IL-12/15/18-preactivated NK cells suppress GvHD in a mouse model of mismatched hematopoietic cell transplantation.

Mismatched hematopoietic cell transplants for treating leukemia are complicated by graft versus host disease (GvHD). Here, we show that adoptively transferred IL-12/15/18-preactivated NK cells suppress GvHD in a mouse model of fully mismatched hematopoietic cell transplantation. These IL-12/15/18-preactivated NK cells maintained Eomesodermin (Eomes) and T-bet expression upon transfer and, while there was no evidence of direct killing of donor T cells or host DCs by the IL-12/15/18-preactivated NK cells, proliferation of donor T cells was inhibited. Strikingly, the graft versus leukemia effect mediated by donor T cells was retained, resulting in improved overall survival of mice that received lymphoma cells, donor allogeneic T cells, and IL-12/15/18-preactivated NK cells. These results suggest that IL-12/15/18-preactivated NK cells may be useful in improving immunotherapy of mismatched hematopoietic cell transplantation. Compared with previously proposed protocols, our findings suggest that in vitro NK-cell preactivation with this cytokine cocktail offers the significant advantage that cytokines do not need to be administered systemically to sustain NK-cell activity, thus avoiding toxicity.This research was supported by the Cambridge NIHR BRC Cell Phenotyping Hub and by research grants from the Wellcome Trust and from Leukaemia & Lymphoma Research to F.C.; C.M.H. was supported by a studentship from the Infection, Immunity and Inflammation PhD Programme funded by the Wellcome Trust.This is the final version of the article. It first appeared from Wiley via http://dx.doi.org/10.1002/eji.20144520

0235 : In vivo overexpression of a cardiac sodium channel mutant in mice

Loss-of-function mutations in the cardiac Na+ channel α-subunit gene, SCN5A, cause Brugada syndrome (BrS), a hereditary disease characterized by ventricular fibrillation and sudden cardiac death. We previously evidenced, in HEK cells, the dominant-negative effect of the R104W BrS mutation in Nav1.5, inducing the retention of the wild-type (WT) channel and the proteasomal degradation of the mutant protein. To explore this dominant-negative effect in vivo, we created a murine model using adeno-associated viruses (AAV).We used a dual AAV vector strategy combining viral DNA recombination and trans-splicing. One-week old mice were injected with two AAV serotypes capsid 9: one, packaging the cardiac specific troponin-T promoter, the 5’ half of hSCN5A, the 5’ donor site of a synthetic intron and a highly recombinogenic sequence; and another, packaging the same recombinogenic sequence, the 3’ acceptor site of the synthetic intron, the 3’ half of hSCN5A, the gfp gene as a reporter, and the SV40 polyA signal. Six weeks after injection, the hSCN5A full-gene expression and the percentage of transduced cardiac cells were assessed by qPCR, western blot (WB) analysis and immunohistochemistry on transduced heart tissues. The Na+ current was recorded by the patchclamp technique in isolated cardiomyocytes.Both WT and mutant human Nav1.5 transcripts and proteins were observed by RT-qPCR, WB and immunohistochemistry on injected-mice heart tissues. Patch-clamp recordings in WT-channel injected mice evidenced a two-fold increase of the Na+ current. In contrast, the cardiac Na+ current of R104Winjected mice was impaired (i.e. the current density was decreased by 45% and the activation was shifted by -4mV).Our data suggest that the trans-splicing and viral DNA recombination strategy using AAV9 serotype and a cardiac-specific promoter is successful to overexpress WT or mutant Na+ channels in mouse hearts. This approach allowed us to modulate the cardiac Na+ current in adult mice

Immunomodulation of Selective Naive T Cell Functions by p110δ Inactivation Improves the Outcome of Mismatched Cell Transplantation.

Allogeneic hematopoietic stem cell transplantation (HSCT) can treat certain hematologic malignancies due to the graft versus leukemia (GvL) effect but is complicated by graft versus host disease (GvHD). Expression of the p110δ catalytic subunit of the phosphoinositide 3-kinase pathway is restricted to leukocytes, where it regulates proliferation, migration, and cytokine production. Here, in a mouse model of fully mismatched hematopoietic cell transplantation (HCT), we show that genetic inactivation of p110δ in T cells leads to milder GvHD, whereas GvL is preserved. Inactivation of p110δ in human lymphocytes reduced T cell allorecognition. We demonstrate that both allostimulation and granzyme B expression were dependent on p110δ in naive T cells, which are the main mediators of GvHD, whereas memory T cells were unaffected. Strikingly, p110δ is not mandatory for either naive or memory T cells to mediate GvL. Therefore, immunomodulation of selective naive T cell functions by p110δ inactivation improves the outcome of allogeneic HSCT.This work was supported by grants from Biotechnology and Biological Sciences Research Council, the Wellcome Trust (088621/Z/09/Z), and Leukaemia & Lymphoma Research (13010) to F.C.This is the final published version. It originally appeared in Cell Reports,10, 702–710 February 10, 2015, DOI: 10.1016/j.celrep.2015.01.002

Rescue of fragile X syndrome phenotypes in Fmr1 KO mice by a BKCa channel opener molecule.

International audienc

Composition, Development, and Function of Uterine Innate Lymphoid Cells.

Innate lymphoid cells (ILCs), including NK cells, contribute to barrier immunity and tissue homeostasis. In addition to the role of uterine NK cells in placentation and fetal growth, other uterine ILCs (uILCs) are likely to play roles in uterine physiology and pathology. In this article, we report on the composition of uILCs in the endometrium during the luteal phase and in the decidua during early pregnancy. Whereas nonkiller uILC1s and uILC2s are barely detectable in mouse and not detected in humans, a sizeable population of uILC3s is found in human endometrium and decidua, which are mostly NCR(+) and partially overlap with previously described IL-22-producing uterine NK cells. Development of mouse uILC3 is Nfil3 independent, suggesting unique features of uILCs. Indeed, although the cytokine production profile of mouse uILCs recapitulates that described in other tissues, IL-5, IL-17, and IL-22 are constitutively produced by uILC2s and uILC3s. This study lays the foundation to understand how ILCs function in the specialized uterine mucosa, both in tissue homeostasis and barrier immunity and during pregnancy.Work supported by grants from the Wellcome Trust, the Medical Research Council, the British Heart Foundation and the Leukaemia & Lymphoma Research to FC. EB is the recipient of a Centre for Trophoblast Research Graduate Studentship. SB is the recipient of a Marie Curie FP7 Fellowship.This is the final version of the article. It first appeared from the American Association of Immunologists via http://dx.doi.org/10.4049/​jimmunol.150068

Development of a Human Cytomegalovirus (HCMV)-Based Therapeutic Cancer Vaccine Uncovers a Previously Unsuspected Viral Block of MHC Class I Antigen Presentation

Human cytomegalovirus (HCMV) induces a uniquely high frequency of virus-specific effector/memory CD8+ T-cells, a phenomenon termed “memory inflation”. Thus, HCMV-based vaccines are particularly interesting in order to stimulate a sustained and strong cellular immune response against cancer. Glioblastoma multiforme (GBM) is the most aggressive primary brain tumor with high lethality and inevitable relapse. The current standard treatment does not significantly improve the desperate situation underlining the urgent need to develop novel approaches. Although HCMV is highly fastidious with regard to species and cell type, GBM cell lines are susceptible to HCMV. In order to generate HCMV-based therapeutic vaccine candidates, we deleted all HCMV-encoded proteins (immunoevasins) that interfere with MHC class I presentation. The aim being to use the viral vector as an adjuvant for presentation of endogenous tumor antigens, the presentation of high levels of vector-encoded neoantigens and finally the repurposing of bystander HCMV-specific CD8+ T cells to fight the tumor. As neoantigen, we exemplarily used the E6 and E7 proteins of human papillomavirus type 16 (HPV-16) as a non-transforming fusion protein (E6/E7) that covers all relevant antigenic peptides. Surprisingly, GBM cells infected with E6/E7-expressing HCMV-vectors failed to stimulate E6-specific T cells despite high level expression of E6/E7 protein. Further experiments revealed that MHC class I presentation of E6/E7 is impaired by the HCMV-vector although it lacks all known immunoevasins. We also generated HCMV-based vectors that express E6-derived peptide fused to HCMV proteins. GBM cells infected with these vectors efficiently stimulated E6-specific T cells. Thus, fusion of antigenic sequences to HCMV proteins is required for efficient presentation via MHC class I molecules during infection. Taken together, these results provide the preclinical basis for development of HCMV-based vaccines and also reveal a novel HCMV-encoded block of MHC class I presentation

IL-22-expressing murine lymphocytes display plasticity and pathogenicity in reporter mice

IL-22 has multiple activities ranging from tissue repair to inflammation. To characterize the pathogenicity and plasticity of cells that produce IL-22 a novel reporter mouse strain was generated. Homeostatic IL-22 reporter expression was observed in intestinal lymphoid cells identified as CD4 T cells and ILC3 cells. In a model of inflammatory bowel disease (IBD), CD4 T cells strongly expressed the IL-22 reporter in mesenteric lymph node. To examine plasticity of IL-22+ T cells, they were purified after generation in vitro or in vivo from inflamed colon, then cultured under Th1, Th2 or Th17 conditions. In vitro-generated IL-22+ CD4 T cells showed relatively durable IL-22 expression under Th1 or Th2 conditions, whereas in vivo generated cells rapidly lost IL-22 expression under these conditions. In vitro-generated cells could not be diverted to express Th1 or Th2 cytokines despite the expression of master regulators. In vivo generated cells could be diverted, at very low frequency, to express Th1 or Th2 cytokines. Both in vitro- and in vivo-generated cells could be induced in vitro to express high levels of IL-17A and IL-17F, assigning them to a Th17 biased class. IL-27 potently downregulated IL-22 expression. To examine IL-22+ T cell pathogenicity, in vitro generated cells were transferred into Rag1-/- mice, retaining modest reporter expression and inducing moderate colitis. In contrast, IL-22 expressers from colitic mice, transferred into secondary hosts, lost reporter expression, acquired high Tbet and modest IFN and IL-17 expression and induced severe colitis. These findings are consistent with a model of strong polarization under optimal in vitro conditions, but a plastic state of T cells in vivo

iNKT cell development is orchestrated by different branches of TGF-β signaling

Invariant natural killer T (iNKT) cells constitute a distinct subset of T lymphocytes exhibiting important immune-regulatory functions. Although various steps of their differentiation have been well characterized, the factors controlling their development remain poorly documented. Here, we show that TGF-β controls the differentiation program of iNKT cells. We demonstrate that TGF-β signaling carefully and specifically orchestrates several steps of iNKT cell development. In vivo, this multifaceted role of TGF-β involves the concerted action of different pathways of TGF-β signaling. Whereas the Tif-1γ branch controls lineage expansion, the Smad4 branch maintains the maturation stage that is initially repressed by a Tif-1γ/Smad4-independent branch. Thus, these three different branches of TGF-β signaling function in concert as complementary effectors, allowing TGF-β to fine tune the iNKT cell differentiation program

Migratory and adhesive cues controlling innate-like lymphocyte surveillance of the pathogen-exposed surface of the lymph node

Lymph nodes (LNs) contain innate-like lymphocytes that survey the subcapsular sinus (SCS) and associated macrophages for pathogen entry. The factors promoting this surveillance behavior have not been defined. Here, we report that IL7R(hi)Ccr6(+) lymphocytes in mouse LNs rapidly produce IL17 upon bacterial and fungal challenge. We show that these innate-like lymphocytes are mostly LN resident. Ccr6 is required for their accumulation near the SCS and for efficient IL17 induction. Migration into the SCS intrinsically requires S1pr1, whereas movement from the sinus into the parenchyma involves the integrin LFA1 and its ligand ICAM1. CD169, a sialic acid-binding lectin, helps retain the cells within the sinus, preventing their loss in lymph flow. These findings establish a role for Ccr6 in augmenting innate-like lymphocyte responses to lymph-borne pathogens, and they define requirements for cell movement between parenchyma and SCS in what we speculate is a program of immune surveillance that helps achieve LN barrier immunity. DOI: http://dx.doi.org/10.7554/eLife.18156.00