Evaluation of the Alere I influenza A&B nucleic acid amplification test by use of respiratory specimens collected in viral transport medium.

The Alere i Influenza A&B assay is a newly developed rapid molecular assay which has the potential to generate results within 15 min from sample collection. In this study, we evaluated the Alere i Influenza A&B assay by using salvaged frozen respiratory specimens that were collected in viral transport medium from children ages 10 months to 19 years. Alere i Influenza A&B assay test results were compared with viral culture and ProFlu(+) real-time reverse transcription-PCR (RT-PCR) assay results. We found that the overall sensitivity and specificity of the Alere i Influenza A&B assay were 93.3% and 94.5% for the detection of influenza A virus and 100% and 100% for the detection of influenza B virus, respectively, compared to viral culture. In comparison to ProFlu(+) real-time RT-PCR, overall sensitivity and specificity of the Alere i Influenza A&B assay for the detection of influenza A virus were 88.8% and 98.3% and 100% and 100% for detecting influenza B virus. Overall, the Alere i Influenza A&B assay performed well compared to either virus cell culture or RT-PCR.

Multicenter clinical performance evaluation of BD Veritor™ system for rapid detection of respiratory syncytial virus.

BACKGROUND: BD Veritor™ System for Rapid Detection of Respiratory Syncytial Virus (RSV) is a new-generation lateral flow immunochromatographic assay for objective detection of RSV in respiratory specimens from children. OBJECTIVE: To evaluate the performance of BD Veritor™ System for Rapid Detection of RSV in respiratory specimens collected from pediatric patients. STUDY DESIGN: A prospective, multicenter clinical trial was undertaken at five study sites representing geographically diverse regions of the U.S. to assess the performance of the BD Veritor™ System for Rapid Detection of RSV in comparison to R-mix shell vial culture and ProFlu+ reverse transcription-PCR assay (Gen-Probe/Prodesse). RESULTS: 440 nasopharyngeal washes/aspirates (NPW/A) and 706 nasopharyngeal swab (NPS) specimens from U.S. subjects<20 years of age were collected and tested using the BD Veritor™ System and compared with shell vial culture and real-time RT-PCR results. Analysis of the data indicates the overall sensitivity and specificity for BD Veritor™ System for all sample types combined was 90% and 97.0% versus shell vial culture and 75.5% and 98.7% versus RT-PCR respectively. CONCLUSION: Overall, the BD Veritor™ System for the Rapid Detection of RSV performed well when compared to both viral cell culture and RT-PCR in children.

T cell development requires constraint of the myeloid regulator C/EBP-α by the Notch target and transcriptional repressor Hes1.

Notch signaling induces gene expression of the T cell lineage and discourages alternative fate outcomes. Hematopoietic deficiency in the Notch target Hes1 results in severe T cell lineage defects; however, the underlying mechanism is unknown. We found here that Hes1 constrained myeloid gene-expression programs in T cell progenitor cells, as deletion of the myeloid regulator C/EBP-α restored the development of T cells from Hes1-deficient progenitor cells. Repression of Cebpa by Hes1 required its DNA-binding and Groucho-recruitment domains. Hes1-deficient multipotent progenitor cells showed a developmental bias toward myeloid cells and dendritic cells after Notch signaling, whereas Hes1-deficient lymphoid progenitor cells required additional cytokine signaling for diversion into the myeloid lineage. Our findings establish the importance of constraining developmental programs of the myeloid lineage early in T cell development.

Early T-cell progenitors are the major granulocyte precursors in the adult mouse thymus.

The mouse thymus supports T-cell development, but also contains non-T-cell lineages such as dendritic cells, macrophages, and granulocytes that are necessary for T-cell repertoire selection and apoptotic thymocyte clearance. Early thymic progenitors (ETPs) are not committed to the T-cell lineage, as demonstrated by both in vitro and in vivo assays. Whether ETPs realize non-T-cell lineage potentials in vivo is not well understood and indeed is controversial. In the present study, we investigated whether ETPs are the major precursors of any non-T-lineage cells in the thymus. We analyzed the development of these populations under experimental circumstances in which ETPs are nearly absent due to either abrogated thymic settling or inhibition of early thymic development by genetic ablation of IL-7 receptorα or Hes1. Results obtained using multiple in vivo approaches indicate that the majority of thymic granulocytes derive from ETPs. These data indicate that myelolymphoid progenitors settle the thymus and thus clarify the pathways by which stem cells give rise to downstream blood cell lineages.

Eliciting the T cell fate with Notch.

Multipotent progenitors arrive at the thymus via the blood. Constraining the non-T cell fates of these progenitors while promoting the T cell fate is a major task of the thymus. Notch appears to be the initial trigger for a developmental program that eventually results in T cell lineage commitment. Several downstream targets of Notch are known, but the specific roles of each are poorly understood. A greater understanding of how Notch and other thymic signals direct progenitors to a T cell fate could be useful for translational work. For example, such work could eventually allow for the generation of fully competent T cells in vitro that could supplement the waning T cell numbers and function in the elderly and boost T cell-mediated immunity in patients with immunodeficiency and after stem cell transplantation.

CCR7 and CCR9 together recruit hematopoietic progenitors to the adult thymus.

T lymphopoiesis requires settling of the thymus by bone marrow-derived precursors throughout adult life. Progenitor entry into the thymus is selective, but the molecular basis of this selectivity is incompletely understood. The chemokine receptor CCR9 has been demonstrated to be important in this process. However, progenitors lacking CCR9 can still enter the thymus, suggesting a role for additional molecules. Here we report that the chemokine receptor CCR7 is also required for efficient thymic settling. CCR7 is selectively expressed on bone marrow progenitors previously shown to have the capacity to settle the thymus, and CCR7(-/-) progenitors are defective in settling the thymus. We further demonstrate that CCR7 sustains thymic settling in the absence of CCR9. Mice deficient for both CCR7 and CCR9 have severe reductions in the number of early thymic progenitors, and in competitive assays CCR7(-/-)CCR9(-/-) double knockout progenitors are almost completely restricted from thymic settling. However, these mice possess near-normal thymic cellularity. Compensatory expansion of intrathymic populations can account for at least a part of this recovery. Together our results illustrate the critical role of chemokine receptor signaling in thymic settling and help to clarify the cellular identity of the physiologic thymic settling progenitors.

Untangling the T branch of the hematopoiesis tree.

T cells develop in the thymus. Previous work suggested an early separation of lymphoid from myeloerythroid lineages during hematopoiesis and hypothesized the thymus was settled exclusively by lymphoid-restricted hematopoietic progenitors. Recent data have instead established the existence of lymphoid-myeloid progenitors, which possess lymphoid and myeloid lineage potentials but lack erythroid potential. Myeloid and lymphoid potentials are present at the clonal level in early thymic progenitors, confirming that progenitors settling the thymus include lymphoid-myeloid progenitors. These results revise our view of the T lineage branch of hematopoiesis and focus attention on the generation, circulation, and homing of lymphoid-myeloid progenitors to the thymus.

Progenitor migration to the thymus and T cell lineage commitment.

T cells developing in the thymus are ultimately derived from bone marrow (BM) hematopoietic stem cells (HSCs). An understanding of the developmental steps between HSCs and T cells is important for gaining insight into cancers of the T lineage, improving T cell reconstitution after BM transplantation, and also to help ameliorate immunological defects in aging. In this article, we summarize our current understanding of the inter-related fields of early T cell development and thymic aging, and briefly discuss major unresolved questions in this field.

Putting ThPOK in place.

ThPOK is necessary for the differentiation of CD4+ helper T cells. Three new studies indicate that, unexpectedly, ThPOK is required only after initial specification to the CD4+ lineage.

The earliest thymic progenitors for T cells possess myeloid lineage potential.

There exists controversy over the nature of haematopoietic progenitors of T cells. Most T cells develop in the thymus, but the lineage potential of thymus-colonizing progenitors is unknown. One approach to resolving this question is to determine the lineage potentials of the earliest thymic progenitors (ETPs). Previous work has shown that ETPs possess T and natural killer lymphoid potentials, and rare subsets of ETPs also possess B lymphoid potential, suggesting an origin from lymphoid-restricted progenitor cells. However, whether ETPs also possess myeloid potential is unknown. Here we show that nearly all ETPs in adult mice possess both T and myeloid potential in clonal assays. The existence of progenitors possessing T and myeloid potential within the thymus is incompatible with the current dominant model of haematopoiesis, in which T cells are proposed to arise from lymphoid-. Our results indicate that alternative models for lineage commitment during haematopoiesis must be considered.

Innocuous IFNgamma induced by adjuvant-free antigen restores normoglycemia in NOD mice through inhibition of IL-17 production.

The role of Th17 cells in type I diabetes (TID) remains largely unknown. Glutamic acid decarboxylase (GAD) sequence 206-220 (designated GAD2) represents a late-stage epitope, but GAD2-specific T cell receptor transgenic T cells producing interferon gamma (IFNgamma) protect against passive TID. Because IFNgamma is known to inhibit Th17 cells, effective presentation of GAD2 peptide under noninflammatory conditions may protect against TID at advanced disease stages. To test this premise, GAD2 was genetically incorporated into an immunoglobulin (Ig) molecule to magnify tolerance, and the resulting Ig-GAD2 was tested against TID at different stages of the disease. The findings indicated that Ig-GAD2 could not prevent TID at the preinsulitis phase, but delayed TID at the insulitis stage. More importantly, Ig-GAD2 sustained both clearance of pancreatic cell infiltration and beta-cell division and restored normoglycemia when given to hyperglycemic mice at the prediabetic stage. This was dependent on the induction of splenic IFNgamma that inhibited interleukin (IL)-17 production. In fact, neutralization of IFNgamma led to a significant increase in the frequency of Th17 cells, and the treatment became nonprotective. Thus, IFNgamma induced by an adjuvant free antigen, contrary to its usual inflammatory function, restores normoglycemia, most likely by localized bystander suppression of pathogenic IL-17-producing cells.

In trans T cell tolerance diminishes autoantibody responses and exacerbates experimental allergic encephalomyelitis.

A number of Ag-specific approaches have been developed that ameliorate experimental allergic encephalomyelitis (EAE), an animal model for the human autoimmune disease multiple sclerosis. Translation to humans, however, remains a consideration, justifying the search for more insight into the mechanism underlying restoration of self-tolerance. Ig-proteolipid protein (PLP) 1 and Ig-myelin oligodendrocyte glycoprotein (MOG) are Ig chimeras carrying the encephalitogenic PLP 139-151 and MOG 35-55 amino acid sequence, respectively. Ig-PLP1 ameliorates EAE in SJL/J (H-2(s)) mice while Ig-MOG modulates the disease in C57BL/6 (H-2(b)) animals. In this study, we asked whether the chimeras would suppress EAE in F(1) mice expressing both parental MHC alleles and representing a polymorphism with more relevance to human circumstances. The results show that Ig-MOG modulates both PLP1 and MOG peptide-induced EAE in the F(1) mice, whereas Ig-PLP1 counters PLP1 EAE but exacerbates MOG-induced disease. This in trans aggravation of MOG EAE by Ig-PLP1 operates through induction of PLP1-specific T cells producing IL-5 that sustained inhibition of MOG-specific Abs leading to exacerbation of EAE. Thus, in trans T cell tolerance, which should be operative in polymorphic systems, can aggravate rather than ameliorate autoimmunity. This phenomenon possibly takes place through interference with protective humoral immunity.

Early effector T cells producing significant IFN-gamma develop into memory.

Currently, transition of T cells from effector to memory is believed to occur as a consequence of exposure to residual suboptimal Ag found in lymphoid tissues at the waning end of the effector phase and microbial clearance. This led to the interpretation that memory arises from slightly activated late effectors producing reduced amounts of IFN-gamma. In this study, we show that CD4 T cells from the early stage of the effector phase in which both the Ag and activation are optimal also transit to memory. Moreover, early effector T cells that have undergone four divisions expressed significant IL-7R, produced IFN-gamma, and yielded rapid and robust memory responses. Cells that divided three times that had marginal IL-7R expression and no IFN-gamma raised base level homeostatic memory, whereas those that have undergone only two divisions and produced IFN-gamma yielded conditioned memory despite low IL-7R expression. Thus, highly activated early effectors generated under short exposure to optimal Ag in vivo develop into memory, and such transition is dependent on a significant production of the cell's signature cytokine, IFN-gamma.

neonatally primed lymph node, but not splenic T cells, display a Gly-Gly motif within the TCR beta-chain complementarity-determining region 3 that controls affinity and may affect lymphoid organ retention.

Ig-proteolipid protein 1 (Ig-PLP1) is an Ig chimera expressing the encephalitogenic PLP1 peptide corresponding to amino acid residues 139-151 of PLP. Newborn mice given Ig-PLP1 in saline on the day of birth and challenged 7 wk later with PLP1 peptide in CFA develop an organ-specific neonatal immunity that confers resistance against experimental allergic encephalomyelitis. The T cell responses in these animals are comprised of Th2 cells in the lymph node and anergic Th1 lymphocytes in the spleen. Intriguingly, the anergic splenic T cells, although nonproliferative and unable to produce IFN-gamma or IL-4, secrete significant amounts of IL-2. Studies were performed to determine whether the two populations display any structural differences in the TCR H chain variable region that could contribute to the differential affinity and retention in different organs. Responsive Th2 lymph node T cells and anergic splenic lymphocytes were immortalized, and the structures of their TCR Vbeta were determined. The results show that Vbeta and Jbeta usage was random, but the CDR3 regions of the lymph node cells had a conserved Gly-Gly motif. Analysis of TCR affinity/avidity correlated the Gly-Gly motif with lower affinity and retention of the Th2 cells in the lymph node. Also, it is suggested that a higher TCR affinity may be a contributing factor for the development of the neonatal Th1 response in the spleen.

Specific T regulatory cells display broad suppressive functions against experimental allergic encephalomyelitis upon activation with cognate antigen.

To date, very few Ag-based regimens have been defined that could expand T regulatory (Treg) cells to reverse autoimmunity. Additional understanding of Treg function with respect to specificity and broad suppression should help overcome these limitations. Ig-proteolipid protein (PLP)1, an Ig carrying a PLP1 peptide corresponding to amino acid residues 139-151 of PLP, displayed potent tolerogenic functions and proved effective against experimental allergic encephalomyelitis (EAE). In this study, we took advantage of the Ig-PLP1 system and the PLP1-specific TCR transgenic 5B6 mouse to define a regimen that could expand Ag-specific Treg cells in vivo and tested for effectiveness against autoimmunity involving diverse T cell specificities. The findings indicate that in vivo exposure to aggregated Ig-PLP1 drives PLP1-specific 5B6 TCR transgenic cells to evolve as Treg cells expressing CD25, CTLA-4, and Foxp3 and producing IL-10. These Treg cells were able to suppress PLP1 peptide-induced EAE in both SJL/J and F(1) (SJL/J x C57BL/6) mice. However, despite being effective against disease induced with a CNS homogenate, the Treg cells were unable to counter EAE induced by a myelin basic protein or a myelin oligodendrocyte glycoprotein peptide. Nevertheless, activation with Ag before transfer into the host mice supports suppression of both myelin oligodendrocyte glycoprotein- and myelin basic protein peptide-induced EAE. Thus, it is suggested that activation of Treg cells by the cognate autoantigen is necessary for operation of broad suppressive functions.

IL-10 diminishes CTLA-4 expression on islet-resident T cells and sustains their activation rather than tolerance.

IL-10, a powerful anti-Th1 cytokine, has shown paradoxical effects against diabetes. The mechanism underlying such variable function remains largely undefined. An approach for controlled mobilization of endogenous IL-10 was applied to the NOD mouse and indicated that IL-10 encounter with diabetogenic T cells within the islets sustains activation, while encounter occurring peripheral to the islets induces tolerance. Insulin beta-chain (INSbeta) 9-23 peptide was expressed on an Ig, and the aggregated (agg) form of the resulting Ig-INSbeta triggered IL-10 production by APCs, and expanded IL-10-producing T regulatory cells. Consequently, agg Ig-INSbeta delayed diabetes effectively in young NOD mice whose pathogenic T cells remain peripheral to the islets. However, agg Ig-INSbeta was unable to suppress the disease in 10-wk-old insulitis-positive animals whose diabetogenic T cells have populated the islets. This is not due to irreversibility of the disease because soluble Ig-INSbeta did delay diabetes in these older mice. Evidence is provided indicating that upon migration to the islet, T cells were activated and up-regulated CTLA-4 expression. IL-10, however, reverses such up-regulation, abolishing CTLA-4-inhibitory functions and sustaining activation of the islet T lymphocytes. Therefore, IL-10 supports T cell tolerance in the periphery, but its interplay with CTLA-4 sustains activation within the islets. As a result, IL-10 displays opposite functions against diabetes in young vs older insulitis-positive mice.

A sudden decline in active membrane-bound TGF-beta impairs both T regulatory cell function and protection against autoimmune diabetes.

Autoimmunity presumably manifests as a consequence of a shortfall in the maintenance of peripheral tolerance by CD4(+)CD25(+) T regulatory cells (Tregs). However, the mechanism underlying the functional impairment of Tregs remains largely undefined. In this study a glutamic acid decarboxylase (GAD) diabetogenic epitope was expressed on an Ig to enhance tolerogenic function, and the resulting Ig-GAD expanded Tregs in both young and older insulitis-positive, nonobese diabetic (NOD) mice, but delayed autoimmune diabetes only in the former. Interestingly, Tregs induced at 4 wk of age had significant active membrane-bound TGF-beta (mTGF-beta) and sustained protection against diabetes, whereas Tregs expanded during insulitis had minimal mTGF-beta and could not protect against diabetes. The Tregs probably operate suppressive function through mTGF-beta, because Ab blockade of mTGF-beta nullifies protection against diabetes. Surprisingly, young Tregs that modulated pathogenic T cells maintained stable frequency over time in the protected animals, but decreased their mTGF-beta at the age of 8 wk. More strikingly, these 8-wk-old mTGF-beta-negative Tregs, which were previously protective, became unable to confer resistance against diabetes. Thus, a developmental decline in active mTGF-beta nullifies Treg function, leading to a break in tolerance and the onset of diabetes.

IL-4 utilizes an alternative receptor to drive apoptosis of Th1 cells and skews neonatal immunity toward Th2.

Primary neonatal Th1 cells develop alongside of Th2 upon priming of the newborn but undergo apoptosis upon recall with antigen. These Th1 cells were isolated, and their death was correlated with elevated IL-13Ralpha1 chain expression. Strikingly, neutralization of Th2s' IL-4 reduced apoptosis, sustained recall responses, and the live Th1 cells displayed a decrease in IL-13Ralpha1 expression. Blockade of IL-13Ralpha1 or IL-4Ralpha also restores recall and secondary Th1 responses. Adult T cells primed within the neonatal environment did not upregulate IL-13Ralpha1 chain or undergo apoptosis and developed recall Th1 responses. These observations indicate that developmental expression of IL-13Ralpha1 along with IL-4Ralpha provides a receptor through which IL-4 induces death of Th1 cells and skews neonatal immunity toward Th2.

Break of neonatal Th1 tolerance and exacerbation of experimental allergic encephalomyelitis by interference with B7 costimulation.

Ig-PLP1 is an Ig chimera expressing proteolipid protein-1 (PLP1) peptide corresponding to aa residues 139-151 of PLP. Newborn mice given Ig-PLP1 in saline on the day of birth and challenged 7 wk later with PLP1 peptide in CFA develop an organ-specific neonatal immunity that confers resistance against experimental allergic encephalomyelitis. The T cell responses in these animals comprise Th2 cells in the lymph node and anergic Th1 lymphocytes in the spleen. Intriguingly, the anergic splenic T cells, although nonproliferative and unable to produce IFN-gamma or IL-4, secrete significant amounts of IL-2. In this work, studies were performed to determine whether costimulation through B7 molecules plays any role in the unusual form of splenic Th1 anergy. The results show that engagement of either B7.1 or B7.2 with anti-B7 Abs during induction of EAE in adult mice that were neonatally tolerized with Ig-PLP1 restores and exacerbates disease severity. At the cellular level, the anergic splenic T cells regain the ability to proliferate and produce IFN-gamma when stimulated with Ag in the presence of either anti-B7.1 or anti-B7.2 Ab. However, such restoration was abolished when both B7.1 and B7.2 molecules were engaged simultaneously, indicating that costimulation is necessary for reactivation. Surprisingly, both anti-B7.1 and anti-B7.2 Abs triggered splenic dendritic cells to produce IL-12, a key cytokine required for restoration of the anergic T cells. Thus, recovery from neonatally induced T cell anergy requires B7 molecules to serve double functions, namely, costimulation and induction of cytokine production by APCs.