IL-1ß+ macrophages fuel pathogenic inflammation in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is a lethal disease with high resistance to therapies1. Inflammatory and immunomodulatory signals co-exist in the pancreatic tumour microenvironment, leading to dysregulated repair and cytotoxic responses. Tumour-associated macrophages (TAMs) have key roles in PDAC2, but their diversity has prevented therapeutic exploitation. Here we combined single-cell and spatial genomics with functional experiments to unravel macrophage functions in pancreatic cancer. We uncovered an inflammatory loop between tumour cells and interleukin-1ß (IL-1ß)-expressing TAMs, a subset of macrophages elicited by a local synergy between prostaglandin E2 (PGE2) and tumour necrosis factor (TNF). Physical proximity with IL-1ß+ TAMs was associated with inflammatory reprogramming and acquisition of pathogenic properties by a subset of PDAC cells. This occurrence was an early event in pancreatic tumorigenesis and led to persistent transcriptional changes associated with disease progression and poor outcomes for patients. Blocking PGE2 or IL-1ß activity elicited TAM reprogramming and antagonized tumour cell-intrinsic and -extrinsic inflammation, leading to PDAC control in vivo. Targeting the PGE2-IL-1ß axis may enable preventive or therapeutic strategies for reprogramming of immune dynamics in pancreatic cancer.

Loss of ribonuclease DIS3 hampers genome integrity in myeloma by disrupting DNA:RNA hybrid metabolism.

The ribonuclease DIS3 is one of the most frequently mutated genes in the hematological cancer multiple myeloma, yet the basis of its tumor suppressor function in this disease remains unclear. Herein, exploiting the TCGA dataset, we found that DIS3 plays a prominent role in the DNA damage response. DIS3 inactivation causes genomic instability by increasing mutational load, and a pervasive accumulation of DNA:RNA hybrids that induces genomic DNA double-strand breaks (DSBs). DNA:RNA hybrid accumulation also prevents binding of the homologous recombination (HR) machinery to double-strand breaks, hampering DSB repair. DIS3-inactivated cells become sensitive to PARP inhibitors, suggestive of a defect in homologous recombination repair. Accordingly, multiple myeloma patient cells mutated for DIS3 harbor an increased mutational burden and a pervasive overexpression of pro-inflammatory interferon, correlating with the accumulation of DNA:RNA hybrids. We propose DIS3 loss in myeloma to be a driving force for tumorigenesis via DNA:RNA hybrid-dependent enhanced genome instability and increased mutational rate. At the same time, DIS3 loss represents a liability that might be therapeutically exploited in patients whose cancer cells harbor DIS3 mutations.

The transcriptional regulator Sin3A balances IL-17A and Foxp3 expression in primary CD4 T cells.

The Sin3 transcriptional regulator homolog A (Sin3A) is the core member of a multiprotein chromatin-modifying complex. Its inactivation at the CD4/CD8 double-negative stage halts further thymocyte development. Among various functions, Sin3A regulates STAT3 transcriptional activity, central to the differentiation of Th17 cells active in inflammatory disorders and opportunistic infections. To further investigate the consequences of conditional Sin3A inactivation in more mature precursors and post-thymic T cell, we have generated CD4-Cre and CD4-CreERT2 Sin3AF/F mice. Sin3A inactivation in vivo hinders both thymocyte development and peripheral T-cell survival. In vitro, in Th17 skewing conditions, Sin3A-deficient cells proliferate and acquire memory markers and yet fail to properly upregulate Il17a, Il23r, and Il22. Instead, IL-2+ and FOXP3+ are mostly enriched for, and their inhibition partially rescues IL-17A+ T cells. Notably, Sin3A deletion also causes an enrichment of genes implicated in the mTORC1 signaling pathway, overt STAT3 activation, and aberrant cytoplasmic RORγt accumulation. Thus, together our data unveil a previously unappreciated role for Sin3A in shaping critical signaling events central to the acquisition of immunoregulatory T-cell phenotypes.

Flow cytometry data mining by cytoChain identifies determinants of exhaustion and stemness in TCR-engineered T cells.

The phenotype of infused cells is a major determinant of Adoptive T-cell therapy (ACT) efficacy. Yet, the difficulty in deciphering multiparametric cytometry data limited the fine characterization of cellular products. To allow the analysis of dynamic and complex flow cytometry samples, we developed cytoChain, a novel dataset mining tool and a new analytical workflow. CytoChain was challenged to compare state-of-the-art and innovative culture conditions to generate stem-like memory cells (TSCM ) suitable for ACT. Noticeably, the combination of IL-7/15 and superoxides scavenging sustained the emergence of a previously unidentified nonexhausted Fit-TSCM signature, overlooked by manual gating and endowed with superior expansion potential. CytoChain proficiently traced back this population in independent datasets, and in T-cell receptor engineered lymphocytes. CytoChain flexibility and function were then further validated on a published dataset from circulating T cells in COVID-19 patients. Collectively, our results support the use of cytoChain to identify novel, functionally critical immunophenotypes for ACT and patients immunomonitoring.

Corneal endothelial cell reduction and increased Neurokinin-1 receptor expression in a graft-versus-host disease preclinical model.

The aim of this work was to assess corneal endothelial morphology in a well-established acute graft-versus-host disease (GVHD) murine model and to quantify the expression of neurokinin-1 receptor (NK1R) in the corneal endothelium during ocular GVHD (oGVHD). Pre-conditioning was performed in BALB/c using myeloablative total body irradiation. Subsequently, allogeneic bone marrow transplantation was performed without (BM) or with mature T cells (BM + T). Corneal transparency was monitored with in vivo biomicroscopy. After sacrifice, corneal thickness and endothelial cell number were measured, and the expression of NK1R was investigated in the corneal endothelium through immunofluorescence and quantified by immunohistochemistry. Mice presenting oGVHD showed a significant reduction in endothelial cell number compared to control animals (p < 0.0001). NK1R expression was significantly increased in oGVHD mice endothelium (p < 0.05). Corneal transparency and thickness were unchanged in all groups. Our results suggest that oGVHD affects the corneal endothelium, inducing a reduction of the cell number, and that this is associated with increased expression of the pro-inflammatory marker NK1R.

Topical neurokinin-1 receptor antagonist Fosaprepitant ameliorates ocular graft-versus-host disease in a preclinical mouse model.

PURPOSE: to assess the effect of topical administration of the Neurokin-1 receptor (NK1R) antagonist Fosaprepitant in a pre-clinical model of ocular Graft-versus-Host disease (GVHD). METHODS: BALB/c mice were pre-conditioned by myeloablative total body irradiation and subjected to allogeneic bone marrow transplantation and mature T cell infusion (BM + T). BM-transplanted mice (BM) were used as controls. Ocular GVHD was specifically assessed by quantifying corneal epithelial damage, tear secretion, blepharitis and phimosis, 3 times/week for 28 days post-transplantation. A group of BM + T mice received Fosaprepitant 10 mg/mL, 6 times/day, topically, from day 7-29 after transplantation. After sacrifice, the expression of NK1R, CD45, CD3, and CXCL10 was quantified in the cornea, conjunctiva, and lacrimal gland by immunohistochemistry. RESULTS: BM + T mice developed corneal epithelial damage (day 0-29, p < 0.001), blepharitis (day 0-29, p < 0.001), and phimosis (day 0-29, p < 0.01), and experienced decreased tear secretion (day 21, p < 0.01) compared to controls. NK1R was found upregulated in corneal epithelium (p < 0.01) and lacrimal gland (p < 0.01) of BM + T mice. Fosaprepitant administration significantly reduced corneal epithelial damage (p < 0.05), CD45+ (p < 0.05) and CD3+ (p < 0.01) immune cell infiltration in the cornea and conjunctiva (p < 0.001 and p < 0.001, respectively). In addition, Fosaprepitant reduced the expression of CXCL10 in the cornea (p < 0.05) and in the lacrimal gland (p < 0.05). CONCLUSIONS: Our results suggest that NK1R represents a novel druggable pathway for the therapy of ocular GVHD.

Enhancement of doxorubicin anti-cancer activity by vascular targeting using IsoDGR/cytokine-coated nanogold.

BACKGROUND: Gold nanospheres tagged with peptides containing isoDGR (isoAsp-Gly-Arg), an αvß3 integrin binding motif, represent efficient carriers for delivering pro-inflammatory cytokines to the tumor vasculature. We prepared bi- or trifunctional nanoparticles bearing tumor necrosis factor-α (TNF) and/or interleukin-12 (IL12) plus a peptide containing isoDGR, and we tested their anti-cancer effects, alone or in combination with doxorubicin, in tumor-bearing mice. RESULTS: In vitro biochemical studies showed that both nanodrugs were monodispersed and functional in terms of binding to TNF and IL12 receptors and to αvß3. In vivo studies performed in a murine model of fibrosarcoma showed that low doses of bifunctional nanoparticles bearing isoDGR and TNF (corresponding to few nanoparticles per cell) delayed tumor growth and increased the efficacy of doxorubicin without worsening its toxicity. Similar effects were obtained using trifunctional nanoparticles loaded with isoDGR, TNF and IL12. Mechanistic studies showed that nanoparticles bearing isoDGR and TNF could increase doxorubicin penetration in tumors a few hours after injection and caused vascular damage at later time points. CONCLUSION: IsoDGR-coated gold nanospheres can be exploited as a versatile platform for single- or multi-cytokine delivery to cells of the tumor vasculature. Extremely low doses of isoDGR-coated nanodrugs functionalized with TNF or TNF plus IL12 can enhance doxorubicin anti-tumor activity.

Boosting Interleukin-12 Antitumor Activity and Synergism with Immunotherapy by Targeted Delivery with isoDGR-Tagged Nanogold.

The clinical use of interleukin-12 (IL12), a cytokine endowed with potent immunotherapeutic anticancer activity, is limited by systemic toxicity. The hypothesis is addressed that gold nanoparticles tagged with a tumor-homing peptide containing isoDGR, an αvß3-integrin binding motif, can be exploited for delivering IL12 to tumors and improving its therapeutic index. To this aim, gold nanospheres are functionalized with the head-to-tail cyclized-peptide CGisoDGRG (Iso1) and murine IL12. The resulting nanodrug (Iso1/Au/IL12) is monodispersed, stable, and bifunctional in terms of αvß3 and IL12-receptor recognition. Low-dose Iso1/Au/IL12, equivalent to 18-75 pg of IL12, induces antitumor effects in murine models of fibrosarcomas and mammary adenocarcinomas, with no evidence of toxicity. Equivalent doses of Au/IL12 (a nanodrug lacking Iso1) fail to delay tumor growth, whereas 15 000 pg of free IL12 is necessary to achieve similar effects. Iso1/Au/IL12 significantly increases tumor infiltration by innate immune cells, such as NK and iNKT cells, monocytes, and neutrophils. NK cell depletion completely inhibits its antitumor effects. Low-dose Iso1/Au/IL12 can also increase the therapeutic efficacy of adoptive T-cell therapy in mice with autochthonous prostate cancer. These findings indicate that coupling IL12 to isoDGR-tagged nanogold is a valid strategy for enhancing its therapeutic index and sustaining adoptive T-cell therapy.

NY-ESO-1 TCR single edited stem and central memory T cells to treat multiple myeloma without graft-versus-host disease.

Transfer of T-cell receptors (TCRs) specific for tumor-associated antigens is a promising approach for cancer immunotherapy. We developed the TCR gene editing technology that is based on the knockout of the endogenous TCR α and ß genes, followed by the introduction of tumor-specific TCR genes, and that proved safer and more effective than conventional TCR gene transfer. Although successful, complete editing requires extensive cell manipulation and 4 transduction procedures. Here we propose a novel and clinically feasible TCR "single editing" (SE) approach, based on the disruption of the endogenous TCR α chain only, followed by the transfer of genes encoding for a tumor-specific TCR. We validated SE with the clinical grade HLA-A2 restricted NY-ESO-1157-165-specific TCR. SE allowed the rapid production of high numbers of tumor-specific T cells, with optimal TCR expression and preferential stem memory and central memory phenotype. Similarly to unedited T cells redirected by TCR gene transfer (TCR transferred [TR]), SE T cells efficiently killed NY-ESO-1pos targets; however, although TR cells proved highly alloreactive, SE cells showed a favorable safety profile. Accordingly, when infused in NSG mice previously engrafted with myeloma, SE cells mediated tumor rejection without inducing xenogeneic graft-versus-host disease, thus resulting in significantly higher survival than that observed in mice treated with TR cells. Overall, single TCR gene editing represents a clinically feasible approach that is able to increase the safety and efficacy of cancer adoptive immunotherapy.

Interleukin-7 Unveils Pathogen-Specific T Cells by Enhancing Antigen-Recall Responses.

Background: Interleukin (IL)-7 promotes the generation, expansion, and survival of memory T cells. Previous mouse and human studies showed that IL-7 can support immune cell reconstitution in lymphopenic conditions, expand tumor-reactive T cells for adoptive immunotherapy, and enhance effector cytokine expression by autoreactive T cells. Whether pathogen-reactive T cells also benefit from IL-7 exposure remains unknown. Methods: In this study, we investigated this issue in cultures of peripheral blood mononuclear cells (PBMCs) derived from patients infected with various endemic pathogens. After short-term exposure to IL-7, we measured PBMC responses to antigens derived from pathogens, such as Mycobacterium tuberculosis, Candida albicans, and cytomegalovirus, and to the superantigen Staphylococcus aureus enterotoxin B. Results: We found that IL-7 favored the expansion and, in some instances, the uncovering of pathogen-reactive CD4 T cells, by promoting pathogen-specific interferon-γ, IL-2, and tumor necrosis factor recall responses. Conclusions: Our findings indicate that IL-7 unveils and supports reactivation of pathogen-specific T cells with possible diagnostic, prognostic, and therapeutic significance of clinical value, especially in conditions of pathogen persistence and chronic infection.

Mycophenolate mofetil for the treatment of children with immune thrombocytopenia and Evans syndrome. A retrospective data review from the Italian association of paediatric haematology/oncology.

Mycophenolate mofetil (MMF) has been shown to be effective in children with immune thrombocytopenia (ITP) and Evans syndrome (ES), but data from larger series and details on the timing of the response are lacking. We evaluated 56 children treated with MMF for ITP (n = 40) or ES (n = 16), which was primary or secondary to autoimmune lymphoproliferative syndrome -related syndrome (ARS). Thirty-five of the 54 evaluable patients (65%) achieved a partial (18%) or complete (46%) response after a median (range) of 20 (7-137) and 37 (7-192) d, respectively. ITP and ES patients responded in 58% and 81% of cases (P = not significant, ns), with complete response in 32% and 81% (P = 0·01), respectively. 60% and 73% of children with primary disease and ARS responded (P = ns) with complete response in 34% and 68% of cases (P = 0·01), respectively. Six of 35 (17%) children relapsed after a median of 283 d (range 189-1036). Limited toxicity was observed in four patients. The median durations of treatment and follow-up were seven and 12·7 months, respectively. This is the largest reported cohort of patients treated with MMF for ITP/ES. The results show that MMF is effective and safe and provides a relatively quick response, suggesting that it has a potential role as an alternative to more aggressive and expensive second/further-line treatments.

Adoptive T-cell therapy for cancer: The era of engineered T cells.

Tumors originate from a number of genetic events that deregulate homeostatic mechanisms controlling normal cell behavior. The immune system, devoted to patrol the organism against pathogenic events, can identify transformed cells, and in several cases cause their elimination. It is however clear that several mechanisms encompassing both central and peripheral tolerance limit antitumor immunity, often resulting into progressive diseases. Adoptive T-cell therapy with either allogeneic or autologous T cells can transfer therapeutic immunity. To date, genetic engineering of T cells appears to be a powerful tool for shaping tumor immunity. In this review, we discuss the most recent achievements in the areas of suicide gene therapy, and TCR-modified T cells and chimeric antigen receptor gene-modified T cells. We provide an overview of current strategies aimed at improving the safety and efficacy of these approaches, with an outlook on prospective developments.

Metabolic determinants of the immune modulatory function of neural stem cells.

BACKGROUND: Neural stem cells (NSCs) display tissue trophic and immune modulatory therapeutic activities after transplantation in central nervous system disorders. The intercellular interplay between stem cells and target immune cells is increased in NSCs exposed to inflammatory cues. Here, we hypothesize that inflammatory cytokine signalling leads to metabolic reprogramming of NSCs regulating some of their immune modulatory effects. METHODS: NSC lines were prepared from the subventricular zone (SVZ) of 7-12-week-old mice. Whole secretome-based screening and analysis of intracellular small metabolites was performed in NSCs exposed to cocktails of either Th1-like (IFN-γ, 500 U/ml; TNF-α, 200 U/ml; IL-1ß, 100 U/ml) or Th2-like (IL-4, IL-5 and IL-13; 10 ng/ml) inflammatory cytokines for 16 h in vitro. Isotopologues distribution of arginine and downstream metabolites was assessed by liquid chromatography/mass spectrometry in NSCs incubated with U-(13)C6 L-arginine in the presence or absence of Th1 or Th2 cocktails (Th1 NSCs or Th2 NSCs). The expression of arginase I and II was investigated in vitro in Th1 NSCs and Th2 NSCs and in vivo in the SVZ of mice with experimental autoimmune encephalomyelitis, as prototypical model of Th1 cell-driven brain inflammatory disease. The effects of the inflammatory cytokine signalling were studied in NSC-lymph node cells (LNC) co-cultures by flow cytometry-based analysis of cell proliferation following pan-arginase inhibition with N(ω)-hydroxy-nor-arginine (nor-NOHA). RESULTS: Cytokine-primed NSCs showed significantly higher anti-proliferative effect in co-cultures vs. control NSCs. Metabolomic analysis of intracellular metabolites revealed alteration of arginine metabolism and increased extracellular arginase I activity in cytokine-primed NSCs. Arginase inhibition by nor-NOHA partly rescued the anti-proliferative effects of cytokine-primed NSCs. CONCLUSIONS: Our work underlines the use of metabolic profiling as hypothesis-generating tools that helps unravelling how stem cell-mediated mechanisms of tissue restoration become affected by local inflammatory responses. Among different therapeutic candidates, we identify arginase signalling as novel metabolic determinant of the NSC-to-immune system communication.

IL-17 protects T cells from apoptosis and contributes to development of ALPS-like phenotypes.

In autoimmune/lymphoproliferative syndrome (ALPS), defective Fas death receptor function causes lymphadenomegaly/splenomegaly, the expansion of T-cell receptor αß(+) CD4/CD8 double-negative T cells, and frequent development of hematologic autoimmunity. Dianzani autoimmune lymphoproliferative disease (DALD) has a similar phenotype but lacks the expansion of double-negative T cells. This work shows that patients with ALPS and DALD have high serum levels of interleukin 17A (IL-17A), IL-17F, and IL-17AF, which are involved in several autoimmune diseases, and that their T cells show increased secretion of these cytokines upon activation in vitro. The following data indicate that these cytokines may contribute to ALPS and DALD: (1) recombinant IL-17A and IL-17F significantly inhibit Fas-induced cell death in Fas-sensitive T cells from healthy donors; (2) this inhibitory effect is also induced by the patients' serum and is reversed by anti-IL-17A antibodies; (3) IL-17A neutralization substantially increases Fas-induced cell death in T cells from ALPS and DALD patients in vitro; and (4) treatment with anti-IL-17A antibodies ameliorates the autoimmune manifestations and, at a lesser extent, the lymphoproliferative phenotype and prolongs survival in MRLlpr/lpr mice, which are an animal model of ALPS. These data suggest that IL-17A and IL-17F could be targeted therapeutically to improve Fas function in ALPS and DALD.

Trick to treat: tricking the thymus to treat cancer.

In this issue of Blood, Schmitt et al address the biology and safety of T cells engineered to express T-cell receptor (TCR) variants endowed with enhanced affinity for tumor-associated antigens.

IL-7 and IL-15 instruct the generation of human memory stem T cells from naive precursors.

Long-living memory stem T cells (T(SCM)) with the ability to self-renew and the plasticity to differentiate into potent effectors could be valuable weapons in adoptive T-cell therapy against cancer. Nonetheless, procedures to specifically target this T-cell population remain elusive. Here, we show that it is possible to differentiate in vitro, expand, and gene modify in clinically compliant conditions CD8(+) T(SCM) lymphocytes starting from naive precursors. Requirements for the generation of this T-cell subset, described as CD62L(+)CCR7(+)CD45RA(+)CD45R0(+)IL-7Rα(+)CD95(+), are CD3/CD28 engagement and culture with IL-7 and IL-15. Accordingly, T(SCM) accumulates early after hematopoietic stem cell transplantation. The gene expression signature and functional phenotype define this population as a distinct memory T-lymphocyte subset, intermediate between naive and central memory cells. When transplanted in immunodeficient mice, gene-modified naive-derived T(SCM) prove superior to other memory lymphocytes for the ability to expand and differentiate into effectors able to mediate a potent xenogeneic GVHD. Furthermore, gene-modified T(SCM) are the only T-cell subset able to expand and mediate GVHD on serial transplantation, suggesting self-renewal capacity in a clinically relevant setting. These findings provide novel insights into the origin and requirements for T(SCM) generation and pave the way for their clinical rapid exploitation in adoptive cell therapy.

Targeting TNF-α to neoangiogenic vessels enhances lymphocyte infiltration in tumors and increases the therapeutic potential of immunotherapy.

Abnormal tumor vasculature impairs T lymphocyte adhesion to endothelial cells and lymphocyte extravasation into neoplastic tissues, limiting the therapeutic potential of both active and adoptive immunotherapies. We have found that treatment of tumor-bearing mice with NGR-TNF, a Cys-Asn-Gly-Arg-Cys peptide-TNF fusion product capable of altering the endothelial barrier function and improving drug penetration in tumors, associated with the intratumor upregulation of leukocyte-endothelial cell adhesion molecules, the release of proinflammatory cytokines and chemokines, and the infiltration of tumor-specific effector CD8(+) T cells. As a result, NGR-TNF enhanced the therapeutic activity of adoptive and active immunotherapy, delaying tumor growth and prolonging survival. Furthermore, we have found that therapeutic effects of these combinations can be further increased by the addition of chemotherapy. Thus, these findings might be relevant for the design of novel immunotherapeutic approaches for cancer patients.

Non-canonical antigens are the largest fraction of peptides presented by MHC class I in mismatch repair deficient murine colorectal cancer.

BACKGROUND: Immunotherapy based on checkpoint inhibitors is highly effective in mismatch repair deficient (MMRd) colorectal cancer (CRC). These tumors carry a high number of mutations, which are predicted to translate into a wide array of neoepitopes; however, a systematic classification of the neoantigen repertoire in MMRd CRC is lacking. Mass spectrometry peptidomics has demonstrated the existence of MHC class I associated peptides (MAPs) originating from non-coding DNA regions. Based on these premises we investigated DNA genomic regions responsible for generating MMRd-induced peptides. METHODS: We exploited mouse CRC models in which the MMR gene Mlh1 was genetically inactivated. Isogenic cell lines CT26 Mlh1+/+ and Mlh1-/- were inoculated in immunocompromised and immunocompetent mice. Whole genome and RNA sequencing data were generated from samples obtained before and after injection in murine hosts. First, peptide databases were built from transcriptomes of isogenic cell lines. We then compiled a database of peptides lost after tumor cells injection in immunocompetent mice, likely due to immune editing. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) and matched next-generation sequencing databases were employed to identify the DNA regions from which the immune-targeted MAPs originated. Finally, we adopted in vitro T cell assays to verify whether MAP-specific T cells were part of the in vivo immune response against Mlh1-/- cells. RESULTS: Whole genome sequencing analyses revealed an unbalanced distribution of immune edited alterations across the genome in Mlh1-/- cells grown in immunocompetent mice. Specifically, untranslated (UTR) and coding regions exhibited the largest fraction of mutations leading to highly immunogenic peptides. Moreover, the integrated computational and LC-MS/MS analyses revealed that MAPs originate mainly from atypical translational events in both Mlh1+/+ and Mlh1-/- tumor cells. In addition, mutated MAPs-derived from UTRs and out-of-frame translation of coding regions-were highly enriched in Mlh1-/- cells. The MAPs trigger T-cell activation in mice primed with Mlh1-/- cells. CONCLUSIONS: Our results suggest that-in comparison to MMR proficient CRC-MMRd tumors generate a significantly higher number of non-canonical mutated peptides able to elicit T cell responses. These results reveal the importance of evaluating the diversity of neoepitope repertoire in MMRd tumors.

A signaling loop of REST, TSC2 and ß-catenin governs proliferation and function of PC12 neural cells.

The RE-1-specific silencing transcription factor (REST or NRSF) is a transcription repressor that orchestrates differentiation and also operates in differentiated neurons and neurosecretory cells (neural cells). Its role in proliferation has been investigated so far only in rapidly growing tumors, with conflicting results: suppression in non-neural tumors, stimulation in medulloblastomas. Working with two clones of chromaffin-neuronal PC12 cells, which express different levels of REST, and using genetic complementation and knockdown approaches, we show that REST also promotes proliferation in differentiated neural cells. Mechanistically, this occurs by a signaling pathway involving REST, the GTPase-activating protein tuberin (TSC2) and the transcription co-factor ß-catenin. In PC12 cells, raised expression of REST correlates with reduced TSC2 levels, nuclear accumulation and co-transcriptional activation of ß-catenin, and increased expression of its target oncogenes Myc and Ccnd1, which might account for the proliferation advantage and the distinct morphology. Rest transcription is also increased, unveiling the existence of a self-sustaining, feed-forward REST-TSC2-ß-catenin signaling loop that is also operative in another neural cell model, NT2/D1 cells. Transfection of REST, knockdown of TSC2 or forced expression of active ß-catenin recapitulated the biochemical, functional and morphological properties of the high-expressing REST clone in wild-type PC12 cells. Upregulation of REST promoted proliferation and phenotypic changes, thus hindering neurosecretion. The new REST-TSC2-ß-catenin signaling paradigm might have an important role in various aspects of neural cell physiology and pathology, including the regulation of proliferation and neurosecretion.

Concurrent allorecognition has a limited impact on posttransplant vaccination.

Transplantation of allogeneic hematopoietic stem cells with or without immunocompetent lymphocytes has proved a successful strategy in the treatment of hematological malignancies. We have recently shown that this approach can also cure mouse prostate cancer, provided that it is combined with tumor-specific vaccination. Whether the response to alloantigens acts by providing helper function to enhance vaccine-specific responses or in other ways impinges on vaccine immunogenicity remains to be clarified, and this question is of clinical relevance. In this study, we have addressed this issue by comparing the immunogenicity of dendritic cells pulsed with a peptide derived from a tumor/viral model Ag in recipients of donor cells either syngeneic to the host or differing for either Y-encoded or multiple minor H antigens. We report that vaccination elicits comparable proliferation and differentiation of peptide-specific CD8(+) T cells despite concurrent expansion and differentiation of minor H antigen-specific IFN-γ effector T cells. Depletion of alloreactive CD4(+) T cells reduced alloreactivity but not vaccine-induced CD8(+) T cell priming, suggesting that alloresponses do not provide helper functions in peripheral lymphoid tissues. Vaccine-mediated T cell priming was also preserved in the case of multiple minor H antigen disparities, prone to graft-versus-host disease. Thus, in the context of nonmyeloablative allotransplantation aimed at restoring an effective tumor-specific T cell repertoire, minor H antigen-specific T cells do not interfere with vaccine-induced T cell priming, supporting the notion that posttransplant vaccination is a valuable strategy to boost tumor and pathogen-specific protective immunity.