(726f) Sustained Release Systems to Locally Expand Regulatory T Cell Populations and Suppress Inflammation

Aberrant inflammation
leading to tissue destruction is responsible for transplant rejection,
autoimmunity, and inflammatory diseases. 
Traditional therapies involve harsh regimens of systemic
immunosuppression, which leave patients vulnerable to opportunistic
infections.  Consequently, there is
considerable interest in developing novel methods to localize suppression to
inflamed tissue.¹  One such therapeutic approach
involves increasing the presence of suppressive regulatory T cells (Treg) at
local sites,² which typically requires systemic infusion of ex vivo cultured cells.³  As inspiration for an alternative method
to increase Treg locally, we consider a naturally occurring mechanism employed
by the immune system.  Specifically,
tolerogenic dendritic cells induce the differentiation of naive T cells to Treg
by establishing a local immunosuppressive milieu, comprised of secreted
cytokines (IL-2 and TGF-β).⁴  In an inflammatory microenvironment,
these factors may be insufficient to prevent differentiation of naive T cells
to pro-inflammatory effector T cells; however, the immunosuppressant drug
rapamycin (rapa) has been shown to preferentially
suppress generation and proliferation of effector T cells, but not Treg.⁵  With this rationale, we have developed
and tested biomimetic sustained release microparticle (MP) formulations of various
factors, including IL-2, TGF-β, and rapa (collectively
FactorMP), which can dramatically increase populations of Treg in vitro and suppress local inflammation
in a murine model of contact hypersensitivity.

To
fabricate sustained release cytokine/drug formulations, IL-2, TGF-β, and rapamycin (rapa)
were encapsulated in poly(lactic-co-glycolic acid)
(PLGA) using emulsion-evaporation techniques, and the resulting FactorMP were
characterized, as described elsewhere.⁶  Empty PLGA MP (BlankMP) were used as
controls.  For in vitro Treg induction assays, na?ve CD4 T cells were cultured in
the presence of the soluble or encapsulated cytokines and drugs.  At day 4, cell phenotypes were analyzed
by flow cytometry, and suppressive function was assessed by a standard mixed
leukocyte reaction.  For the contact
hypersensitivity suppression study, either FactorMP or BlankMP were injected
into the footpads of mice at days 
-11 and -6.  On day -5, mice
were sensitized with dinitrofluorobenzene
(DNFB).  A contact hypersensitivity
response was elicited on day 1 by painting one footpad with DNFB, and
measurements of footpad thickness (indicative of inflammation) were taken at
24, 48, and 72 hours post-elicitation. 
Subsequently, draining lymph nodes were isolated and T cell populations
analyzed by flow cytometry.

IL-2,
TGF-β, and rapa were
successfully encapsulated in PLGA MP, with sizes ranging from 16.7±6.3 to 25.5±7.5μm
(large enough to remain at an injection site).  Each MP formulation provided sustained
release of the encapsulated factor for at least 3 weeks in vitro, creating a sustained immunosuppressive, local
microenvironment.  Similar in vitro Treg induction efficiencies
were observed for naive CD4 T cells cultured in the presence of FactorMP or
soluble factors.  Notably, the
presence of soluble rapa or rapaMP, significantly (p<0.05) enhanced induction
efficiency from 57±18% (with IL-2 and TGF-β alone) to 71±15%.  Importantly, induced Treg expressed
canonical Treg surface markers (CD25 and GITR), as well as FoxP3, a
characteristic Treg transcription factor. 
Furthermore, FactorMP-induced Treg were
functionally suppressive in a mixed leukocyte reaction.  Finally, data from a contact
hypersensitivity model (Figure 1) suggests that establishment of a local
immunosuppressive milieu by FactorMP can decrease footpad swelling (local
inflammation), presumably by increasing local Treg numbers.

In
conclusion, we have successfully fabricated MP that provide
sustained release of Treg-inducing factors.  These FactorMP significantly increase
numbers of functional Treg in vitro.  Preliminary results also suggest that they
can suppress inflammation in vivo.  Ultimately, this FactorMP system may have
the potential to treat a variety of inflammatory and autoimmune diseases, as
well as transplant rejection.

[1] Thomson AW (ed). Immunology and Medicine: Therapeutic
Immunosuppression (2001) Springer; 29:1-512.

[2]
Sakaguchi S et al. Immunol Rev (2001) 182:18-32.          

[3] Raimondi G et al. J Immunol (2010) 184:624-36.            

[4] Horwitz DA et al. Eur J Immunol
(2008) 38:901-937.      

[5] Battaglia M et al. Blood
(2005) 105:4743-4748.

[6] Jhunjhunwala S et al. J Control Release (2012) 159:78-84.