miR-155 augments CD8(+) T-cell antitumor activity in lymphoreplete hosts by enhancing responsiveness to homeostatic gamma(c) cytokines

Lymphodepleting regimens are used before adoptive immunotherapy to augment the antitumor efficacy of transferred T cells by removing endogenous homeostatic "cytokine sinks." These conditioning modalities, however, are often associated with severe toxicities. We found that microRNA-155 (miR-155) enabled tumor-specific CD8(+) T cells to mediate profound antitumor responses in lymphoreplete hosts that were not potentiated by immune-ablation. miR-155 enhanced T-cell responsiveness to limited amounts of homeostatic gamma c cytokines, resulting in delayed cellular contraction and sustained cytokine production. miR-155 restrained the expression of the inositol 5-phosphatase Ship1, an inhibitor of the serine-threonine protein kinase Akt, and multiple negative regulators of signal transducer and activator of transcription 5 (Stat5), including suppressor of cytokine signaling 1 (Socs1) and the protein tyrosine phosphatase Ptpn2. Expression of constitutively active Stat5a recapitulated the survival advantages conferred by miR-155, whereas constitutive Akt activation promoted sustained effector functions. Our results indicate that overexpression of miR-155 in tumor-specific T cells can be used to increase the effectiveness of adoptive immunotherapies in a cell-intrinsic manner without the need for life-threatening, lymphodepleting maneuvers.112922Ysciescopu

Characterisation of Neutropenia-Associated Neutrophil Elastase Mutations in a Murine Differentiation Model <i>In Vitro</i> and <i>In Vivo</i>

<div><p>Severe congenital neutropenia (SCN) is characterised by a differentiation block in the bone marrow and low neutrophil numbers in the peripheral blood, which correlates with increased risk of bacterial infections. Several underlying gene defects have been identified in SCN patients. Mutations in the neutrophil elastase <i>(ELANE)</i> gene are frequently found in SCN and cyclic neutropenia. Both mislocalization and misfolding of mutant neutrophil elastase protein resulting in ER stress and subsequent induction of the unfolded protein response (UPR) have been proposed to be responsible for neutrophil survival and maturation defects. However, the detailed molecular mechanisms still remain unclear, in part due to the lack of appropriate in vitro and in vivo models. Here we used a system of neutrophil differentiation from immortalised progenitor lines by conditional expression of Hoxb8, permitting the generation of mature near-primary neutrophils <i>in vitro</i> and <i>in vivo</i>. NE-deficient Hoxb8 progenitors were reconstituted with murine and human forms of typical NE mutants representative of SCN and cyclic neutropenia, and differentiation of the cells was analysed <i>in vitro</i> and <i>in vivo</i>. ER stress induction by NE mutations could be recapitulated during neutrophil differentiation in all NE mutant-reconstituted Hoxb8 cells. Despite ER stress induction, no change in survival, maturation or function of differentiating cells expressing either murine or human NE mutants was observed. Further analysis of <i>in vivo</i> differentiation of Hoxb8 cells in a murine model of adoptive transfer did not reveal any defects in survival or differentiation in the mouse. Although the Hoxb8 system has been found to be useful for dissection of defects in neutrophil development, our findings indicate that the use of murine systems for analysis of NE-mutation-associated pathogenesis is complicated by differences between humans and mice in the physiology of granulopoiesis, which may go beyond possible differences in expression and activity of neutrophil elastase itself.</p></div

Characterisation of differentiating Hoxb8 cells reconstituted with human wt or mutant NE.

<p>NE^-/- Hoxb8 neutrophils transduced with empty vector pMIGR1 (pMiGR1), human neutrophil elastase (hNE), or hNE mutant G185R (hNEG185R) (genetic background C57BL/6) were induced to undergo differentiation by estrogen withdrawal for 4 days. (A) Cell numbers of differentiating neutrophils were measured daily by automated counting with a CASY cell counter after initial seeding of 300,000 cells/well in a 6-well plate at day 0. Data are mean/SD of 3 independent experiments. (B) Cell death analysis of Hoxb8 neutrophils cultured from day 0 to day 4 of differentiation as described above. Viability was determined using a CASY cell counter. Data are mean/SD of 3 independent experiments. (C) Western blot analysis of human NE (hNE) and BIP (GRP78/HASP5) expression in cell lysates of Hoxb8 cells at day 2 of differentiation. Hoxb8 cells transduced with empty vector (pMIGR1), human wt NE (hNE) or human NE mutant G185R (hNEG185R) were induced to undergo differentiation <i>in vitro</i> by estrogen withdrawal. Differentiating cells were harvested on day 2. Samples were directly lysed in Laemmli buffer and heated at 95°C for 5 min. Samples were separated by SDS-PAGE, transferred onto nitrocellulose membranes and probed with antibodies against mNE and BIP followed by detection using enhanced chemoluminescence. GAPDH served as loading control.</p

<i>In vivo</i> differentiation of Hoxb8 cells.

<p>(A-D) Flow cytometry analysis of samples from bone marrow and peripheral blood on day 6 after transplantation of irradiated mice with Hoxb8 Ne^-/- progenitors (C57BL/6 background) transduced with empty vector pMIGR1 (ev), human neutrophil elastase (hNE) or human neutrophil elastase mutation G185R (hNEG185R). (A) Representative FACS plots of peripheral blood and bone marrow samples analysed for GFP positivity after gating on live, DAPI-negative cells. (B) Quantitative analysis of all samples from peripheral blood and bone marrow analysed as in (A). (C) Representative FACS plots of peripheral blood and bone marrow samples showing percentage of Gr-1⁺CD11b⁺ cells of all live GFP-positive cells. (D) Quantitative analysis of Gr-1 and CD11b expression of all samples from peripheral blood and bone marrow analysed as in C. Percentage of Gr-1⁺CD11b⁺ cells of total live GFP-positive cells is shown. Each symbol represents one mouse. Horizontal bars: mean of each distribution. Data were obtained from two independent <i>in vivo</i> experiments including a total of n = 5 mice for empty vector control, n = 3 for hNE and n = 6 for hNEG185R.</p

Cytokine secretion by day 4 differentiated Hoxb8 neutrophils upon LPS stimulation.

<p>NE^-/- Hoxb8 cell lines transduced with empty vector pMIGR1 (pMiGR1), murine neutrophil elastase (mNE), human neutrophil elastase (hNE), or mNE mutants G185R (mNeG185R) and S97L (mNES97L) (genetic background 129/Sv) were differentiated for 4 days and then stimulated with 1 μg/ml LPS. (A, B) The cytokines IL-6 (A) and TNF (B) were measured by ELISA in supernatants harvested after 16h of LPS-stimulation. Data are mean/SD of three independent experiments and dual ELISA of each stimulation. Unstimulated medium control values are 0-6pg/ml.</p

NE expression, secretion and ER stress induction in transfected 293FT cells.

<p>Supernatants and cell lysates from 293FT cells were analysed 64h after transfection with either empty vector (pMiGR1), murine neutrophil elastase (mNE), human neutrophil elastase (hNE) or mNE/hNE mutants G185R (mNEG185R/hNEG185R) or S97L (mNES97L/ hNES97L). (A) expression of murine neutrophil elastase (mNE) and the ER-stress markers BIP (GRP78/HASP5) and PDI in cell lysates and supernatants, (B) expression of human neutrophil elastase (hNE) in cell lysates and supernatant (the same samples as in (A) were rerun on a separate gel and probed with a human-specific anti-hNE antibody). Samples corresponding to 20μg cell lysate or 20μl of supernatant were separated by SDS-PAGE, transferred onto nitrocellulose membranes and probed for the antibodies indicated. GAPDH served as loading control. Data represent one of two independent experiments.</p

Characterisation of differentiating Hoxb8 cells reconstituted with murine wt or mutant NE.

<p>NE^-/- Hoxb8 neutrophils transduced with empty vector pMIGR1 (pMiGR1), murine neutrophil elastase (mNE), or mNE mutants G185R (mNeG185R) or S97L (mNeS97L) (genetic background 129/SV) were induced to undergo differentiation by estrogen withdrawal for 4 days. (A) Proliferation of differentiating neutrophils as measured daily by manual counting (Neubauer chamber) after initial seeding of 300,000 cells/well in a 6-well plate at day 0. Data are mean/SD of 3 independent experiments. (B) Cell death analysis of Hoxb8 neutrophils cultured from day 0 to day 4 of differentiation as above. Cell death was determined by propidium iodide (PI) staining for loss of cell membrane integrity. Shown are % PI-positive cells. Data are mean/SD of 4 independent experiments. (C) Western blot showing murine neutrophil elastase (mNE), BIP (GRP78/HASP5) and PDI in cell lysates of murine Hoxb8 progenitors (NP) and day 1 to day 4 (D1-D4) in vitro differentiated neutrophils. Samples were separated by SDS-PAGE, transferred onto nitrocellulose membranes and probed with antibodies against mNE and BIP followed by detection using enhanced chemoluminescence. GAPDH served as as loading control. Data shown are representative of 2 independent experiments.</p

<i>Roscovitine causes</i> the loss of Mcl-1-protein but not -mRNA in differentiated neutrophils.

<p>(A) Western blot showing expression of Bcl-2 family proteins in day 4 differentiated Bcl-2-transgenic neutrophils after various times of roscovitine (25 µM) treatment. Mcl-1 expression is also shown in roscovitine-treated WT and Noxa-/- differentiated neutrophils.Tubulin or GAPDH serves as a loading control. Two experiments were performed with very similar results. (B) Western blot showing expression of Mcl-1 in day 4 differentiated Bcl2- transgenic neutrophils upon treatment for various times with roscovitine (Ros, 25 µM) and/or the proteasome-inhibitor MG-132 (0.2 µM). Expression of GAPDH was used as a loading control. The Western blot is representative of 2 independent experiments. (C) Graph shows noxa and mcl-1 transcript levels in day 4 differentiated Bcl-2- transgenic neutrophils treated with roscovitine for 4h. Data are mean/SEM of 3 independent experiments. Statistical significance was calculated using Student’s t-test (ns, non-significant; p > 0.05; **, p<0,01).</p