NK Cells from RAG- or DCLRE1C-Deficient Patients Inhibit HCMV

The recombination-activating genes (RAGs) and the DNA cross-link repair 1C gene (DCLRE1C) encode the enzymes RAG1, RAG2 and Artemis. They are critical components of the V(D)J recombination machinery. V(D)J recombination is well known as a prerequisite for the development and antigen diversity of T and B cells. New findings suggested that RAG deficiency impacts the cellular fitness and function of murine NK cells. It is not known whether NK cells from severe combined immunodeficiency (SCID) patients with defective RAGs or DCLRE1C (RAGs−/DCLRE1C−-NK) are active against virus infections. Here, we evaluated the anti-HCMV activity of RAGs−/DCLRE1C−-NK cells. NK cells from six SCID patients were functional in inhibiting HCMV transmission between cells in vitro. We also investigated the expansion of HCMV-induced NK cell subset in the RAG- or DCLRE1C-deficient patients. A dynamic expansion of NKG2C+ NK cells in one RAG-2-deficient patient was observed post HCMV acute infection. Our study firstly reveals the antiviral activity of human RAGs−/ DCLRE1C−-NK cells

Regulated expression of nuclear receptor RORγt confers distinct functional fates to NK cell receptor-expressing RORγt(+) innate lymphocytes

Whether the recently identified innate lymphocyte population coexpressing natural killer cell receptors (NKRs) and the nuclear receptor RORγt is part of the NK or lymphoid tissue inducer (LTi) cell lineage remains unclear. By using adoptive transfer of genetically tagged LTi-like cells, we demonstrate that NKR⁻RORγt(+) innate lymphocytes but not NK cells were direct progenitors to NKR(+)RORγt(+) cells in vivo. Genetic lineage tracing revealed that the differentiation of LTi-like cells was characterized by the stable upregulation of NKRs and a progressive loss of RORγt expression. Whereas interleukin-7 (IL-7) and intestinal microbiota stabilized RORγt expression within such NKR-LTi cells, IL-12 and IL-15 accelerated RORγt loss. RORγt(+) NKR-LTi cells produced IL-22, whereas RORγt⁻ NKR-LTi cells released IFN-γ and were potent inducers of colitis. Thus, the RORγt gradient in NKR-LTi cells serves as a tunable rheostat for their functional program. Our data also define a previously unappreciated role of RORγt⁻ NKR-LTi cells for the onset or maintenance of inflammatory bowel diseases

Regulated expression of nuclear receptor ROR t confers distinct functional fates to NK cell receptor-expressing ROR t(+) innate lymphocytes

Whether the recently identified innate lymphocyte population coexpressing natural killer cell receptors (NKRs) and the nuclear receptor ROR t is part of the NK or lymphoid tissue inducer (LTi) cell lineage remains unclear. By using adoptive transfer of genetically tagged LTi-like cells, we demonstrate that NKR ? ROR t(+) innate lymphocytes but not NK cells were direct progenitors to NKR(+)ROR t(+) cells in vivo. Genetic lineage tracing revealed that the differentiation of LTi-like cells was characterized by the stable upregulation of NKRs and a progressive loss of ROR t expression. Whereas interleukin-7 (IL-7) and intestinal microbiota stabilized ROR t expression within such NKR-LTi cells, IL-12 and IL-15 accelerated ROR t loss. ROR t(+) NKR-LTi cells produced IL-22, whereas ROR t ? NKR-LTi cells released IFN- and were potent inducers of colitis. Thus, the ROR t gradient in NKR-LTi cells serves as a tunable rheostat for their functional program. Our data also define a previously unappreciated role of ROR t ? NKR-LTi cells for the onset or maintenance of inflammatory bowel diseases

Comparison of outcomes of hematopoietic stem cell transplantation without chemotherapy conditioning by using matched sibling and unrelated donors for treatment of severe combined immunodeficiency

BACKGROUND: Patients with severe combined immunodeficiency disease who have matched sibling donors (MSDs) can proceed to hematopoietic cell transplantation (HCT) without conditioning chemotherapy. OBJECTIVE: We sought to determine whether the results of HCT without chemotherapy-based conditioning from matched unrelated donors (URDs), either from volunteer adults or umbilical cord blood, are comparable with those from MSDs. METHODS: We performed a multicenter survey of severe combined immunodeficiency transplantation centers in North America, Europe, and Australia to compile retrospective data on patients who have undergone unconditioned HCT from either URDs (n = 37) or MSDs (n = 66). RESULTS: Most patients undergoing URD HCT (92%) achieved donor T-cell engraftment compared with 97% for those with MSDs; however, estimated 5-year overall and event-free survival were worse for URD recipients (71% and 60%, respectively) compared with MSD recipients (92% and 89%, respectively; P < .01 for both). URD recipients who received pre-HCT serotherapy had similar 5-year overall survival (100%) to MSD recipients. The incidences of grade II to IV acute and chronic graft-versus-host disease were higher in URD (50% and 39%, respectively) compared with MSD (22% and 5%, respectively) recipients (P < .01 for both). In the surviving patients there was no difference in T-cell reconstitution at the last follow-up between the URD and MSD recipients; however, MSD recipients were more likely to achieve B-cell reconstitution (72% vs 17%, P < .001). CONCLUSION: Unconditioned URD HCT achieves excellent rates of donor T-cell engraftment similar to that seen in MSD recipients, and reconstitution rates are adequate. However, only a minority will have myeloid and B-cell reconstitution, and attention must be paid to graft-versus-host disease prophylaxis. This approach might be safer in children ineligible for intense regimens to spare the potential complications of chemotherapy

Comparison of outcomes of hematopoietic stem cell transplantation without chemotherapy conditioning by using matched sibling and unrelated donors for treatment of severe combined immunodeficiency.

BackgroundPatients with severe combined immunodeficiency disease who have matched sibling donors (MSDs) can proceed to hematopoietic cell transplantation (HCT) without conditioning chemotherapy.ObjectiveWe sought to determine whether the results of HCT without chemotherapy-based conditioning from matched unrelated donors (URDs), either from volunteer adults or umbilical cord blood, are comparable with those from MSDs.MethodsWe performed a multicenter survey of severe combined immunodeficiency transplantation centers in North America, Europe, and Australia to compile retrospective data on patients who have undergone unconditioned HCT from either URDs (n = 37) or MSDs (n = 66).ResultsMost patients undergoing URD HCT (92%) achieved donor T-cell engraftment compared with 97% for those with MSDs; however, estimated 5-year overall and event-free survival were worse for URD recipients (71% and 60%, respectively) compared with MSD recipients (92% and 89%, respectively; P &lt; .01 for both). URD recipients who received pre-HCT serotherapy had similar 5-year overall survival (100%) to MSD recipients. The incidences of grade II to IV acute and chronic graft-versus-host disease were higher in URD (50% and 39%, respectively) compared with MSD (22% and 5%, respectively) recipients (P &lt; .01 for both). In the surviving patients there was no difference in T-cell reconstitution at the last follow-up between the URD and MSD recipients; however, MSD recipients were more likely to achieve B-cell reconstitution (72% vs 17%, P &lt; .001).ConclusionUnconditioned URD HCT achieves excellent rates of donor T-cell engraftment similar to that seen in MSD recipients, and reconstitution rates are adequate. However, only a minority will have myeloid and B-cell reconstitution, and attention must be paid to graft-versus-host disease prophylaxis. This approach might be safer in children ineligible for intense regimens to spare the potential complications of chemotherapy

Validation of a novel, fully integrated and flexible microarray benchtop facility for gene expression profiling

Here we describe a novel microarray platform that integrates all functions needed to perform any array-based experiment in a compact instrument on the researcher’s laboratory benchtop. Oligonucle otide probes are synthesized in situ via a light- activated process within the channels of a three-dimensional microfluidic reaction carrier. Arrays can be designed and produced within hours according to the user’s requirements. They are processed in a fully automatic workflow. We have characterized this new platform with regard to dynamic range, discrimination power, reproducibility and accuracy of biological results. The instrument detects sample RNAs present at a frequency of 1:100 000. Detection is quantitative over more than two orders of magnitude. Experiments on four identical arrays with 6398 features each revealed a mean coefficient of variation (CV) value of 0.09 for the 6398 unprocessed raw intensities indicating high reproducibility. In a more elaborate experiment targeting 1125 yeast genes from an unbiased selection, a mean CV of 0.11 on the fold change level was found. Analyzing the transcriptional response of yeast to osmotic shock, we found that biological data acquired on our platform are in good agreement with data from Affymetrix GeneChips, quantitative real-time PCR and—albeit somewhat less clearly—to data from spotted cDNA arrays obtained from the literature

Human RIPK1 deficiency causes combined immunodeficiency and inflammatory bowel diseases

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