HSP70 Enhances Immunosuppressive Function of CD4⁺CD25⁺FoxP3⁺ T Regulatory Cells and Cytotoxicity in CD4⁺CD25⁻ T Cells

<div><p>Human CD4⁺CD25⁺FoxP3⁺ T regulatory cells (Tregs) control effector T cells and play a central role in peripheral tolerance and immune homeostasis. Heat shock protein 70 (HSP70) is a major immunomodulatory molecule, but its effect on the functions of Tregs is not well understood. To investigate target-dependent and –independent Treg functions, we studied cytokine expression, regulation of proliferation and cytotoxicity after exposure of Tregs to HSP70. HSP70-treated Tregs significantly inhibited proliferation of CD4⁺CD25⁻ target cells and downregulated the secretion of the proinflammatory cytokines IFN-γ and TNF-α. By contrast, HSP70 increased the secretion of Treg suppressor cytokines IL-10 and TGF-β. Treatment with HSP70 enhanced the cytotoxic properties of Tregs only to a minor extent (4-fold), but led to stronger responses in CD4⁺CD25⁻ cells (42-fold). HSP70-induced modulation of T-cell responses was further enhanced by combined treatment with HSP70 plus IL-2. Treatment of Tregs with HSP70 led to phosphorylation of PI3K/AKT and the MAPKs JNK and p38, but not that of ERK1/2. Exposure of Tregs to specific inhibitors of PI3K/AKT and the MAPKs JNK and p38 reduced the immunosuppressive function of HSP70-treated Tregs as indicated by the modified secretion of specific target cell (IFN-γ, TNF-α) and suppressor cytokines (IL-10, TGF-β). Taken together, the data show that HSP70 enhances the suppressive capacity of Tregs to neutralize target immune cells. Thus HSP70-enhanced suppression of Tregs may prevent exaggerated immune responses and may play a major role in maintaining immune homeostasis.</p> </div

HSP70 mediates phosphorylation of AKT, p38, JNK and ERK1/2 in CD4⁺CD25⁺ T cells and suppressive capacity of these cells could be influenced by the respective inhibitors.

<p>CD4⁺CD25⁺ Treg cells were incubated alone, with IL-2 (200 U/ml), HSP70 (10 µg/ml) or both for 10 min washed and exposed to anti-CD3 antibodies (OKT3, 1 µg/ml) for 5, 10, 20 and 30 minutes. Phosphorylation for intracellular kinases (<b>A</b>) phospo-AKT [p-AKT Ser⁴⁷³], (<b>B</b>) phospo-JNK [p-JNK Thr¹⁸³/Tyr¹⁸⁵], (<b>C</b>) phospo-p38 MAPK [p-p38 MAPK Thr¹⁸⁰/Tyr¹⁸²] and (<b>D</b>) phospo-ERK1/2 [p-ERK1/2 Thr²⁰²/Tyr²⁰⁴/Thr¹⁸⁵/Tyr¹⁸⁷] was determined by the bead-based multiplex assay (Luminex xMAP technology). Untreated CD4⁺CD25⁺ T cells were adjusted to 1.00 by the use of the Bio-Plex Manager 6.0 software and used to calculate the ratios. Furthermore, CD4⁺CD25⁺ T cells were treated with 5 µM of the following intracellular signal transduction inhibitors: (<b>E</b>) Wortmannin, (<b>F</b>) JNK or (<b>G</b>) SB 203580 for 15 min before incubation alone, with IL-2 (200 U/ml), HSP70 (10 µg/ml) or both for 2 h. Cells were then co-cultured with CD4⁺CD25⁻ T cells (E∶T ratio 1∶5) on 96-well plates coated with anti-CD3 antibodies (OKT3, 1 µg/ml) in serum-free medium for 48 h. Supernatants were analyzed for IFN-γ, TNF-α, IL-10 and TGF-β. Results of four independent experiments, expressed as mean fold increases or decreases in comparison to results obtained for experiments without preactivation of Tregs and without inhibitor treatment (control).</p

CD4⁺CD25⁺ treatment with HSP70 inhibits CD4⁺CD25⁻ target T-cell proliferation.

<p>CD4⁺CD25⁺ T cells were incubated with IL-2 (200 U/ml), HSP70 (10 µg/ml) or both for 2 h, washed and co-cultured with CFSE-labeled CD4⁺CD25⁻ target T cells (E∶T ratio 1∶10) on 96-well plates coated with anti-CD3 antibodies (OKT3, 1 µg/ml) in serum-free medium. For control CD4⁺CD25⁻ target T cells were culture in the absence of Tregs. After 5 days of incubation, cell proliferation was determined by FACS analysis. (<b>A</b>) One representative experiment of CD4⁺CD25⁻ T-cell proliferation under different CD4⁺CD25⁺ stimulation conditions. (<b>B</b>) Results of six independent experiments, expressed as mean ± SD. p-values (* p<0.05, ** p<0.01 or ***p<0.001) are indicated with asterisks. Proliferation levels of CD4⁺CD25⁻ target cells without Treg co-cultivation was defined as 100% (control, no suppression).</p

Uptake of HSP70 by CD4⁺CD25⁺ Tregs and HSP70-dependent expression of Ki-67 and caspase-3.

<p>Representative fluorescence microscopy results for isolated Tregs incubated for 2 h (<b>A</b>) without or with (<b>B</b>) HSP70-FITC. Shown are the immunofluorescence microscopy results for DAPI (blue), FITC (green) and PE (red) staining. Purified CD4⁺CD25⁺ and CD4⁺CD25⁻ T cells were incubated with IL-2 (200 U/ml), HSP70 (10 µg/ml) or both for 2 h, washed and transferred to 96-well plates coated with anti-CD3 antibodies (OKT3, 1 µg/ml) in serum-free medium. After 20 h, Ki-67 and caspase-3 expression was determined by FACS analysis. (<b>C</b>) Results of one representative Ki-67-experiment out of six in CD4⁺CD25⁺ T cells under different stimulation conditions. Results of six independent experiments using CD4⁺CD25⁺ cells isolated from six different donors for (<b>D</b>) Ki-67 and (<b>E</b>) caspase-3, expressed as mean ± SD. p-values (* p<0.05, ** p<0.01 or ***p<0.001) are indicated with asterisks.</p

Augmentation of cytokine secretion by CD4⁺CD25⁺ and CD4⁺CD25⁻ T cells after HSP70 treatment.

<p>CD4⁺CD25⁺ and CD4⁺CD25⁻ T cells were incubated with IL-2 (200 U/ml), HSP70 (10 µg/ml) or both for 2 h, washed and transferred to 96-well plates coated with anti-CD3 antibodies (OKT3, 1 µg/ml) in serum-free medium. After 48 h of incubation, the supernatants were analyzed for (<b>A</b>) IL-10, (<b>B</b>) TGF-β, (<b>C</b>) IFN-γ and (<b>D</b>) TNF-α. Results of four independent experiments, expressed as mean ± SD. p-values (* p<0.05, ** p<0.01 or ***p<0.001) are indicated with asterisks.</p

CD4⁺CD25⁺-induced inhibition of target T-cell proliferation is cytokine-dependent.

<p>CD4⁺CD25⁺ T cells were incubated with IL-2 (200 U/ml), HSP70 (10 µg/ml) or both for 2 h, washed and co-cultured with CFSE-labeled CD4⁺CD25⁻ target T cells (E∶T ratio 1∶10) on 96-well plates coated with anti-CD3 antibodies (OKT3, 1 µg/ml) in serum-free medium. For control CD4⁺CD25⁻ target T cells were culture in the absence of Tregs. After 5 days of incubation, the supernatants were analyzed for (<b>A</b>) IL-10, (<b>B</b>) TGF-β, (<b>C</b>) IFN-γ and (<b>D</b>) TNF-α. Results of six independent experiments, expressed as mean ± SD. p-values (* p<0.05, ** p<0.01 or ***p<0.001) are indicated with asterisks.</p

Secretion of cytotoxic effector molecule granzyme B of both CD4⁺CD25⁺ and CD4⁺CD25⁻ T-cell subsets is target-independent enhanced by HSP70.

<p>(<b>A</b>) Granzyme B mRNA levels in CD4⁺CD25⁺ T cells or independently stimulated CD4⁺CD25⁻ T cells were assessed by real-time PCR after 48 h of stimulation on anti-CD3 (OKT3, 1 µg/ml) coated plates in serum-free medium. Analysis was performed for comparison between the different CD4⁺CD25⁺ and CD4⁺CD25⁻ T-cell subsets. (<b>B</b>) Secretion of granzyme B was assessed by ELISA. Data from four independent experiments (mRNA) and two independent experiments (protein levels), expressed as mean ± SD. Comparison between groups was performed using t-tests. Statistically significant differences are indicated with asterisks (* p<0.05, ** p<0.01 or ***p<0.001).</p