Chimera of IL‑2 Linked to Light Chain of anti-IL‑2 mAb Mimics IL-2/anti-IL‑2 mAb Complexes Both Structurally and Functionally

IL-2/anti-IL-2 mAb immunocomplexes were described to have dramatically higher activity than free IL-2 <i>in vivo</i>. We designed protein chimera consisting of IL-2 linked to light chain of anti-IL-2 mAb S4B6 through flexible oligopeptide spacer (Gly₄Ser)₃. This protein chimera mimics the structure of IL-2/S4B6 mAb immunocomplexes but eliminates general disadvantages of immunocomplexes like possible excess of either IL-2 or anti-IL-2 mAb and their dissociation to antibody and IL-2 at low concentrations. This novel kind of protein chimera is characterized by an intramolecular interaction between IL-2 and binding site of S4B6 mAb similarly as in IL-2/S4B6 mAb immunocomplexes. Our protein chimera has biological activity comparable to IL-2/S4B6 mAb immunocomplexes <i>in vitro</i>, as shown by stimulation of proliferation of purified and activated OT-I CD8⁺ T cells. The protein chimera exerts higher stimulatory activity to drive expansion of purified CFSE-labeled OT-I CD8⁺ T cells activated by an injection of a low dose of SIINFEKL peptide than IL-2/S4B6 mAb immunocomplexes <i>in vivo</i>

<i>Bordetella</i> Adenylate Cyclase Toxin Differentially Modulates Toll-Like Receptor-Stimulated Activation, Migration and T Cell Stimulatory Capacity of Dendritic Cells

<div><p>Adenylate cyclase toxin (CyaA) is a key virulence factor of the whooping cough agent <i>Bordetella pertussis</i>. The toxin targets CD11b-expressing phagocytes and delivers into their cytosol an adenylyl cyclase (AC) enzyme that subverts cellular signaling by increasing cAMP levels. In the present study, we analyzed the modulatory effects of CyaA on adhesive, migratory and antigen presenting properties of Toll-like receptor (TLR)-activated murine and human dendritic cells (DCs). cAMP signaling of CyaA enhanced TLR-induced dissolution of cell adhesive contacts and migration of DCs towards the lymph node-homing chemokines CCL19 and CCL21 <i>in vitro</i>. Moreover, we examined in detail the capacity of toxin-treated DCs to induce CD4⁺ and CD8⁺ T cell responses. Exposure to CyaA decreased the capacity of LPS-stimulated DCs to present soluble protein antigen to CD4⁺ T cells independently of modulation of co-stimulatory molecules and cytokine production, and enhanced their capacity to promote CD4⁺CD25⁺Foxp3⁺ T regulatory cells <i>in vitro</i>. In addition, CyaA decreased the capacity of LPS-stimulated DCs to induce CD8⁺ T cell proliferation and limited the induction of IFN-γ producing CD8⁺ T cells while enhancing IL-10 and IL-17-production. These results indicate that through activation of cAMP signaling, the CyaA may be mobilizing DCs impaired in T cell stimulatory capacity and arrival of such DCs into draining lymph nodes may than contribute to delay and subversion of host immune responses during <i>B. pertussis</i> infection.</p></div

CyaA decreases the capacity of TLR-stimulated DCs to present soluble antigen to CD4⁺ T cells.

<p>BMDCs were left untreated, incubated with LPS (100 ng/ml) alone or in combination with CyaA or CyaA-AC⁻ at 10 ng/ml in the presence of OVA protein at 2.5 µg/ml or OVA_323–339 peptide (5 µg/ml) for 4 h prior to washing and co-cultivation with naïve CFSE-labeled OT-II CD4⁺ T cells. T cell proliferation was determined by flow cytometry after 72 h as a dilution of CFSE. (A) Histograms are representative of n = 4. (B) Quantitative analysis of A where the percentage of undivided LPS-treated cells (medium) was set to 100% (* <i>p</i><0.05). (C) Expansion of adoptively transferred CFSE-labeled CD4⁺ T cells <i>in vivo</i> was determined after 72 h by flow cytometry as a fold of expansion of 2×10⁶ counted spleen cells where 1 represents the non-stimulated adoptively transferred CD4⁺ T cells (control). Dot plots are representative of n = 3. (D, E) CyaA inhibits macropinocytosis but not receptor-mediated endocytosis and antigen (Ag) degradation in LPS-treated DCs. DCs were left untreated, incubated with LPS alone or in combinantion with 10 ng/ml of toxins or chloroquine (100 µM) for 30 min. (D) Lucifer Yellow (500 µg/ml), transferrin-Alexa647 or OVA-Alexa647 (both 5 µg/ml) were subsequently added for 30 min. The Ag uptake in living CD11c⁺ cells was determined by flow cytometry. (E) A mixture of OVA-Alexa647 (5 µg/ml, marker for Ag uptake) and OVA-DQ (5 µg/ml, marker for Ag uptake and degradation) were added for 30 min. The processed OVA-DQ was determined from gated CD11c⁺OVA-DQ⁺OVA-Alexa647⁺ DCs and calculated as a ratio of MFI OVA-DQ/OVA-Alexa647. Values represent means ± SEM of n = 5 where Ags taken up by LPS-treated DC (medium) was set to 100% of MFI (ratio 1) (* <i>p</i><0.05).</p

CyaA accelerates cell detachment and migration of TLR-activated DCs.

<p>(A) Impedance measurements using the real-time cell electronic sensing system xCelligence were used to determine MDDC adhesion and spreading. MDDCs were seeded on fibronectin-coated sensors and were left untreated (medium), or treated with LPS (1 µg/ml) alone, or in combination with CyaA or CyaA-AC⁻ at 10 ng/ml for 24 h. The representative experiment is shown (A) as well as quantitative analysis of 4 donors at time point of 12 h (B) where cell index (CI) of LPS-treated DCs at 12 h was normalized to 1.0. (C) Migration of DCs treated with toxins and LPS (for 24 h) towards CCL19 or CCL21 (both 200 ng/ml) in transwell plates was determined by flow cytometry after additional 14 h (MDDCs) or 4 h (BMDCs) of incubation at 37°C. Values represent the means ± SEM of n = 4 or 5 donors, respectively (* <i>p</i><0.05) where the number of transmigrated LPS-treated DCs (medium) was set to 1. (D) CCR7 expression on DCs was determined by flow cytometry after 24 h and 48 h. Values represent the means ± SEM of n = 3–5 or 5 donors, respectively (* <i>p</i><0.05).</p

Exposure to low concentrations of CyaA does induce cell death in TLR-activated DCs.

<p>(A) BMDCs and MDDCs were left untreated or incubated with CyaA at 10 ng/ml for 30 min. The intracellular level of cAMP was determined by ELISA. (B, C) DCs were left untreated (medium), or incubated with LPS (1 µg/ml MDDCs or 100 ng/ml BMDCs), CyaA or CyaA-AC⁻ at 10 ng/ml alone or in their combination for 18 h (BMDCs) or 24 h (MDDCs) (D) BMDCs were incubated with LPS and CyaA or CyaA-AC⁻ at 100 ng/ml or 300 ng/ml alone or in their combination for 18 h and stained with Annexin V-FITC and Hoechst 33258. Values represent the means ± SEM of n = 3–5 or 5–6 donors, respectively (* <i>p</i><0.05).</p

CyaA commits TLR-stimulated DCs to expand CD4⁺CD25⁺Foxp3⁺ T regulatory cells <i>in vitro</i>.

<p>(A) BMDCs were left untreated, incubated with LPS (100 ng/ml) alone or in combination with CyaA or CyaA-AC⁻ at 10 ng/ml in the presence of OVA at 2.5 µg/ml for 4 h prior to washing and co-cultivation with naïve CFSE-labeled OT-II CD4⁺ T cells. After 72 h the number of CD4⁺CD25⁺Foxp3⁺ T cells was determined by flow cytometry. Dot plots show one representative experiment and quantitative analysis represent means ± SEM of n = 4 (* <i>p</i><0.05). (B) MDDCs were incubated with LPS (1 µg/ml) alone or in combination with CyaA or CyaA-AC⁻ at 10 ng/ml. After 24 h cells were used as stimulators of naïve allogeneic T cells at DC : T cell ratio of 1 : 10. The expansion of human CD4⁺CD25⁺Foxp3⁺ T regulatory cells was determined after 7 days. The representative experiment is shown and quantitative analysis represent means ± SEM of n = 5 (* <i>p</i><0.05).</p

CyaA reduces the capacity of TLR-stimulated DCs to induce CD8⁺ T cell proliferation.

<p>BMDCs were left untreated, incubated with LPS (100 ng/ml) alone or in combination with 10 ng/ml of CyaA or CyaA-AC⁻ in the presence of OVA protein at 5 µg/ml or OVA_257–264 peptide (1 ng/ml) for 4 h prior to co-cultivation with naïve CFSE-labeled OT-I CD8⁺ T cells. T cell proliferation was determined by flow cytometry after 72 h as a dilution of CFSE. (A) Histograms are representative of n = 4. (B) Quantitative analysis of A where the percentage of undivided LPS-treated cells (medium) was set to 100% (* <i>p</i><0.05). (C) Expansion of adoptively transferred CFSE-labeled CD8⁺ T cells <i>in vivo</i> after 72 h was determined by flow cytometry as a fold of expansion of 2×10⁶ spleen cells counted where 1 represents the non-stimulated adoptively transferred CD8⁺ T cells (control). Dot plots are representative of n = 3 (D) MDDCs were incubated with LPS (1 µg/ml) alone or in combination with CyaA or CyaA-AC⁻ at 10 ng/ml for 24 h and then loaded with influenza matrix peptide. The induction of specific IFN-γ producting CD8⁺ T cells was determined after 7 days by flow cytometry. Dot plots show one representative experiment and quantitative analysis represent means ± SEM of n = 6 (* <i>p</i><0.05). (E) BMDCs were left untreated, incubated with LPS (100 ng/ml) alone or in combination with CyaA or CyaA-AC⁻ at 10 ng/ml or inhibitor of proteasome lactacystin (10 µM) for 30 min. After cell lysis, 50 µg of cellular proteins was mixed with fluorogenic proteasomal peptide substrates (100 µM) and incubated 90 min at 37°C. Values represent means ± SEM of n = 4 where the amount of processed substrates by LPS-treated DCs (medium) was set to 100% (* <i>p</i><0.05). (F) The production of IL-17, IL-10 and IFN-γ in BMDC-CD8⁺ T cell culture supernatant after 72 h was determined by ELISA. Mean values ± SEM are representative of n = 3 (* <i>p</i><0.05).</p