Human cytomegalovirus (CMV) infections and relapse of disease remain major problems after allogeneic stem cell transplantation (allo-SCT), in particular in combination with CMV-negative donors or cordblood transplantations. Recent data suggest a paradoxical association between CMV reactivation after allo-SCT and reduced leukemic relapse. Given the potential of Vδ2-negative γδT cells to recognize CMV-infected cells and tumor cells, the molecular biology of distinct γδT-cell subsets expanding during CMV reactivation after allo-SCT was investigated. Vδ2(neg) γδT-cell expansions after CMV reactivation were observed not only with conventional but also cordblood donors. Expanded γδT cells were capable of recognizing both CMV-infected cells and primary leukemic blasts. CMV and leukemia reactivity were restricted to the same clonal population, whereas other Vδ2(neg) T cells interact with dendritic cells (DCs). Cloned Vδ1 T-cell receptors (TCRs) mediated leukemia reactivity and DC interactions, but surprisingly not CMV reactivity. Interestingly, CD8αα expression appeared to be a signature of γδT cells after CMV exposure. However, functionally, CD8αα was primarily important in combination with selected leukemia-reactive Vδ1 TCRs, demonstrating for the first time a co-stimulatory role of CD8αα for distinct γδTCRs. Based on these observations, we advocate the exploration of adoptive transfer of unmodified Vδ2(neg) γδT cells after allo-SCT to tackle CMV reactivation and residual leukemic blasts, as well as application of leukemia-reactive Vδ1 TCR-engineered T cells as alternative therapeutic tools.

Major limitations of currently investigated␣␤T cells redirected against cancer by transfer of tumor-specific ␣␤TCR arise from their low affinity, MHC restriction, and risk to mediate self-reactivity after pairing with endogenous ␣ or ␤TCR chains. Therefore, the ability of a defined ␥9␦2TCR to redirect ␣␤T cells selectively against tumor cells was tested and its molecular interaction with a variety of targets investigated. Functional analysis revealed that a ␥9␦2TCR efficiently reprograms both CD4 ؉ and CD8 ؉ IntroductionThe major challenge in the field of adoptive immunotherapy is the generation of tumor-reactive ␣␤T cells which can be applied to a broad variety of cancer patients. To facilitate the rapid generation of tumor-reactive ␣␤T cells, it has been proposed that ␣␤T cells can be reprogrammed with genes encoding for a tumor-specific ␣␤TCR or a chimeric receptor. 1 Several such receptors are already being used to redirect ␣␤T cells in phase 1 clinical trials. 1,2 However, reprogramming ␣␤T cells with defined ␣␤TCRs is substantially hampered by their restriction to HLA types, thus limiting the number of patients who can be treated with one ␣␤TCR. In addition, pairing of introduced with endogenous ␣␤TCR chains can induce life-threatening autoreactivity. 3,4 One attractive alternative to mediate a selective antitumor reactivity with a high-affinity TCR might arise from the ability of ␥␦T cells to mediate antitumor reactivity while ignoring a healthy environment. [5][6][7] Isolated ␥9␦2T cells efficiently kill tumor cells of hematologic malignancies and from solid tumors. 7 However, the function and proliferation capacity of ␥␦T cells is frequently heavily impaired in cancer patients 8 making autologous ␥␦T cells less attractive for immune interventions. On the other hand, as end-stage cancer patients can easily elicit ␣␤T-cell immune responses against, for example, viral Ags, 9,10 ␣␤T cells might serve as carriers for broadly tumor-reactive ␥␦TCRs.The recognition of mevalonate metabolites (phosphoantigens) 11 which are overexpressed in a broad range of tumor cells has been suggested as an important mechanism by which multiple ␥9␦2TCR can sense malignant transformation as the recognition involves TCR domains which are conserved in most ␥9␦2TCRs. [12][13][14] In addition, ␥9␦2TCR G115 has been also suggested to bind to a complex of Apolipoprotein AI (ApoAI) and F1-ATPase, 15 a complex mitochondrial enzyme found on the surface of many malignant cells. 16 This knowledge might allow a rational design of ␥␦T cell-based immunotherapies. Therefore, we investigated whether a defined ␥9␦2TCR can be efficiently expressed in ␣␤T cells, mediate tumor-specific proliferation of ␣␤T cells, and redirect both effector CD8 ϩ and helper CD4 ϩ ␣␤T-cell subsets against a broad panel of tumor cell lines while ignoring normal cells in vitro and in vivo. MethodsCell lines, Abs, the retroviral transduction and expansion of ␣␤T cells, functional T-cell assays 11,[17][18][19][20] as well as the animal model used are described in supp...

Adenosine deaminase acting on RNA 1 (ADAR1) is the master RNA editor, catalyzing the deamination of adenosine to inosine. RNA editing is vital for preventing abnormal activation of cytosolic nucleic acid sensing pathways by self-double-stranded RNAs. Here we determine, by parallel analysis of RNA secondary structure sequencing (PARS-seq), the global RNA secondary structure changes in ADAR1 deficient cells. Surprisingly, ADAR1 silencing resulted in a lower global double-stranded to single-stranded RNA ratio, suggesting that A-to-I editing can stabilize a large subset of imperfect RNA duplexes. The duplexes destabilized by editing are composed of vastly complementary inverted Alus found in untranslated regions of genes performing vital biological processes, including housekeeping functions and type-I interferon responses. They are predominantly cytoplasmic and generally demonstrate higher ribosomal occupancy. Our findings imply that the editing effect on RNA secondary structure is context dependent and underline the intricate regulatory role of ADAR1 on global RNA secondary structure.

Adenosine deaminase acting on RNA (ADAR) 1 binds and edits double-stranded (ds) RNA secondary structures found mainly within untranslated regions of many transcripts. In the current research, our aim was to study the role of ADAR1 in liver homeostasis. As previous studies show a conserved immunoregulatory function for ADAR1 in mammalians, we focused on its role in preventing chronic hepatic inflammation and the associated activation of hepatic stellate cells to produce extracellular matrix and promote fibrosis. We show that hepatocytes specific ADAR1 knock out (KO) mice display massive liver damage with multifocal inflammation and fibrogenesis. The bioinformatics analysis of the microarray gene-expression datasets of ADAR1 KO livers reveled a type-I interferons signature and an enrichment for immune response genes compared to control littermate livers. Furthermore, we found that in vitro silencing of ADAR1 expression in HepG2 cells leads to enhanced transcription of NFκB target genes, foremost of the pro-inflammatory cytokines IL6 and IL8. We also discovered immune cell-independent paracrine signaling among ADAR1-depleted HepG2 cells and hepatic stellate cells, leading to the activation of the latter cell type to adopt a profibrogenic phenotype. This paracrine communication dependent mainly on the production and secretion of the cytokine IL6 induced by ADAR1 silencing in hepatocytes. Thus, our findings shed a new light on the vital regulatory role of ADAR1 in hepatic immune homeostasis, chiefly its inhibitory function on the crosstalk between the NFκB and type-I interferons signaling cascades, restraining the development of liver inflammation and fibrosis.

The pathogenesis of juvenile idiopathic arthritis (JIA) is thought to involve multiple components of the cellular immune system, including subsets of γδ T cells. In this study, we conducted experiments to define the functional roles of one of the major synovial fluid (SF) T cell subsets, Vγ9+Vδ2+ (Vγ9+) T cells, in JIA. We found that as opposed to CD4+ T cells, equally high percentages (∼35%) of Vγ9+ T cells in SF and peripheral blood (PB) produced TNF-α and IFN-γ. Furthermore, stimulation with isopentenyl pyrophosphate (IPP), a metabolite in the mevalonate pathway, which is a specific potent Ag for Vγ9Jγ1.2+ T cells, similarly amplified cytokine secretion by SF and PB Vγ9+ T cells. Significantly, the SF subset expressed higher levels of CD69 in situ, suggesting their recent activation. Furthermore, 24-h coculturing with SF-derived fibroblasts enhanced CD69 on the SF > PB Vγ9+ T cells, a phenomenon strongly augmented by zoledronate, a farnesyl pyrophosphate synthase inhibitor that increases endogenous intracellular IPP. Importantly, although Vγ9+ T cell proliferation in response to IPP was significantly lower in SF than PBMC cultures, it could be enhanced by depleting SF CD4+CD25+FOXP3+ cells (regulatory T cells). Furthermore, coculture with the Vγ9+ T cells in medium containing zoledronate or IPP strongly increased SF-derived fibroblasts' apoptosis. The findings that IPP-responsive proinflammatory synovial Vγ9+ T cells for which proliferation is partly controlled by regulatory T cells can recognize and become activated by SF fibroblasts and then induce their apoptosis suggest their crucial role in the pathogenesis and control of synovial inflammation.

Human γδ T cells, which play innate and adaptive, protective as well as destructive, roles in the immune response, were discovered in 1986, but the clinical significance of alterations of the levels of these cells in the peripheral blood in human diseases has not been comprehensively reviewed. Here, we review patterns of easily measurable changes of this subset of T cells in peripheral blood from relevant publications in PubMed and their correlations with specific disease categories, specific diagnoses within disease categories, and prognostic outcomes. These collective data suggest that enumeration of γδ T cells and their subsets in the peripheral blood of patients could be a useful tool to evaluate diagnosis and prognosis in the clinical setting.

Failure to precisely repair DNA damage in self-renewing Hematopoietic Stem and early Progenitor Cells (HSPCs) can disrupt normal hematopoiesis and promote leukemogenesis. Although HSPCs are widely considered a target of ionizing radiation (IR)-induced hematopoietic injury, definitive data regarding cell death, DNA repair, and genomic stability in these rare quiescent cells are scarce. We found that irradiated HSPCs, but not lineage-committed progenitors (CPs), undergo rapid ATM-dependent apoptosis, which is suppressed upon interaction with bone-marrow stroma cells. Using DNA repair reporters to quantify mutagenic Non-Homologous End Joining (NHEJ) processes, we found that HSPCs exhibit reduced NHEJ activities in comparison with CPs. HSPC-stroma interactions did not affect the NHEJ capacity of HSPCs, emphasizing its cell autonomous regulation. We noted diminished expression of multiple double strand break (DSB) repair transcripts along with more persistent 53BP1 foci in irradiated HSPCs in comparison with CPs, which can account for low NHEJ activity and its distinct control in HSPCs. Finally, we documented clonal chromosomal aberrations in 10% of IR-surviving HSPCs. Taken together, our results revealed potential mechanisms contributing to the inherent susceptibility of human HSPC to the cytotoxic and mutagenic effects of DNA damage.

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