level of IFN-γ secreted (Figure 3E).
Lastly, CNP-Chromogranin A significantly decreased the level of IFN-γ secreted upon
splenocyte culture in the presence of chromogranin A359–372 and IGRP437–460 (Figure 3G–
H). Therefore, splenocytes from CNP-T1D treated mice additively presented the functional
changes for splenocytes from mice that had been treated with CNP-Insulin, CNP-GAD65,
or CNP-Chromogranin A alone. These findings indicate that CNP-T1D induced the broadest
overall tolerogenic phenotype in the self-reactive T cells, and this modulation of the Ag-
specific T cell response to T1D-asscoaited peptides correlates with the disease course data
(Figure 3A).
CNP-T1D induces a regulatory phenotype within the spleen, pancreatic lymph nodes, and
pancreas
Author Manuscript
To confirm the ex vivo findings following treatment with CNP-T1D versus treatment with
the individual proteins alone, splenocytes were analyzed by flow cytometry (Figure 4A).
CNP-T1D or CNP-Chromogranin A treatment significantly increased the number of total
CD4+ T cells, macrophages, and monocytes increased within the spleen, as compared
to Unloaded CNP treated mice. When comparing the phenotype of the CD4+ T cells,
macrophages, and monocytes within the spleen, CNP-T1D treatment significantly increased
the number of FoxP3+ and PD-1+ CD4+ T cells (Figure 4B–C), as well as increasing
the number of PD-L1+ and CD206+ macrophages and PD-L1+ monocytes (Figure 4B, E–
F). In contrast, CNP-GAD65 treatment, which also increased the level of secreted IL-10
similar to CNP-T1D treatment (Figure 3C–G), increased the number of IFN-γ+ and IL-17+
CD4+ T cells within the spleen (Figure 4C). Additionally, the finding that CNP-GAD65
and CNP-T1D induced an increase in the level of IL-10 secreted, while not significantly
increasing the number of IL-10+/CD4+ T cells within the spleen suggests that treatment
Author Manuscript
induced an increase in the amount of IL-10 secreted per IL-10+/CD4+ T cell at the 9 weeks
of age time point. These present data correlate with our previously published data in the
BDC2.5 transfer disease into NOD-scid mice (16). Taken together, the data show that while
mice treated with the individual diabetogenic protein containing CNPs may induce some
of the same regulatory alterations as CNP-T1D, it is only the mice that were treated with
CNP-T1D that have the combined effect of decreasing IFN-γ and increasing IL-10.

To further assess the immune cell population differences, flow cytometry analysis of the
Author Manuscript
spleen, pancreatic lymph nodes, and pancreas was carried out at a later stage of disease (12
weeks of age, when controls begin to develop overt T1D) in NOD mice that had been treated
with Unloaded CNP, CNP-T1D or the individual recombinant diabetogenic proteins at 6, 7,
and 11 weeks of age. CNP-T1D treatment increased the number of total immune cells within
the spleen, while significantly decreasing the number of total immune cells within both the
pancreatic lymph nodes and the pancreas (Figure 5A). When compared to the individual
proteins, only CNP-T1D treatment significantly increased the number of CD4+ T cells,
macrophages, and monocytes within the spleen (Figure 5B). The same pattern was found
at 9 weeks of age, which is after only two doses of CNP-T1D (Figure 4B). Additionally,
CNP-T1D significantly decreased the number of CD4+ T cells and CD8+ T cells within the
pancreatic lymph nodes and pancreas (Figure 5C and D). These findings indicate a shift of
immune cells away from the disease-associated target tissue, i.e., the pancreas.
Author Manuscript
We also assessed the phenotype of the CD4+ T cells in the various organs. The percentage
of FoxP3+, CTLA-4+, and PD-1+ CD4+ T cells increased in both the spleen and pancreas
of CNP-T1D treated mice (Figure 5E–G). The present data show that CNP-GAD65 and
CNP-T1D treatment increased the percentage of IL-10+ Treg cells within the spleen,
pancreatic lymph nodes, and pancreas, while CNP-Chromogranin-A only increased the
percentage within the pancreas (Figure 5E–G and Supp. Figure 2). When considered with
the CNP-T1D-induced decrease in the number of total immune cells and CD4+ T cells
within the pancreas as compared to the Unloaded CNP treated NOD mice, this equates to
a significant decrease in the number of IFN-γ+ and IL-17+ CD4+ T cells present within
the pancreata along with an increase in Treg cells. This finding is consistent with the
histopathology scoring data presented in Figure 2C and F. Together, the present findings
suggest that CNP-T1D treatment induces a regulatory phenotype in an Ag-specific manner
Author Manuscript
for multiple T cell specificities, and thereby inhibits the inflammatory immune response
within the pancreas.
Discussion
Previous studies have attempted tolerogenic treatment of T1D by targeting individual
diabetogenic antigens (Ags), with limited success (8, 32–35). Indeed our attempts to
prevent/delay onset of T1D in wildtype NOD mice using PLGA nanoparticles coupled with
or encapsulating a number of dominant natural and hybrid diabetogenic peptide epitopes
(Figure 1A and B) were unsuccessful even though these formulations were previously shown
to robustly inhibit monospecific T1D induced by transfer of transgenic CD4+ T cells and
CD8+ T cells into NOD.scid recipients (16, 18, 36). It is well established that the genesis of
Author Manuscript
T1D in humans and NOD mice is characterized by the presence of autoreactive T cell and
antibody (Ab) responses to multiple pancreatic β cell Ags which arise well before the onset
of overt hyperglycemia (5, 25–27) likely via epitope spreading (6–9). Thus, to determine if
simultaneous tolerogenic targeting of multiple CD4+ T cell and CD8+ T cell diabetogenic
epitopes may be required to block development of T1D in NOD mice, we assessed disease
in mice treated with PLGA nanoparticles encapsulating either recombinant insulin, GAD65
or chromogranin A, alone or in combination (CNP-T1D), beginning at 6 weeks of age.
Indeed, disease was only blocked in mice receiving CNP-T1D, but not in mice receiving

nanoparticles encapsulating any of the individual diabetogenic proteins (Figures 2A–C and
Author Manuscript
3A). This indicates that simultaneous targeting of multiple autoepitopes is required for
efficient disease protection in a multi-determinant autoimmune disease to allow development
of a critical mass of regulatory responses. Further, effective tolerance required 2–3 doses of
CNP-T1D administered at 6, 7 and 12 weeks of age (Figure 2D).
In support of the requirement for multiple diabetogenic peptides to be contained within

the Ag-specific tolerance treatment, e.g., CNP-T1D, we detected low, but significant
and reproducible, pro-inflammatory IFN-γ production by NOD T cells in response to
at least eight different diabetogenic peptide epitopes within six different β cell proteins
(chromogranin, insulin, GAD65, IGRP, HSP60 and ZnT8) by 6 weeks of age (Figure
1C), a time point well prior to development of overt T1D which onsets between 13–16
weeks of age in our vivarium. Additionally, responses to seven of the eight peptides
show a trend toward an increase by 9 weeks of age and no responses were seen in
Author Manuscript
6 week-old T1D-resistant C57BL/6 mice. The breadth of β cell autoepitopes targeted

in NOD mice is perhaps not surprising as epitope spreading has been previously well-
documented in various experimental autoimmune disease models including experimental
autoimmune encephalomyelitis (EAE) (37) and T1D in NOD mice (38, 39). Epitope
spreading can occur to different epitopes within the same self-Ag (intramolecular epitope
spreading) or to epitopes on a different self-Ags (intermolecular epitope spreading) and has
critical implications to tolerance-based immunotherapy as the effectiveness would require
induction of unresponsiveness to multiple autoepitopes in pre-established disease (7).
Relevant to disease therapy and the location of initial autoreactive CD4+ T cell activation,
we have shown in relapsing EAE induced by priming SJL/J mice with PLP178–191/CFA
that naïve CFSE-labeled PLP139–151-specific transgenic T cells which drive the primary
relapse (intramolecular epitope spreading) are initially activated directly within the central
Author Manuscript
nervous system (CNS) target organ and not in the draining cervical lymph nodes or other
peripheral lymphoid organs (40). The spread epitope-specific T cells are activated by
peripherally-derived myeloid dendritic cells (mDCs) which infiltrate the CNS during the
acute disease episode (41). Thus, epitope spreading takes place in a hierarchical order
of peptide dominance and activation of the spread epitope-specific CD4+ T cells occurs
within the inflamed target organ and can be inhibited by tolerance induction to the spread
epitope (23). Therefore, blockade of disease-associated tissue inflammation and destruction
by Ag-specific tolerance may also inhibit the activation of spread epitope-specific T cells
that are responsive to peptides not contained within CNP-T1D. We have also shown that
early life oligodendrocyte ablation using diptheria toxin A (DTA) in the C57BL/6 DTA
mouse model (43) leads to a later life fatal, secondary demyelinating disease driven by
myelin oligodendrocyte glycoprotein (MOG)-specific T cells developing approximately 30
Author Manuscript
weeks after recovering from initial oligodendrocyte loss and demyelination. Importantly,
MOG35–55-specific tolerance induced by MOG35–55-encapsulating PLGA nanoparticles
treatment blocks the progression of this late-onset disease (44). Thus, Ag-specific CNP
treatment can function as a potent therapeutic treatment in chronic autoimmunity induced by
either inflammatory T cells (EAE) or secondary to non-inflammatory cell death (DTA mice)
in the target organ.

In addition to assessing the Ag-specificity of CNP-T1D treatment, our studies demonstrate

Author Manuscript
the durability of CNP-T1D-induced tolerance. Mice treated with CNP-T1D at 6, 7, and

11 weeks of age remained disease free through 25 weeks of age, i.e., at least 13.5 weeks
post third dose administration (Figure 2A). Tolerance durability was assessed by the level
of blood glucose remaining <250 mg/dL and a marked reduction in inflammatory cell
infiltration into the pancreas leading to preserved islet architecture. We have previously
demonstrated the durability of nanoparticle-induced Ag-specific tolerance and its ability
to halt previously established autoimmune disease in myelin peptide/CFA-induced EAE
models utilizing a single peptide to induce CD4+ T cell-mediated autoimmunity (15, 23). In
addition to the ability of CNP-T1D to protect from T1D development in NOD mice treated
at 6 weeks of age, we notably show similar protection in NOD mice treated beginning at
10 weeks of age a time equivalent to stage 2 T1D in humans. For tolerance approaches to
effectively restore insulin function, patients will require a critical mass of functional β cells.
T1D is staged based on presence of β cell-specific Abs and clinical signs of disease: stage
Author Manuscript
1 T1D patients have two or more unique β cell-specific Abs with normoglycemia and no
clinical symptoms; stage 2 T1D patients progress to dysglycemia and no clinical symptoms,
and stage 3 T1D patients progress to dysglycemia and symptomatic disease (45). The
combination of T cell and B cell activation results in β cell loss and inflammatory lesions
within pancreatic islets (insulitis). Studies in NOD mice (28) and humans (29, 30) suggest
that 40–85% of the β cell mass must be destroyed by onset of diabetes (stage 3 in humans).
Therefore, tolerogenic therapies administered prior to complete destruction of the β cell
mass will serve the greatest benefit for patients. The utilization of the present multi-protein
tolerance induction platform may also prove to be efficacious in the preservation of β cell
transplants. If true, then therapy would not be limited to pre-diabetic and early onset T1D
patients, but also useful for T1D patients with late-stage disease.
Author Manuscript
In addition to the profound protection from T1D development, CNP-T1D treatment inhibited
proinflammatory IFN-γ secretion and increased anti-inflammatory IL-10 secretion, as
compared to the control Unloaded CNP treated mice (Figure 3B–J). CNP-T1D treatment
significantly inhibited the secretion of IFN-γ upon culture of splenocytes in the presence of
the primary epitopes contained within CNP-T1D, e.g., insulin, GAD65, and chromogranin
A, as well as the response to a non-targeted bystander epitope on IGRP. Additionally,
CNP-T1D treatment significantly increased the secreted level of the regulatory cytokine,
IL-10, upon culture of splenocytes in all ex vivo activation conditions. These results suggest
that CNP-T1D treatment induced a regulatory phenotype within the spleen. Tolerance thus
appears to be driven by both intrinsic Ag-specific anergy and resultant prevention of T1D by
increasing the numbers of FoxP3+ regulatory CD4+ T (Treg) cells, IL-10 producing CD4+
T cells (Tr1), and PD-1+ T cells in the spleen as well as PD-L1+ and CD206+ macrophages
Author Manuscript
(Figures 4C, 4E, 4F, and 5E) (14). CNP-T1D treatment significantly decreased the number
of both total CD45hi cells, CD4+ T cells, and CD8+ T cells within the pancreas (Figure
5A and 5D) supporting the hypothesis that while not every potential diabetogenic protein
was contained within the CNP-T1D nanoparticle, T cells specific for spread epitopes were
not activated following treatment. This hypothesis is supported by the CNP-T1D-induced
decrease in the level of IFN-γ secreted upon culture of splenocytes in the presence of
IGRP206–214, not contained within CNP-T1D (Figure 1C). Additionally, of the CD4+ T cells

still present within the pancreata of CNP-T1D treatment NOD mice, an increased percentage
of these CD4+ T cells were of a regulatory/tolerogenic phenotype (FoxP3+, CTLA-4+,
Author Manuscript
PD-1+, and IL-10+) (Figure 5G). Likewise, an increased percentage of CD4+ T cells within
the pancreatic lymph nodes of CNP-T1D treated NOD mice were CTLA-4+ (Figure 5F).
These data demonstrate that CNP-T1D induces tolerance to multiple diabetogenic epitopes
in the spontaneous NOD model by inducing regulatory T cell responses. Notably, we have
recently published that protection in NOD.scid recipients of activated transgenic BDC2.5 T
cells induced by tolerization with PLGA nanoparticle encapsulating the chromogranin p31
mimotope peptide, was reversed by both depletion of Treg cells using anti-CD25 (16) and
administration of anti-IL-10 (46).
The major advantage of our nanoparticle tolerance delivery system is that induction and
maintenance of unresponsiveness is due to the activation of autoantigen-specific Treg
cells. The majority of current FDA approved therapies for the treatment of autoimmune
Author Manuscript
disease focus on the reduction of disease symptoms using anti-inflammatory drugs, e.g.,
corticosteroids, or biologicals, e.g., tumor necrosis factor (TNF-α) inhibitors (47). However,
long-term use of these drugs is associated with adverse side effects such as increased
susceptibility to opportunistic infections and tumors (48). Additional T1D therapies in
current development, e.g., anti-CD3, are broadly suppressive and transient and do not treat
the root cause of disease by specifically targeting only autoantigen-specific pathogenic T
cells. Treg cells are recognized to play a central role in the maintenance of peripheral self-
tolerance. Experimental evidence from preclinical animal models has shown that transfer of
natural Treg cells (nTregs) can regulate immune responses to self-Ags (49–51). To date the
translation of in vitro expanded Treg cells for treatment of autoimmune diseases has been
hampered by issues such as production efficiency, stability, and the risks of pan suppression.
Antigen-specific Treg cells have been shown to preferentially suppress Ag-specific CD4+ T
Author Manuscript
cell responses, and to have enhanced disease-associated regulatory function in comparison

to polyclonal Treg cells. For example, similar to other findings (52–54), we have shown
that 10-fold fewer induced PLP139–151-specific Treg cells (2×105) (55) were sufficient to
suppress PLP139–151-induced EAE in SJL/J mice as compared with polyclonal natural Treg
cells where a minimum of 2×106 cells were required (49).
Immune tolerance to circulating Ags and cells is carried out in the spleen and liver (56,
57), as these organs filter the blood and contain phagocytic myeloid cells which evolved to
clear and dispose of the large number of apoptotic cells generated by normal cell turnover in
the hematopoietic system. Efferocytosis is the process by which dead, dying, and apoptotic
cells are cleared from the blood, and this process is vital for the maintenance of immune
homeostasis. It has been hypothesized that dysregulation of this process may be involved in
Author Manuscript
several autoimmune disease states (58, 59). We have previously shown that Ag-encapsulated
carboxylated PLGA nanoparticles serve as surrogates for apoptotic bodies and following
intravenous infusion are specifically uptaken by APCs in the splenic marginal zone and
liver via the macrophage receptor with collagenous structure (MARCO) scavenger receptor
(60) which we have shown is necessary for induction of carboxylated PLGA nanoparticle
tolerance (15). MARCO has been shown to be responsible for uptake of apoptotic cells
(61) and negatively charged nanoparticles (62). Following MARCO engagement, the APCs
upregulate PD-L1 and secrete IL-10 and TGFβ and the individual encapsulated diabetogenic

proteins within CNP-T1D are processed and multiple epitopes from each protein are
Author Manuscript
presented to both activated and naïve Ag-specific T cells in the tolerogenic environment
resulting in anergy induction (23) and the activation of iTreg cells and Tr1 cells (14). The
Treg cells then inhibit the autoimmune T cell responses restoring overall immunologic
homeostasis.
In summary, CNP-T1D treatment effectively and efficiently prevents induction and

progression of spontaneous T1D in NOD mice preventing autoimmune destruction of
insulin-producing pancreatic β cells in an Ag-specific manner. Compared to other tolerance
inducing approaches tested in NOD mice (63–66), CNP-T1D- induced tolerance requires
fewer treatments and generates more durable protection. Mechanistically, CNP-T1D induces
the activation of Treg cells and Tr1 cells which regulate diabetogenic T cell responses
to both epitopes contained within the encapsulated β cell proteins as well as bystander
suppression, i.e., blocking IFN-γ production by diabetogenic T cells specific for non-
Author Manuscript
targeted Ags (IGRP and ZnT8). CNP-T1D appears to be a highly promising approach
for translation to testing in human T1D in light of the ease of GMP production of
Ag-encapsulating PLGA nanoparticles and the demonstrated success of this Ag delivery
platform in inducing gliadin-specific tolerance in a phase 1/2a human celiac disease clinical
trial (20).
Supplementary Material
Refer to Web version on PubMed Central for supplementary material.
Acknowledgements:
We gratefully acknowledge the Northwestern Mouse Histopathology & Phenotyping Core for their services. We
Author Manuscript
acknowledge the support from all members of the Miller laboratory.
Funding:
This research was supported by grants to S.D.M. from COUR Pharmaceutical Development Company, Inc.; the
National Institutes of Health (R01 AI155678), the Juvenile Diabetes Foundation (2-SRA-2018-566-S-B), the David
and Amy Fulton Foundation, and the Cramer Family Foundation. T.N. was supported by JDRF Postdoctoral
Fellowship 3-PDF-2018-582-A-N.
Abbreviations:
Ab antibody
Ag antigen
APCs antigen-presenting cells

Author Manuscript
CNP-T1D COUR nanoparticles encapsulating insulin, chromogranin A and

GAD65
EAE experimental autoimmune encephalomyelitis
CNS central nervous system
ECDI ethylene carbodiimide

GAD65 glutamic acid decarboxylase 65: IA-2islet antigen-2

Author Manuscript
IGRP islet specific glucose 6 phosphate catalytic subunit related protein
IMP immune modifying nanoparticle
MARCO macrophage receptor with collagenous structure
MOG myelin oligodendrocyte glycoprotein
NP nanoparticles
PLGA poly(lactic-co-glycolic acid)
T1D type 1 diabetes
TIMP tolerance inducing microparticles

Author Manuscript
Treg cell regulatory T cells
ZnT8 zinc transporter 8
References
1. 2020. National Diabetes Statistics Report, 2020. Centers for Disease Control and Prevention, U.S.
Dept of Health and Human Services, Atlanta, GA.
2. Mobasseri M, Shirmohammadi M, Amiri T, Vahed N, Hosseini Fard H, and Ghojazadeh M. 2020.
Prevalence and incidence of type 1 diabetes in the world: a systematic review and meta-analysis.
Health Promot. Perspect 10: 98–115. [PubMed: 32296622]
3. Pathak V, Pathak NM, O’Neill CL, Guduric-Fuchs J, and Medina RJ. 2019. Therapies for type 1
diabetes: Current scenario and future perspectives. Clin. Med. Insights Endocrinol. Diabetes 12:
1179551419844521.
Author Manuscript
4. Nakayama M, Abiru N, Moriyama H, Babaya N, Liu E, Miao D, Yu L, Wegmann DR, Hutton JC,
Elliott JF, and Eisenbarth GS. 2005. Prime role for an insulin epitope in the development of type 1
diabetes in NOD mice. Nature 435: 220–223. [PubMed: 15889095]
5. Di Lorenzo TP, Peakman M, and Roep BO. 2007. Translational mini-review series on type 1
diabetes: Systematic analysis of T cell epitopes in autoimmune diabetes. Clin. Exp. Immunol 148:
1–16. [PubMed: 17349009]
6. Culina S, Boitard C, and Mallone R. 2011. Antigen-based immune therapeutics for type 1 diabetes:
magic bullets or ordinary blanks? Clin. Dev. Immunol 2011;2011:286248: 286248. [PubMed:
21647401]
7. Vanderlugt CL, and Miller SD. 2002. Epitope spreading in immune-mediated diseases: implications
for immunotherapy. Nat. Rev. Immunol 2: 85–95. [PubMed: 11910899]
8. Luo X, Herold KC, and Miller SD. 2010. Immunotherapy of type 1 diabetes: where are we and
where should we be going? Immunity 32: 488–499. [PubMed: 20412759]
9. Prasad S, Kohm AP, McMahon JS, Luo X, and Miller SD. 2012. Pathogenesis of NOD diabetes is
Author Manuscript
initiated by reactivity to the insulin B chain 9–23 epitope and involves functional epitope spreading.
J. Autoimm 39: 347–353.
10. Stadinski BD, Delong T, Reisdorph N, Reisdorph R, Powell RL, Armstrong M, Piganelli JD,
Barbour G, Bradley B, Crawford F, Marrack P, Mahata SK, Kappler JW, and Haskins K. 2010.
Chromogranin A is an autoantigen in type 1 diabetes. Nat. Immunol 11: 225–231. [PubMed:
20139986]
11. Wenzlau JM, Juhl K, Yu L, Moua O, Sarkar SA, Gottlieb P, Rewers M, Eisenbarth GS, Jensen
J, Davidson HW, and Hutton JC. 2007. The cation efflux transporter ZnT8 (Slc30A8) is a major

autoantigen in human type 1 diabetes. Proc. Natl. Acad. Sci. USA 104: 17040–17045. [PubMed:
17942684]
Author Manuscript
12. Nikoopour E, Sandrock C, Huszarik K, Krougly O, Lee-Chan E, Masteller EL, Bluestone JA, and
Singh B. 2011. Cutting edge: vasostatin-1-derived peptide ChgA29–42 is an antigenic epitope of
diabetogenic BDC2.5 T cells in nonobese diabetic mice. J. Immunol 186: 3831–3835. [PubMed:
21357258]
13. Miller SD, Turley DM, and Podojil JR. 2007. Antigen-specific tolerance strategies for the
prevention and treatment of autoimmune disease. Nat. Rev. Immunol 7: 665–677. [PubMed:
17690713]
14. Pearson RM, Podojil JR, Shea LD, King NJC, Miller SD, and Getts DR. 2019.
Overcoming challenges in treating autoimmuntity: Development of tolerogenic immune-modifying
nanoparticles. Nanomed 18: 282–291.
15. Getts DR, Martin AJ, McCarthy DP, Terry RL, Hunter ZN, Yap WT, Getts MT, Pleiss M, Luo X,
King NJ, Shea LD, and Miller SD. 2012. Microparticles bearing encephalitogenic peptides induce
T-cell tolerance and ameliorate experimental autoimmune encephalomyelitis. Nat. Biotechnol 30:
1217–1224. [PubMed: 23159881]
Author Manuscript
16. Prasad S, Neef T, Xu D, Podojil JR, Getts DR, Shea LD, and Miller SD. 2018. Tolerogenic
Ag-PLG nanoparticles induce Tregs to suppress activated diabetogenic CD4 and CD8 T cells. J.
Autoimmun 89: 112–124. [PubMed: 29258717]
17. Freitag TL, Podojil JR, Pearson RM, Fokta FJ, Sahl C, Messing M, Andersson LC, Leskinen
K, Saavalainen P, Hoover LI, Huang K, Phippard D, Maleki S, King NJC, Shea LD, Miller SD,
Meri SK, and Getts DR. 2020. Gliadin nanoparticles induce immune tolerance to gliadin in mouse
models of celiac disease. Gastroenterol 158: 1667–1681.e1612.
18. Jamison BL, Neef T, Goodspeed A, Bradley B, Baker RL, Miller SD, and Haskins K. 2019.
Nanoparticles containing an insulin-ChgA hybrid peptide protect from transfer of autoimmune
diabetes by shifting the balance between effector T cells and regulatory T cells. J. Immunol 203:
48–57. [PubMed: 31109955]
19. Smarr CB, Hsu CL, Byrne AJ, Miller SD, and Bryce PJ. 2011. Antigen-fixed leukocytes tolerize
Th2 responses in mouse models of allergy. J. Immunol 187: 5090–5098. [PubMed: 21976774]
20. Kelly CP, Murray JA, Leffler DA, Getts DR, Bledsoe AC, Smithson G, First MR, Morris A, Boyne
M, Elhofy A, Wu TT, Podojil JR, Miller SD, and Group TAKS. 2021. TAK-101 nanoparticles
Author Manuscript
induce gluten-specific tolerance in celiac disease: A randomized, double-blind, placebo-controlled

study. Gastroenterol 161: 66–80 e68.
21. Thayer TC, Wilson SB, and Mathews CE. 2010. Use of nonobese diabetic mice to understand
human type 1 diabetes. Endocrinol. Metab. Clin. North Am 39: 541–561. [PubMed: 20723819]
22. Smarr CB, Yap WT, Neef TP, Pearson RM, Hunter ZN, Ifergan I, Getts DR, Bryce PJ, Shea LD,
and Miller SD. 2016. Biodegradable antigen-associated PLG nanoparticles tolerize Th2-mediated
allergic airway inflammation pre- and postsensitization. Proc. Natl. Acad. Sci. USA 113: 5059–
5064. [PubMed: 27091976]
23. Hunter Z, McCarthy DP, Yap WT, Harp CT, Getts DR, Shea LD, and Miller SD. 2014. A
biodegradable nanoparticle platform for the induction of antigen-specific immune tolerance for
treatment of autoimmune disease. ACS Nano 8: 2148–2160. [PubMed: 24559284]
24. Carrero JA, Calderon B, Towfic F, Artyomov MN, and Unanue ER. 2013. Defining the
transcriptional and cellular landscape of type 1 diabetes in the NOD mouse. PLoS One 8: e59701.
[PubMed: 23555752]
Author Manuscript
25. Lo B, Swafford AD, Shafer-Weaver KA, Jerome LF, Rakhlin L, Mathern DR, Callahan CA, Jiang
P, Davison LJ, Stevens HE, Lucas CL, White J, von Borstel R, Todd JA, and Lenardo MJ. 2011.
Antibodies against insulin measured by electrochemiluminescence predicts insulitis severity and
disease onset in non-obese diabetic mice and can distinguish human type 1 diabetes status. J.
Transl. Med 9:203.doi:10.1186/1479-5876-9-203. [PubMed: 22123298]
26. Trembleau S, Penna G, Gregori S, Magistrelli G, Isacchi A, and Adorini L. 2000. Early
Th1 response in unprimed nonobese diabetic mice to the tyrosine phosphatase-like insulinoma-
associated protein 2, an autoantigen in type 1 diabetes. J. Immunol 165: 6748–6755. [PubMed:
11120794]

27. Tian J, Gregori S, Adorini L, and Kaufman DL. 2001. The frequency of high avidity T cells
determines the hierarchy of determinant spreading. J. Immunol 166: 7144–7150. [PubMed:
Author Manuscript
11390460]
28. Sreenan S, Pick AJ, Levisetti M, Baldwin AC, Pugh W, and Polonsky KS. 1999. Increased
beta-cell proliferation and reduced mass before diabetes onset in the nonobese diabetic mouse.
Diabetes 48: 989–996. [PubMed: 10331402]
29. Foulis AK, Liddle CN, Farquharson MA, Richmond JA, and Weir RS. 1986. The histopathology of
the pancreas in type 1 (insulin-dependent) diabetes mellitus: a 25-year review of deaths in patients
under 20 years of age in the United Kingdom. Diabetologia 29: 267–274. [PubMed: 3522324]
30. Klinke DJ 2nd. 2008. Extent of beta cell destruction is important but insufficient to predict the
onset of type 1 diabetes mellitus. PLoS One 3: e1374. [PubMed: 18167535]
31. Patterson SJ, Pesenacker AM, Wang AY, Gillies J, Mojibian M, Morishita K, Tan R, Kieffer TJ,
Verchere CB, Panagiotopoulos C, and Levings MK. 2016. T regulatory cell chemokine production
mediates pathogenic T cell attraction and suppression. J. Clin. Invest 126: 1039–1051. [PubMed:
26854929]
32. Robert S, Gysemans C, Takiishi T, Korf H, Spagnuolo I, Sebastiani G, Van Huynegem K,
Author Manuscript
Steidler L, Caluwaerts S, Demetter P, Wasserfall CH, Atkinson MA, Dotta F, Rottiers P, Van
Belle TL, and Mathieu C. 2014. Oral delivery of glutamic acid decarboxylase (GAD)-65 and
IL10 by Lactococcus lactis reverses diabetes in recent-onset NOD mice. Diabetes 63: 2876–2887.
[PubMed: 24677716]
33. Yamamoto T, Yamato E, Tashiro F, Sato T, Noso S, Ikegami H, Tamura S, Yanagawa Y, and
Miyazaki JI. 2004. Development of autoimmune diabetes in glutamic acid decarboxylase 65
(GAD65) knockout NOD mice. Diabetologia 47: 221–224. [PubMed: 14676944]
34. Brezar V, Culina S, Gagnerault MC, and Mallone R. 2012. Short-term subcutaneous insulin
treatment delays but does not prevent diabetes in NOD mice. Eur. J. Immunol 42: 1553–1561.
[PubMed: 22678909]
35. Jhala G, Selck C, Chee J, Kwong CJ, Pappas EG, Thomas HE, Kay TWH, and Krishnamurthy B.
2021. Tolerance to proinsulin-1 reduces autoimmune diabetes in NOD mice. Front. Immunol 12:
645817. [PubMed: 33841427]
36. Jamison BL, DiLisio JE, Beard KS, Neef T, Bradley B, Goodman J, Gill RG, Miller SD, Baker
RL, and Haskins K. 2022. Tolerogenic delivery of a hybrid insulin peptide markedly prolongs
Author Manuscript
islet graft survival in the non-obese diabetic (NOD) mouse. Diabetes 71: 483–496. [PubMed:
35007324]
37. McRae BL, Vanderlugt CL, Dal Canto MC, and Miller SD. 1995. Functional evidence for epitope
spreading in the relapsing pathology of experimental autoimmune encephalomyelitis. J. Exp. Med
182: 75–85. [PubMed: 7540658]
38. Kaufman DL, Clare-Salzler M, Tian J, Forsthuber T, Ting GS, Robinson P, Atkinson MA, Sercarz
EE, Tobin AJ, and Lehmann PV. 1993. Spontaneous loss of T-cell tolerance to glutamic acid
decarboxylase in murine insulin-dependent diabetes. Nature 366: 69–72. [PubMed: 7694152]
39. Tisch R, Yang XD, Singer SM, Liblau RS, Fugger L, and McDevitt HO. 1993. Immune response
to glutamic acid decarboxylase correlates with insulitis in non-obese diabetic mice. Nature 366:
72–75. [PubMed: 8232539]
40. McMahon EJ, Bailey SL, Castenada CV, Waldner H, and Miller SD. 2005. Epitope spreading
initiates in the CNS in two mouse models of multiple sclerosis. Nat. Med 11: 335–339. [PubMed:
15735651]
Author Manuscript
41. Bailey SL, Schreiner B, McMahon EJ, and Miller SD. 2007. CNS myeloid DCs presenting
endogenous myelin peptides ‘preferentially’ polarize CD4+ T(H)-17 cells in relapsing EAE. Nat.
Immunol 8: 172–180. [PubMed: 17206145]
42. Munz C, Lunemann JD, Getts MT, and Miller SD. 2009. Antiviral immune responses: triggers of
or triggered by autoimmunity? Nat. Rev. Immunol 9: 246–258. [PubMed: 19319143]
43. Traka M, Arasi K, Avila RL, Podojil JR, Christakos A, Miller SD, Soliven B, and Popko B. 2010.
A genetic mouse model of adult-onset, pervasive central nervous system demyelination with robust
remyelination. Brain 133: 3017–3029. [PubMed: 20851998]

44. Traka M, Podojil JR, McCarthy DP, Miller SD, and Popko B. 2016. Oligodendrocyte death results
in immune-mediated CNS demyelination. Nat. Neurosci 19: 65–74. [PubMed: 26656646]
Author Manuscript
45. Insel RA, Dunne JL, Atkinson MA, Chiang JL, Dabelea D, Gottlieb PA, Greenbaum CJ, Herold
KC, Krischer JP, Lernmark A, Ratner RE, Rewers MJ, Schatz DA, Skyler JS, Sosenko JM,
and Ziegler AG. 2015. Staging presymptomatic type 1 diabetes: a scientific statement of JDRF,
the Endocrine Society, and the American Diabetes Association. Diabetes Care 38: 1964–1974.
[PubMed: 26404926]
46. Neef T, Ifergan I, Beddow S, Penaloza-MacMaster P, Haskins K, Shea LD, Podojil JR, and
Miller SD. 2021. Tolerance induced by antigen-loaded PLG nanoparticles affects the phenotype
and trafficking of transgenic CD4(+) and CD8(+) T cells. Cells 10:3445. 10.3390/cells10123445
[PubMed: 34943952]
47. Kerschbaumer A, Sepriano A, Smolen JS, van der Heijde D, Dougados M, van Vollenhoven R,
McInnes IB, Bijlsma JWJ, Burmester GR, de Wit M, Falzon L, and Landewe R. 2020. Efficacy
of pharmacological treatment in rheumatoid arthritis: a systematic literature research informing
the 2019 update of the EULAR recommendations for management of rheumatoid arthritis. Ann.
Rheum. Dis 79: 744–759. [PubMed: 32033937]
Author Manuscript
48. Wang W, Zhou H, and Liu L. 2018. Side effects of methotrexate therapy for rheumatoid arthritis: A
systematic review. Eur. J. Med. Chem 158: 502–516. [PubMed: 30243154]
49. Kohm AP, Carpentier PA, Anger HA, and Miller SD. 2002. Cutting edge: CD4+CD25+ regulatory
T cells suppress antigen-specific autoreactive immune responses and central nervous system
inflammation during active experimental autoimmune encephalomyelitis. J. Immunol 169: 4712–
4716. [PubMed: 12391178]
50. Liu H, Hu B, Xu D, and Liew FY. 2003. CD4+CD25+ regulatory T cells cure murine colitis: the
role of IL-10, TGF-beta, and CTLA4. J. Immunol 171: 5012–5017. [PubMed: 14607897]
51. DiPaolo RJ, Glass DD, Bijwaard KE, and Shevach EM. 2005. CD4+CD25+ T cells prevent the
development of organ-specific autoimmune disease by inhibiting the differentiation of autoreactive
effector T cells. J. Immunol 175: 7135–7142. [PubMed: 16301616]
52. Tang Q, Henriksen KJ, Bi M, Finger EB, Szot G, Ye J, Masteller EL, McDevitt H, Bonyhadi
M, and Bluestone JA. 2004. In vitro-expanded antigen-specific regulatory T cells suppress
autoimmune diabetes. J. Exp. Med 199: 1455–1465. [PubMed: 15184499]
53. Tarbell KV, Yamazaki S, Olson K, Toy P, and Steinman RM. 2004. CD25+ CD4+ T cells,
Author Manuscript
expanded with dendritic cells presenting a single autoantigenic peptide, suppress autoimmune
diabetes. J. Exp. Med 199: 1467–1477. [PubMed: 15184500]
54. DiPaolo RJ, Brinster C, Davidson TS, Andersson J, Glass D, and Shevach EM. 2007. Autoantigen-
specific TGFbeta-induced Foxp3+ regulatory T cells prevent autoimmunity by inhibiting dendritic
cells from activating autoreactive T cells. J. Immunol 179: 4685–4693. [PubMed: 17878367]
55. Zhang H, Podojil JR, Chang J, Luo X, and Miller SD. 2010. TGF-beta-induced myelin peptide-
specific regulatory T cells mediate antigen-specific suppression of induction of experimental
autoimmune encephalomyelitis. J. Immunol 184: 6629–6636. [PubMed: 20483764]
56. Tiegs G, and Lohse AW. 2010. Immune tolerance: what is unique about the liver. J. Autoimmun 34:
1–6. [PubMed: 19717280]
57. Hosseini H, Li Y, Kanellakis P, Tay C, Cao A, Tipping P, Bobik A, Toh BH, and Kyaw
T. 2015. Phosphatidylserine liposomes mimic apoptotic cells to attenuate atherosclerosis by
expanding polyreactive IgM producing B1a lymphocytes. Cardiovasc. Res 106: 443–452.
[PubMed: 25681396]
Author Manuscript
58. Abdolmaleki F, Farahani N, Gheibi Hayat SM, Pirro M, Bianconi V, Barreto GE, and Sahebkar
A. 2018. The role of efferocytosis in autoimmune diseases. Front. Immunol 9: 1645. [PubMed:
30083153]
59. Doran AC, Yurdagul A Jr., and Tabas I. 2020. Efferocytosis in health and disease. Nat. Rev.
Immunol 20: 254–267. [PubMed: 31822793]
60. Jing J, Yang IV, Hui L, Patel JA, Evans CM, Prikeris R, Kobzik L, O’Connor BP, and Schwartz
DA. 2013. Role of macrophage receptor with collagenous structure in innate immune tolerance. J.
Immunol 190: 6360–6367. [PubMed: 23667110]

61. Allen JE, and Ruckerl D. 2017. The silent undertakers: Macrophages programmed for
efferocytosis. Immunity 47: 810–812. [PubMed: 29166582]
Author Manuscript
62. Kanno S, Furuyama A, and Hirano S. 2007. A murine scavenger receptor MARCO recognizes
polystyrene nanoparticles. Toxicol. Sci 97: 398–406. [PubMed: 17361018]
63. Pagni PP, Chaplin J, Wijaranakula M, Wesley JD, Granger J, Cracraft J, O’Brien C, Perdue N,
Kumar V, Li S, Ratliff SS, Roach A, Misquith A, Chan CL, Coppieters K, and von Herrath M.
2021. Multicomponent plasmid protects mice from spontaneous autoimmune diabetes. Diabetes
71: 157–169.
64. Yoon YM, Lewis JS, Carstens MR, Campbell-Thompson M, Wasserfall CH, Atkinson MA, and
Keselowsky BG. 2015. A combination hydrogel microparticle-based vaccine prevents type 1
diabetes in non-obese diabetic mice. Sci. Rep 5: 13155. [PubMed: 26279095]
65. Lee JS, Han P, Chaudhury R, Khan S, Bickerton S, McHugh MD, Park HB, Siefert AL, Rea G,
Carballido JM, Horwitz DA, Criscione J, Perica K, Samstein R, Rageb R, Kim D, and Fahmy
TM. 2021. Metabolic and immunomodulatory control of type 1 diabetes via orally delivered
bile-acid-polymer nanocarriers of insulin or rapamycin. Nat. Biomed. Eng 5: 983–997. [PubMed:
34616050]
Author Manuscript
66. Alleva DG, Rezaee M, Yip L, Ren G, Rosenberg J, Concepcion W, Escher A, Shabahang S,
and Thakor AS. 2020. Reversal of hyperglycemia and suppression of type 1 diabetes in the
NOD mouse with apoptotic DNA immunotherapy (ADi), ADi-100. Biomedicines 4;8(3):53. doi:
10.3390/biomedicines8030053.
Author Manuscript
Author Manuscript

Key Findings
Author Manuscript
1. T1D is initiated and driven by multiple diabetogenic CD4+ T cell epitopes.
2. Tolerance to individual epitopes/proteins fails to inhibit T1D development.
3. Tolerance to a cocktail of diabetogenic proteins inhibits T1D development.

Author Manuscript
Author Manuscript
Author Manuscript

Author Manuscript
Author Manuscript
Figure 1: Various diabetogenic peptides induce IFN-γ secretion by splenocytes from NOD mice.
Author Manuscript
(A) Female NOD mice (n=12) were treated with the indicated PLGA particles (2.5 mg/dose)
at 5, 7, 9, and 11 weeks of age. (B) Female NOD mice (n=20) were treated with the
indicated PLGA particles (2.5 mg/dose) at 10, 12, and 14 weeks of age. For A and B,
blood glucose levels were monitored weekly, and mice were scored as diabetic after two
consecutive blood glucose reading >250 mg/dL. The data are presented as the percentage
of mice that were normoglycemic over time. (C) Splenocytes (1×106 cells/well) from 6
week-old NOD, 9 week-old NOD, and 6 week-old C57BL/6 mice (n=3) were cultured in
the presence of anti-CD3 (1 μg/mL), OVA323–339, or the indicated diabetogenic peptides (20
μg/mL). Culture supernatants were collected at 72 hours, and the level of secreted IFN-γ
measured. The data are presented as the mean pg/mL of IFN-γ +/− S.E.M. secreted within
the respective cultures. The calculated p-values are listed above the experimental group
comparisons. Data are representative of one experiment of two individual experiments.
Author Manuscript

Author Manuscript
Author Manuscript
Author Manuscript
Figure 2: CNP-T1D prevents onset and progression of spontaneous T1D and immune cell
infiltration into the pancreatic islets of NOD mice.
(A and E) Female NOD mice (n=10) were treated with either Unloaded CNP or CNP-T1D
(2.5 mg/dose) at 6, 7, and 11 weeks of age (A) or at 10, 11, and 15 weeks of age (E).
Blood glucose levels were monitored bi-weekly, and mice were scored as diabetic after two
consecutive blood glucose reading >250 mg/dL. The data are presented as the percentage
Author Manuscript
of mice that were normoglycemic over time. (B, C, and F) Hematoxylin and eosin (H&E)
staining of pancreata from Unloaded CNP and CNP-T1D treated mice were scored for
the level of immune cell infiltrate in pancreatic islets (B). The data are presented as the
percentage of islets that contained no immune cells, immune cells around the edge of the
islet (peri-insulitis), or the percentage of the islet that contained immune cell infiltration (%
infiltration) +/− S.E.M. (C, and F). (D) Female NOD mice (n=10) were treated at 6, 7, and
11 weeks of age with either Unloaded CNP (3 total doses), 1 dose CNP-T1D plus 2 doses of
Unloaded CNP, 2 doses of CNP-T1D plus 1 dose of Unloaded CNP, or 3 doses of CNP-T1D

(2.5 mg/dose) at 6, 7, and 11 weeks of age. Blood glucose levels were monitored bi-weekly,
Author Manuscript
and mice were scored as diabetic after two consecutive blood glucose reading >250 mg/dL.
The data are presented as the percentage of mice that were normoglycemic over time.
Asterisks (*, **, ***, ****) indicate a statistically significant difference as compared to
Unloaded CNP treated mice, p < 0.05, < 0.01, < 0.001, and < 0.0001 respectively. Data are
representative of one experiment of two individual experiments.
Author Manuscript
Author Manuscript
Author Manuscript

Author Manuscript
Author Manuscript
Figure 3: Effective tolerogenic treatment of spontaneous T1D in NOD mice requires targeting
Author Manuscript
of multiple β cell autoantigens and induces an Ag-specific decrease in the level of IFN-γ and
increase in the level of IL-10 secreted by NOD splenocytes.
(A) Female NOD mice (n=10) were treated with either Unloaded CNP, CNP-Insulin, CNP-
GAD65, CNP-Chromogranin A, or CNP-T1D (2.5 mg/dose) at 6, 7, and 11 weeks of age.
Blood glucose levels were monitored bi-weekly, and mice were scored as diabetic after two
consecutive blood glucose reading >250 mg/dL. The data are presented as the percentage of
mice that were normoglycemic over time. (B) At 9 weeks of age spleens were collected from
a cohort of treated mice, and splenocytes (1×106 cells/well) were cultured in the presence
of the indicated peptides (20 μg/mL). The data are presented as the ratio of IFN-γ (pg/mL)
to IL-10 secreted (pg/mL) (C-J) The specific values for the level of IFN-γ and IL-10 are
presented for the respective peptide stimuli. Culture supernatants were collected at 72 hours,
and the level of secreted IFN-γ and IL-10 measured. The data are presented as the mean
pg/mL of IFN-γ and IL-10 ± S.E.M. secreted within the respective cultures. Asterisks (*,
Author Manuscript
**, ***, ****) indicate a statistically significant difference as compared to Unloaded CNP
treated mice, p < 0.05, < 0.01, < 0.001, and < 0.0001 respectively. Data are representative of
one experiment of two individual experiments.

Author Manuscript
Author Manuscript
Author Manuscript
Figure 4: CNP-T1D increases the number of FoxP3+ CD4+ T cells and regulatory APCs present
within the spleen.
Female NOD mice (n=5) were treated with either Unloaded CNP, CNP-Insulin, CNP-
GAD65, CNP-Chromogranin A, or CNP-T1D (2.5 mg/dose) at 6 and 7 weeks of age,
and splenocytes were analyzed by flow cytometry. (A) Representative flow plots and gating
scheme (singlets -> live cells ->) to get to the CD4+ T cells, CD8+ T cells, macrophages,
and monocytes is presented. (B) The number of the various immune cell populations within
the spleen is presented. (C) The number of CD4+ T cells expressing FoxP3, IL-10, PD-1,
IFN-γ, and IL-17 is presented. (D) The number of CD8+ T cells expressing CD122, PD-1,
IFN-γ, and granzyme is presented. (E and F) The number of macrophages and monocytes
expressing PD-L1, CD206, and CD80 is presented. The data are presented as the mean cell
number ± SEM. Asterisks (*, **, ***, ****) indicate a statistically significant difference
Author Manuscript
as compared to Unloaded CNP treated mice, p < 0.05, < 0.01, < 0.001, and < 0.0001
respectively. Data are representative of one experiment of two individual experiments.

Author Manuscript
Author Manuscript
Author Manuscript
Figure 5: CNP-T1D increases the percentage of regulatory CD4+ T cells within the spleen,
pancreatic lymph nodes, and pancreas.
Female NOD mice (n=5) were treated with either Unloaded CNP, CNP-Insulin, CNP-
GAD65, CNP-Chromogranin A, or CNP-T1D (2.5 mg/dose) at 6, 7, and 11 weeks of age.
At 12 weeks of age spleens, pancreatic lymph nodes, and pancreata were collected for
flow cytometry. (A) The total number of CD45hi cells were calculated for each tissue, as
well as the number +/− S.E.M. of CD4+ T cells, CD8+ T cells, monocytes, macrophages,
and neutrophils within the (B) spleen, (C) pancreatic lymph nodes, and (D) pancreas. The
percentage of CD4+ T cells that expressed FoxP3, CTLA4, PD-1, IL-10, CCL3, IFN-γ, or
Author Manuscript
IL-17, as well as the percentage of FoxP3+/CD4+ T cells that expressed CTLA-4, PD-1,
and IL-10 is presented in heatmaps for the (E) spleen, (F) pancreatic lymph nodes, and
(G) pancreas. Asterisks (*, **, ***, ****) indicate a statistically significant difference
as compared to Unloaded CNP treated mice, p < 0.05, < 0.01, < 0.001, and < 0.0001
respectively. Data are representative of one experiment of two individual experiments.

Art Seminario 25 - 08 - 23

Uploaded by

Document Information

Original Description:

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Art Seminario 25 - 08 - 23

Uploaded by

Copyright:

Available Formats

HHS Public Access

Published in final edited form as:

Tolerogenic Immune Modifying Nanoparticles Encapsulating

Chicago, IL, USA;

disease control (6, 8, 9).

Biodegradable carboxylated PLGA nanoparticles containing encapsulated Ag [tolerance-

J Immunol. Author manuscript; available in PMC 2023 August 01.

With regards to clinical translation, CNP nanoparticles have demonstrated efficacy to

human clinical studies with TAK-101/TIMP-GLIA and demonstrated successful induction

In the present study we sought to address both a fundamental immunological question

chromogranin A proteins known to be potential targets in TID were encapsulated within

J Immunol. Author manuscript; available in PMC 2023 August 01.

Materials and Methods

University of Colorado. See Supp. Table 1 for peptide sequences.

Manufacture of CNP nanoparticles (PLGA nanoparticles)

NOD mice, blood glucose monitoring, and treatment

J Immunol. Author manuscript; available in PMC 2023 August 01.

Pathology Scoring of Pancreata

Tissue processing and flow cytometry

J Immunol. Author manuscript; available in PMC 2023 August 01.

this tolerance therapy to be effective. We previously showed that treatment of NOD.scid

CNP-T1D treatment inhibits the development of T1D in NOD mice

T1D consisted of 0.83 mg of CNP-Insulin, 0.83 mg of CNP-GAD65, 0.83 mg of

J Immunol. Author manuscript; available in PMC 2023 August 01.

humans). Therefore, tolerogenic therapies administered prior to complete destruction of the

To better simulate treatment closer to onset of dysglycemia, we next tested CNP-T1D

compared to nanoparticles encapsulating any one of the individual recombinant proteins

J Immunol. Author manuscript; available in PMC 2023 August 01.

J Immunol. Author manuscript; available in PMC 2023 August 01.

J Immunol. Author manuscript; available in PMC 2023 August 01.

In support of the requirement for multiple diabetogenic peptides to be contained within

6 week-old T1D-resistant C57BL/6 mice. The breadth of β cell autoepitopes targeted

J Immunol. Author manuscript; available in PMC 2023 August 01.

In addition to assessing the Ag-specificity of CNP-T1D treatment, our studies demonstrate

the durability of CNP-T1D-induced tolerance. Mice treated with CNP-T1D at 6, 7, and

J Immunol. Author manuscript; available in PMC 2023 August 01.

cell responses, and to have enhanced disease-associated regulatory function in comparison

J Immunol. Author manuscript; available in PMC 2023 August 01.

In summary, CNP-T1D treatment effectively and efficiently prevents induction and

acknowledge the support from all members of the Miller laboratory.

APCs antigen-presenting cells

CNP-T1D COUR nanoparticles encapsulating insulin, chromogranin A and

EAE experimental autoimmune encephalomyelitis

CNS central nervous system

ECDI ethylene carbodiimide

J Immunol. Author manuscript; available in PMC 2023 August 01.

GAD65 glutamic acid decarboxylase 65: IA-2islet antigen-2

IGRP islet specific glucose 6 phosphate catalytic subunit related protein

IMP immune modifying nanoparticle

MARCO macrophage receptor with collagenous structure

MOG myelin oligodendrocyte glycoprotein

PLGA poly(lactic-co-glycolic acid)

T1D type 1 diabetes

TIMP tolerance inducing microparticles

Treg cell regulatory T cells

ZnT8 zinc transporter 8

J Immunol. Author manuscript; available in PMC 2023 August 01.

induce gluten-specific tolerance in celiac disease: A randomized, double-blind, placebo-controlled

J Immunol. Author manuscript; available in PMC 2023 August 01.

J Immunol. Author manuscript; available in PMC 2023 August 01.

J Immunol. Author manuscript; available in PMC 2023 August 01.

J Immunol. Author manuscript; available in PMC 2023 August 01.