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. 2015 Aug:59:172-81.
doi: 10.1016/j.biomaterials.2015.04.003. Epub 2015 May 15.

Paracrine co-delivery of TGF-β and IL-2 using CD4-targeted nanoparticles for induction and maintenance of regulatory T cells

Michael D McHugh  1 , Jason Park  1 , Ross Uhrich  1 , Wenda Gao  2 , David A Horwitz  3 , Tarek M Fahmy  4
Affiliations

Paracrine co-delivery of TGF-β and IL-2 using CD4-targeted nanoparticles for induction and maintenance of regulatory T cells

Michael D McHugh et al. Biomaterials. 2015 Aug.

Abstract

The cytokine milieu is critical for orchestration of lineage development towards effector T cell (Teff) or regulatory T cell (Treg) subsets implicated in the progression of cancer and autoimmune disease. Importantly, the fitness and survival of the Treg subset is dependent on the cytokines Interleukin-2 (IL-2) and transforming growth factor beta (TGF-β). The production of these cytokines is impaired in autoimmunity increasing the probability of Treg conversion to aggressive effector cells in a proinflammatory microenvironment. Therapy using soluble TGF-β and IL-2 administration is hindered by the cytokines' toxic pleiotropic effects and hence bioavailability to CD4(+) T cell targets. Thus, there is a clear need for a strategy that rectifies the cytokine milieu in autoimmunity and inflammation leading to enhanced Treg stability, frequency and number. Here we show that inert biodegradable nanoparticles (NP) loaded with TGF-β and IL-2 and targeted to CD4(+) cells can induce CD4(+) Tregs in-vitro and expand their number in-vivo. The stability of induced Tregs with cytokine-loaded NP was enhanced leading to retention of their suppressive phenotype even in the presence of proinflammatory cytokines. Our results highlight the importance of a nanocarrier-based approach for stabilizing and expanding Tregs essential for cell-immunotherapy of inflammation and autoimmune disease.

Keywords: Autoimmunity; Drug delivery; Immunomodulation; Interleukin-2; Nanoparticles; TGF-β.

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Figures

Fig. 1
Fig. 1
Characterization of PLGA nanoparticles containing TGF-β and IL-2 (A) Scanning electron microscopy of TGF-β+IL-2 nanoparticles. (B) Quantification of nanoparticle size distribution by Nanosight particle tracking system. (C) Release kinetics of TGF-β (blue circles) and IL-2 (red diamonds), measured by ELISA. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 2
Fig. 2
Targeted nanoparticles bind CD4 cells. (A) After incubating 10 μg DiR-labeled nanoparticles with 1 × 106 mouse splenocytes for 20 min at 37 °C, FACS plots were generated using Amnis Imagestream. (B) Fluorescent imaging illustrates targeting-dependent surface-binding of nanoparticles to CD4+ cells.
Fig. 3
Fig. 3
Nano-encapsulated cytokines generate Foxp3+ CD4 Tregs. (A) Naïve or nTreg-depleted splenocytes were incubated with 5 ng/ml TGF-β and 50 U/ml IL-2 for 4 days and resulting Treg induction was analyzed by FACS. (B) In a separate experiment, mixed splenocytes were cultured with TGF-β and IL-2 in soluble (5 ng/ml and 10 U/ml, respectively) or nano-encapsulated form (0.1 mg nanoparticle/ml) to induce CD25+Foxp3+ Tregs. TGF-β and IL-10 concentrations in the supernatant were measure by ELISA. (C) Representative FACS plots showing Foxp3 induction are gated on CD4+ lymphocytes. (D) Results from 4 independent experiments are plotted (***P < 0.001). (E) Dose responses of nano-encapsulated (green), and soluble (blue) cytokine. Values are percentages of CD4+ lymphocytes. (F) IL-2 dependent CTRL-1842 cells were dosed with nano-encapsulated vs. free IL-2 and proliferation was quantified after 4 days by Coulter Counter. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 4
Fig. 4
Nanoparticle-induced Tregs have enhanced suppressive function. (A) A schematic of the experimental setup shows representative cell numbers over time. Suppressor cells (Thy1.1−) were generated by activating T cells with plate bound anti-CD3 and soluble anti-CD28 with either free IL-2 and TGF-β (10 U/ml and 5 ng/ml, respectively), or nanoparticles (0.1 mg/ml) for 5 days (induction phase). Foxp3+ Tregs (yellow) were quantified by FACS, washed, and added in various relative Treg fractions to CD4+Thy1.1+CD25− splenocytes (responder cells, pink). These cultures were stimulated with anti-CD3/CD28 beads in 96-well flat-bottom plates at a 1:2 bead-to-cell ratio for an additional 4 days (suppression phase). During this period, responder cells proliferated while Foxp3 expression on suppressor cells decreased. (B) Following the suppression phase, suppressor and responder cells were identified by surface expression of Thy1.1 and incorporation of CellTrace Violet as shown in the representative FACS plot. (C) Each generation of proliferated responder cells was gated as shown in the representative histogram, and gated frequencies were used to calculate proliferative index, PI, as described in Table 2. (D) Representative CellTrace Violet dilutions across titrated initial Treg fractions shows that suppressor cells treated with nanoparticles preferentially suppressed responder proliferation. (E) Proliferative index of responder cells co-cultured with suppressor cells generated with soluble cytokines (blue triangles) vs. nano-encapsulated cytokines (green circles) is plotted (*p < 0.05, **p < 0.005, ***p < 0.001). (F) Representative FACS plots compare Foxp3 expression of suppressor cells at the end of the 4-day suppression period, showing elevated Foxp3 expression in the nano-encapsulated group. (G) Foxp3 expression of suppressor cells is plotted as a function of initial Treg fraction. (H) Total numbers of remaining Foxp3+ suppressor cell numbers are plotted as a function of initial Treg fraction. (*p < 0.05, **p < 0.005, ***p < 0.001). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 5
Fig. 5
Nano-encapsulated cytokines enhance Treg stability. (A) Mixed splenocytes were treated with soluble (blue triangles) or nano-encapsulated (green circles) TGF-β and IL-2 for a 3 day induction phased before washing the cells and replating, and Foxp3 expression was monitored over time. Control cells (black circles) were replenished with soluble cytokine after washing. (*p < 0.05 between nano-encapsulated and soluble using a 2-tailed T test on day 9 Foxp3 expression). (B) After a 5 day induction phase, cells were washed and reseeded in the presence of TGF-β (5 nm/ml) and IL-6 (10 ng/ml) for Th17 polarization. FACS plots show CD4 phenotype at day 7. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 6
Fig. 6
Nanoparticles enhance Tregs in-vivo. (A) 2.0 mg coumarin-6 labeled nanoparticles were injected I.P. and tissues were harvested after 5 days. Coumarin-6 was measured by fluorescence microscopy. (B–D) 2.0 mg TGF-β IL-2 CD4-targeted nanoparticles were injected I.P. and mice were sacrificed after 5 days for FACS analysis. (B) Tregs are plotted as percentages of CD4+ T cells (*p < 0.05 vs. untreated controls). (C) Treg numbers per tissue are plotted. (D) Representative FACS plots are gated on CD4+ lymphocytes. Values on the lower left of the gate show Tregs as a percentage of CD4+ cells. Values to the right show Tregs as a percentage of all lymphocytes.
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