A GMCSF-Neuroantigen Tolerogenic Vaccine Elicits Systemic Lymphocytosis of CD4+ CD25high FOXP3+ Regulatory T Cells in Myelin-Specific TCR Transgenic Mice Contingent Upon Low-Efficiency T Cell Antigen Receptor Recognition

Previous studies showed that single-chain fusion proteins comprised of GM-CSF and major encephalitogenic peptides of myelin, when injected subcutaneously in saline, were potent tolerogenic vaccines that suppressed experimental autoimmune encephalomyelitis (EAE) in rats and mice. These tolerogenic vaccines exhibited dominant suppressive activity in inflammatory environments even when emulsified in Complete Freund's Adjuvant (CFA). The current study provides evidence that the mechanism of tolerance was dependent upon vaccine-induced regulatory CD25+ T cells (Tregs), because treatment of mice with the Treg-depleting anti-CD25 mAb PC61 reversed tolerance. To assess tolerogenic mechanisms, we focused on 2D2-FIG mice, which have a transgenic T cell repertoire that recognizes myelin oligodendrocyte glycoprotein peptide MOG35-55 as a low-affinity ligand and the neurofilament medium peptide NFM13-37 as a high-affinity ligand. Notably, a single subcutaneous vaccination of GMCSF-MOG in saline elicited a major population of FOXP3+ Tregs that appeared within 3 days, was sustained over several weeks, expressed canonical Treg markers, and was present systemically at high frequencies in the blood, spleen, and lymph nodes. Subcutaneous and intravenous injections of GMCSF-MOG were equally effective for induction of FOXP3+ Tregs. Repeated booster vaccinations with GMCSF-MOG elicited FOXP3 expression in over 40% of all circulating T cells. Covalent linkage of GM-CSF with MOG35-55 was required for Treg induction whereas vaccination with GM-CSF and MOG35-55 as separate molecules lacked Treg-inductive activity. GMCSF-MOG elicited high levels of Tregs even when administered in immunogenic adjuvants such as CFA or Alum. Conversely, incorporation of GM-CSF and MOG35-55 as separate molecules in CFA did not support Treg induction. The ability of the vaccine to induce Tregs was dependent upon the efficiency of T cell antigen recognition, because vaccination of 2D2-FIG or OTII-FIG mice with the high-affinity ligands GMCSF-NFM or GMCSF-OVA (Ovalbumin323-339), respectively, did not elicit Tregs. Comparison of 2D2-FIG and 2D2-FIG-Rag1−/− strains revealed that GMCSF-MOG may predominantly drive Treg expansion because the kinetics of vaccine-induced Treg emergence was a function of pre-existing Treg levels. In conclusion, these findings indicate that the antigenic domain of the GMCSF-NAg tolerogenic vaccine is critical in setting the balance between regulatory and conventional T cell responses in both quiescent and inflammatory environments

Do the size and landscape context of forest openings influence the abundance and breeding success of shrubland songbirds in southern New England?

Early successional birds have declined in the northeastern United States due to the regeneration of forest on abandoned farm fields and the suppression of natural disturbances that once provided appropriate habitat. These species have become increasingly dependent on early successional habitats generated by such activities as timber harvesting. Recent approaches of timber harvesting, which range from single-tree harvesting to clearcutting, create forest openings of different sizes and configurations embedded in landscapes with different land use patterns. To assess the importance of forest openings created by timber harvesting for shrubland birds, we surveyed birds on 50m radius plots in 34 harvest sites (0.5–21ha). We collected data on multi-scaled habitat variables ranging from plot-level vegetation characteristics to land use patterns within 1km of each study site. We also monitored mating and nesting success of Blue-winged Warblers (Vermivora pinus) in 10 forest openings. The abundance of most shrubland species was influenced by plot-level habitat variables, such as tree density and vegetation height, rather than shrubland area or the composition of land uses in the surrounding landscape. Only Eastern Towhees (Pipilo erythrophthalmus) were more frequent in survey plots in larger forest openings. In contrast, neither abundance nor reproductive activity of Blue-winged Warblers was correlated with the size of the forest opening. Their abundance was negatively related to vegetation height, however. Only 54% of the territorial male Blue-winged Warblers in forest openings were mated. We documented relatively low nest success rates of 21.1% during the egg laying and incubation nest stages, but increased success rates during the later stages of nesting. Our results indicate that even small forest openings with low vegetation provide habitat for Blue-winged Warblers and other shrubland birds. The overall reproductive rate of territorial male Blue-winged Warblers in forests openings was low during the 2 years of the study, however. Further studies are needed to assess the long-term value of this type of habitat for sustaining shrubland bird populations

µChemLab: twenty years of developing CBRNE detection systems with low false alarm rates

Gas Chromatography (GC) is routinely used in the laboratory to temporally separate chemical mixtures into their constituent components for improved chemical identification. This paper will provide a overview of more than twenty years of development of one-dimensional field-portable micro GC systems, highlighting key experimental results that illustrate how a reduction in false alarm rate (FAR) is achieved in real-world environments. Significantly, we will also present recent results on a micro two-dimensional GC (micro GCxGC) technology. This ultra-small system consists of microfabricated columns, NanoElectroMechanical System (NEMS) cantilever resonators for detection, and a valve-based stop-flow modulator. The separation of a 29-component polar mixture in less than 7 seconds is demonstrated along with peak widths in the second dimension ranging from 10-60 ms. For this system, a peak capacity of just over 300 was calculated for separation in about 6 s. This work has important implications for field detection, to drastically reduce FAR and significantly improve chemical selectivity and identification. This separation performance was demonstrated with the NEMS resonator and bench scale FID. But other detectors, suitably fast and sensitive can work as well. Recent research has shown that the identification power of GCxGC-FID can match that of GC-MS. This result indicates a path to improved size, weight, power, and performance in micro GCxGC systems outfitted with relatively non-specific, lightweight detectors. We will briefly discuss the performance of possible options, such as the pulsed discharge helium ionization detector (PDHID) and miniature correlation ion mobility spectrometer (mini-CIMS)

Molecular characterisation and clinical outcome of B-cell precursor acute lymphoblastic leukaemia with IG-MYC rearrangement

Rarely, immunophenotypically immature B-cell precursor acute lymphoblastic leukaemia (BCP-ALL) carries an immunoglobulin-MYC rearrangement (IG-MYC-r). This can result in diagnostic confusion with Burkitt lymphoma/leukaemia and use of unproven individualised treatment schedules. Here we contrast the molecular characteristics of these conditions and investigate historic clinical outcome data. We identified 90 cases registered on a national BCP-ALL clinical trial/registry. Where present, diagnostic material underwent cytogenetic, exome, methylome and transcriptome analysis. Outcome was analysed to define 3-year event free survival (EFS) and overall survival (OS). IG-MYC-r was identified in diverse cytogenetic backgrounds, co-existing with either: established BCP-ALL specific abnormalities (high hyperdiploidy n=3, KMT2A-rearrangement n=6, iAMP21 n=1, BCR-ABL n=1); BCL2/BCL6-rearrangements (n=15); or, most commonly, as the only defining feature (n=64). Within this final group, precursor-like V(D)J breakpoints predominated (8/9) and KRAS mutations were common (5/11). DNA methylation identified a cluster of V(D)J rearranged cases, clearly distinct from Burkitt leukaemia/lymphoma. Children with IG-MYC-r within that subgroup had 3-year EFS of 47% and OS of 60%, representing a high-risk BCP-ALL. To develop effective management strategies this patient group must be allowed access to contemporary, minimal residual disease adapted, prospective clinical trial protocols

A SINGLE-CHAIN GMCSF-MOG TOLEROGENIC VACCINE EXPANDS MOG-SPECIFIC CD25+ FOXP3+ REGULATORY T CELLS THROUGH LOW-EFFICIENCY ANTIGEN RECOGNITION EVENTS TO INHIBIT EXPERIMENTAL AUTOIMMUNE ENCEPHALOMYELITIS

Previous studies showed that tolerogenic vaccines comprised of single-chain GMCSF-neuroantigen (NAg) fusion proteins inhibited experimental autoimmune encephalomyelitis (EAE) in rodents. The studies detailed here provide evidence that GMCSF-NAg vaccines elicited tolerance through the expansion of preexisting NAg-specific regulatory T cells (Tregs) via low-efficiency antigen recognition that was below the CD40/CD40L activation threshold. GMCSF-NAg-induced tolerance was dependent upon vaccine-induced Tregs, because treatment of mice with a Treg-depleting mAb reversed vaccine-induced tolerance. Vaccine-induced T cell responses were investigated using T cell receptor (TCR) transgenic OTII-FIG mice which recognize OVA323-337 as a high-efficiency antigen, and 2D2-FIG mice which recognize MOG35-55 as a low-efficiency antigen and NFM13-37 as a high-efficiency antigen. Subcutaneous vaccination of 2D2-FIG mice with the low-efficiency GMCSF-MOG vaccine elicited a major Treg population that appeared within 3 days, was sustained over several weeks, expressed canonical Treg markers, and was present systemically in the blood, spleen, and lymph nodes. The GMCSF-MOG vaccine required covalent linkage because a vaccine that contained GM-CSF and MOG35-55 as separate molecules did not elicit Treg responses. GMCSF-MOG vaccination elicited Tregs when introduced either subcutaneously or intravenously as well as in the proinflammatory adjuvants CFA and alum. The GMCSF-MOG-induced Tregs were immunosuppressive and prevented the proliferation of MOG35-55-specific T cells. The GMCSF-MOG vaccine not only elicited Tregs but also induced a desensitized MOG35-55-specific (2D2) T cell repertoire because the vaccine decreased the number of 2D2 CD3+ T cells, reduced the overall expression of the 2D2 TCR, and increased the CD4- T cell compartment. The ability of GMCSF-NAg vaccines to induce Tregs was dependent upon the efficiency of T cell antigen recognition, because treatment of OTII-FIG and 2D2-FIG mice with the high-efficiency GMCSF-OVA and GMCSF-NFM vaccines respectively, did not elicit Treg responses. The high-efficiency GMCSF-NFM vaccine induced a vigorous T conventional cell (Tcon) memory response and activated the CD40L/CD40 co-stimulatory pathway. In contrast, the low-efficiency GMCSF-MOG vaccine elicited Tregs and lacked sufficient TCR signal strength to activate CD40L/CD40 pathway. Activation of the CD40L/CD40 pathway using an agonistic anti-CD40 mAb precluded Treg expansion with the low-efficiency GMCSF-MOG vaccine in 2D2-FIG mice. Therefore, the strength of the TCR stimulus and the downstream activation or exclusion of the CD40L/CD40 costimulatory pathway was the switch that controlled Tcon versus Treg responses respectively. Remarkably, the low-efficiency GMCSF-MOG vaccine retained Treg expansive activity when co-administered with the high-efficiency GMCSF-NFM vaccine in 2D2-FIG mice. The GMCSF-MOG vaccine appeared to predominantly drive Treg expansion rather than Treg induction because the emergence of Tregs was delayed in 2D2-FIG-Rag-/- mice which have reduced frequencies of pre-existing Tregs as compared to 2D2-FIG mice. Pre-existing Tregs were also required for tolerance because GMCSF-MOG was encephalitogenic in 2D2-FIG-Rag1-/- mice but not in 2D2-FIG mice. Likewise, GMCSF-MOG was an effective prophylactic in Treg-sufficient C57BL/6 mice and prevented active EAE. Overall, these studies provide evidence that GM-CSF is an effective tolerogenic adjuvant when combined with low-efficiency peptides that fall below the CD40L/CD40 triggering threshold. Thus, a subthreshold CD40L/ CD40 response delimits a critical parameter needed for antigen-specific tolerance and expansion of pre-existing Treg populations.Ph.D

A SINGLE-CHAIN GMCSF-MOG TOLEROGENIC VACCINE EXPANDS MOG-SPECIFIC CD25+ FOXP3+ REGULATORY T CELLS THROUGH LOW-EFFICIENCY ANTIGEN RECOGNITION EVENTS TO INHIBIT EXPERIMENTAL AUTOIMMUNE ENCEPHALOMYELITIS

Previous studies showed that tolerogenic vaccines comprised of single-chain GMCSF-neuroantigen (NAg) fusion proteins inhibited experimental autoimmune encephalomyelitis (EAE) in rodents. The studies detailed here provide evidence that GMCSF-NAg vaccines elicited tolerance through the expansion of preexisting NAg-specific regulatory T cells (Tregs) via low-efficiency antigen recognition that was below the CD40/CD40L activation threshold. GMCSF-NAg-induced tolerance was dependent upon vaccine-induced Tregs , because treatment of mice with a Treg-depleting mAb reversed vaccine-induced tolerance. Vaccine-induced T cell responses were investigated using T cell receptor (TCR) transgenic OTII-FIG mice which recognize OVA323-337 as a high-efficiency antigen , and 2D2-FIG mice which recognize MOG35-55 as a low-efficiency antigen and NFM13-37 as a high-efficiency antigen. Subcutaneous vaccination of 2D2-FIG mice with the low-efficiency GMCSF-MOG vaccine elicited a major Treg population that appeared within 3 days , was sustained over several weeks , expressed canonical Treg markers , and was present systemically in the blood , spleen , and lymph nodes. The GMCSF-MOG vaccine required covalent linkage because a vaccine that contained GM-CSF and MOG35-55 as separate molecules did not elicit Treg responses. GMCSF-MOG vaccination elicited Tregs when introduced either subcutaneously or intravenously as well as in the proinflammatory adjuvants CFA and alum. The GMCSF-MOG-induced Tregs were immunosuppressive and prevented the proliferation of MOG35-55-specific T cells. The GMCSF-MOG vaccine not only elicited Tregs but also induced a desensitized MOG35-55-specific (2D2) T cell repertoire because the vaccine decreased the number of 2D2 CD3+ T cells , reduced the overall expression of the 2D2 TCR , and increased the CD4- T cell compartment. The ability of GMCSF-NAg vaccines to induce Tregs was dependent upon the efficiency of T cell antigen recognition , because treatment of OTII-FIG and 2D2-FIG mice with the high-efficiency GMCSF-OVA and GMCSF-NFM vaccines respectively , did not elicit Treg responses. The high-efficiency GMCSF-NFM vaccine induced a vigorous T conventional cell (Tcon) memory response and activated the CD40L/CD40 co-stimulatory pathway. In contrast , the low-efficiency GMCSF-MOG vaccine elicited Tregs and lacked sufficient TCR signal strength to activate CD40L/CD40 pathway. Activation of the CD40L/CD40 pathway using an agonistic anti-CD40 mAb precluded Treg expansion with the low-efficiency GMCSF-MOG vaccine in 2D2-FIG mice. Therefore , the strength of the TCR stimulus and the downstream activation or exclusion of the CD40L/CD40 costimulatory pathway was the switch that controlled Tcon versus Treg responses respectively. Remarkably , the low-efficiency GMCSF-MOG vaccine retained Treg expansive activity when co-administered with the high-efficiency GMCSF-NFM vaccine in 2D2-FIG mice. The GMCSF-MOG vaccine appeared to predominantly drive Treg expansion rather than Treg induction because the emergence of Tregs was delayed in 2D2-FIG-Rag-/- mice which have reduced frequencies of pre-existing Tregs as compared to 2D2-FIG mice. Pre-existing Tregs were also required for tolerance because GMCSF-MOG was encephalitogenic in 2D2-FIG-Rag1-/- mice but not in 2D2-FIG mice. Likewise , GMCSF-MOG was an effective prophylactic in Treg-sufficient C57BL/6 mice and prevented active EAE. Overall , these studies provide evidence that GM-CSF is an effective tolerogenic adjuvant when combined with low-efficiency peptides that fall below the CD40L/CD40 triggering threshold. Thus , a subthreshold CD40L/ CD40 response delimits a critical parameter needed for antigen-specific tolerance and expansion of pre-existing Treg populations

Human Cytomegalovirus Induces the Activity and Expression of Acetyl-Coenzyme A Carboxylase, a Fatty Acid Biosynthetic Enzyme Whose Inhibition Attenuates Viral Replication▿†

We have previously reported that human cytomegalovirus (HCMV) infection induces large-scale changes to host cell glycolytic, nucleic acid, and phospholipid metabolism. Here we explore the viral mechanisms involved in fatty acid biosynthetic activation. Our results indicate that HCMV targets ACC1, the rate-limiting enzyme of fatty acid biosynthesis, through multiple mechanisms. HCMV infection was found to activate ACC1 expression, increasing the abundance of both ACC1 mRNA and protein. Viral gene expression but not viral DNA replication was found to be necessary for HCMV-mediated induction of ACC1 levels. HCMV infection was also found to increase the proteolytic processing of SREBP-2, a transcription factor whose proteolytic cleavage is known to activate a variety of phospholipid metabolic genes. Processing of SREBP-2 was found to be dependent on mTOR activity; pharmaceutical inhibition of mTOR blocked HCMV-induced SREBP-2 processing and prevented the induction of fatty acid biosynthesis and ACC1 expression. Independent of the increases in ACC1 expression, HCMV infection also induced ACC1's enzymatic activity. Inhibition of ACC1 through either RNA interference (RNAi) or inhibitor treatment was found to attenuate HCMV replication, and HCMV replication was sensitive to ACC1 inhibition even at the later stages of infection, suggesting a late role for fatty acid biosynthesis during HCMV replication. These findings indicate that HCMV infection actively modulates numerous functional aspects of a key metabolic regulatory enzyme that is important for high-titer viral replication

IFN-β Facilitates Neuroantigen-Dependent Induction of CD25+ FOXP3+ Regulatory T Cells That Suppress Experimental Autoimmune Encephalomyelitis

This study introduces a flexible format for tolerogenic vaccination that incorporates IFN-β and neuroantigen (NAg) in the Alum adjuvant. Tolerogenic vaccination required all three components, IFN-β, NAg, and Alum, for inhibition of experimental autoimmune encephalomyelitis (EAE) and induction of tolerance. Vaccination with IFN-β + NAg in Alum ameliorated NAg-specific sensitization and inhibited EAE in C57BL/6 mice in pretreatment and therapeutic regimens. Tolerance induction was specific for the tolerogenic vaccine Ag PLP178-191 or myelin oligodendrocyte glycoprotein (MOG)35--55 in proteolipid protein-- and MOG-induced models of EAE, respectively, and was abrogated by pretreatment with a depleting anti-CD25 mAb. IFN-β/Alum--based vaccination exhibited hallmarks of infectious tolerance, because IFN-β + OVA in Alum--specific vaccination inhibited EAE elicited by OVA + MOG in CFA but not EAE elicited by MOG in CFA. IFN-β + NAg in Alum vaccination elicited elevated numbers and percentages of FOXP3+ T cells in blood and secondary lymphoid organs in 2D2 MOG-specific transgenic mice, and repeated boosters facilitated generation of activated CD44high CD25+ regulatory T cell (Treg) populations. IFN-β and MOG35--55 elicited suppressive FOXP3+ Tregs in vitro in the absence of Alum via a mechanism that was neutralized by anti--TGF-β and that resulted in the induction of an effector CD69+ CTLA-4+ IFNAR+ FOXP3+ Treg subset. In vitro IFN-β + MOG--induced Tregs inhibited EAE when transferred into actively challenged recipients. Unlike IFN-β + NAg in Alum vaccines, vaccination with TGF-β + MOG35-55 in Alum did not increase Treg percentages in vivo. Overall, this study indicates that IFN-β + NAg in Alum vaccination elicits NAg-specific, suppressive CD25+ Tregs that inhibit CNS autoimmune disease. Thus, IFN-β has the activity spectrum that drives selective responses of suppressive FOXP3+ Tregs

Partial cD25 antagonism enables Dominance of antigen-inducible cD25high FOXP3+ regulatory T cells as a Basis for a regulatory T cell- Based adoptive immunotherapy

FOXP3+ regulatory T cells (Tregs) represent a promising platform for effective adoptive immunotherapy of chronic inflammatory disease, including autoimmune diseases such as multiple sclerosis. Successful Treg immunotherapy however requires new technologies to enable long-term expansion of stable, antigen-specific FOXP3+ Tregs in cell culture. Antigen-specific activation of naïve T cells in the presence of TGF-β elicits the initial differentiation of the FOXP3+ lineage, but these Treg lines lack phenotypic stability and rapidly transition to a conventional T cell (Tcon) phenotype during in vitro propagation. Because Tregs and Tcons differentially express CD25, we hypothesized that anti-CD25 monoclonal antibodies (mAbs) would only partially block IL-2 signaling in CD25high FOXP3+ Tregs while completely blocking IL-2 responses of CD25low-intermediate Tcons to enable preferential outgrowth of Tregs during in vitro propagation. Indeed, murine TGF-β- induced MOG-specific Treg lines from 2D2 transgenic mice that were maintained in IL-2 with the anti-CD25 PC61 mAb rapidly acquired and indefinitely maintained a FOXP3high phenotype during long-term in vitro propagation (>90% FOXP3+ Tregs), whereas parallel cultures lacking PC61 rapidly lost FOXP3. These results pertained to TGF-β-inducible "iTregs" because Tregs from 2D2-FIG Rag1−/− mice, which lack thymic or natural Tregs, were stabilized by continuous culture in IL-2 and PC61. MOG-specific and polyclonal Tregs upregulated the Treg-associated markers Neuropilin-1 (NRP1) and Helios (IKZF2). Just as PC61 stabilized FOXP3+ Tregs during expansion in IL-2, TGF-β fully stabilized FOXP3+ Tregs during cellular activation in the presence of dendritic cells and antigen/ mitogen. Adoptive transfer of blastogenic CD25high FOXP3+ Tregs from MOG35-55- specific 2D2 TCR transgenic mice suppressed experimental autoimmune encephalomy- elitis in pretreatment and therapeutic protocols. In conclusion, low IL-2 concentrations coupled with high PC61 concentrations constrained IL-2 signaling to a low-intensity range that enabled dominant stable outgrowth of suppressive CD25high FOXP3+ Tregs. The ability to indefinitely expand stable Treg lines will provide insight into FOXP3+ Treg physiology and will be foundational for Treg-based immunotherapy