A Randomized Controlled Clinical Trial in Healthy Older Adults to Determine Efficacy of Glycine and N-Acetylcysteine Supplementation on Glutathione Redox Status and Oxidative Damage.

Glycine and cysteine are non-essential amino acids that are required to generate glutathione, an intracellular tripeptide that neutralizes reactive oxygen species and prevents tissue damage. During aging glutathione demand is thought to increase, but whether additional dietary intake of glycine and cysteine contributes towards the generation of glutathione in healthy older adults is not well understood. We investigated supplementation with glycine and n-acetylcysteine (GlyNAC) at three different daily doses for 2 weeks (low dose: 2.4 g, medium dose: 4.8 g, or high dose: 7.2 g/day, 1:1 ratio) in a randomized, controlled clinical trial in 114 healthy volunteers. Despite representing a cohort of healthy older adults (age mean = 65 years), we found significantly higher baseline levels of markers of oxidative stress, including that of malondialdehyde (MDA, 0.158 vs. 0.136 µmol/L, p < 0.0001), total cysteine (Cysteine-T, 314.8 vs. 276 µM, p < 0.0001), oxidized glutathione (GSSG, 174.5 vs. 132.3 µmol/L, p < 0.0001), and a lower ratio of reduced to oxidized glutathione (GSH-F:GSSG) (11.78 vs. 15.26, p = 0.0018) compared to a young reference group (age mean = 31.7 years, n = 20). GlyNAC supplementation was safe and well tolerated by the subjects, but did not increase levels of GSH-F:GSSG (end of study, placebo = 12.49 vs. 7.2 g = 12.65, p-value = 0.739) or that of total glutathione (GSH-T) (end of study, placebo = 903.5 vs. 7.2 g = 959.6 mg/L, p-value = 0.278), the primary endpoint of the study. Post-hoc analyses revealed that a subset of subjects characterized by high oxidative stress (above the median for MDA) and low baseline GSH-T status (below the median), who received the medium and high doses of GlyNAC, presented increased glutathione generation (end of study, placebo = 819.7 vs. 4.8g/7.2 g = 905.4 mg/L, p-value = 0.016). In summary GlyNAC supplementation is safe, well tolerated, and may increase glutathione levels in older adults with high glutathione demand. Clinical Trial Registration: https://clinicaltrials.gov/ct2/show/NCT05041179, NCT05041179.

Clinical- and omics-based models of subclinical atherosclerosis in healthy Chinese adults: a cross-sectional exploratory study.

BACKGROUND: Classical risk factors, such as fasting cholesterol, blood pressure (BP), and diabetes status are used today to predict the risk of developing cardiovascular disease (CVD). However, accurate prediction remains limited, particularly in low-risk groups such as women and younger individuals. Growing evidence suggests that biomarker concentrations following consumption of a meal challenge are better and earlier predictors of disease development than biomarker concentrations. OBJECTIVE: To test the hypothesis that postprandial responses of circulating biomarkers differ between healthy subjects with and without subclinical atherosclerosis (SA) in an Asian population at low risk of coronary artery disease (CAD). METHODS: One hundred healthy Chinese subjects (46 women, 54 men) completed the study. Subjects consumed a mixed-meal test and 164 blood biomarkers were analyzed over 6 h by using a combination of chemical and NMR techniques. Models were trained using different methodologies (including logistic regression, elastic net, random forest, sparse partial least square) on a random 75% subset of the data, and their performance was evaluated on the remaining 25%. RESULTS: We found that models based on baseline clinical parameters or fasting biomarkers could not reliably predict SA. By contrast, an omics model based on magnitude and timing of postprandial biomarkers achieved high performance [receiving operating characteristic (ROC) AUC: 91%; 95% CI: 77, 100). Investigation of key features of this model enabled derivation of a considerably simpler model, solely based on postprandial BP and age, with excellent performance (AUC: 91%; 95% CI: 78, 100). CONCLUSION: We report a novel model to detect SA based on postprandial BP and age in a population of Asian subjects at low risk of CAD. The use of this model in large-scale CVD prevention programs should be explored. This trial was registered at ClinicalTrials.gov as NCT03531879.

Hallmarks of improved immunological responses in the vaccination of more physically active elderly females.

Physical inactivity is one of the leading contributors to worldwide morbidity and mortality. The elderly are particularly susceptible since the features of physical inactivity overlap with the outcomes of natural aging - including the propensity to develop cardiovascular diseases, cancer, diabetes mellitus, sarcopenia and cognitive impairment. The age-dependent loss of immune function, or immunosenescence, refers to the progressive depletion of primary immune resources and is linked to the development of many of these conditions. Immunosenescence is primarily driven by chronic immune activation and physical activity interventions have demonstrated the potential to reduce the risk of complications in the elderly by modulating inflammation and augmenting the immune system. Since poor vaccination outcome is a hallmark of immunosenescence, the assessment of vaccine efficacy provides a window to study the immunological effects of regular physical activity. Using an accelerator-based study, we demonstrate in a Singaporean Chinese cohort that elderly women (n=56) who walk more after vaccination display greater post-vaccination expansion of monocytes and plasmablasts in peripheral blood. Active elderly female participants also demonstrated lower baseline levels of IP-10 and Eotaxin, and the upregulation of genes associated with monocyte/macrophage phagocytosis. We further describe postive correlations between the monocyte response and the post-vaccination H1N1 HAI titres of participants. Finally, active elderly women reveal a higher induction of antibodies against Flu B in their 18-month second vaccination follow-up. Altogether, our data are consistent with better immunological outcomes in those who are more physically active and highlight the pertinent contribution of monocyte activity.

DN2 Thymocytes Activate a Specific Robust DNA Damage Response to Ionizing Radiation-Induced DNA Double-Strand Breaks.

For successful bone marrow transplantation (BMT), a preconditioning regime involving chemo and radiotherapy is used that results in DNA damage to both hematopoietic and stromal elements. Following radiation exposure, it is well recognized that a single wave of host-derived thymocytes reconstitutes the irradiated thymus, with donor-derived thymocytes appearing about 7 days post BMT. Our previous studies have demonstrated that, in the presence of donor hematopoietic cells lacking T lineage potential, these host-derived thymocytes are able to generate a polyclonal cohort of functionally mature peripheral T cells numerically comprising ~25% of the peripheral T cell pool of euthymic mice. Importantly, we demonstrated that radioresistant CD44+ CD25+ CD117+ DN2 progenitors were responsible for this thymic auto-reconstitution. Until recently, the mechanisms underlying the radioresistance of DN2 progenitors were unknown. Herein, we have used the in vitro "Plastic Thymus" culture system to perform a detailed investigation of the mechanisms responsible for the high radioresistance of DN2 cells compared with radiosensitive hematopoietic stem cells. Our results indicate that several aspects of DN2 biology, such as (i) rapid DNA damage response (DDR) activation in response to ionizing radiation-induced DNA damage, (ii) efficient repair of DNA double-strand breaks, and (iii) induction of a protective G1/S checkpoint contribute to promoting DN2 cell survival post-irradiation. We have previously shown that hypoxia increases the radioresistance of bone marrow stromal cells in vitro, at least in part by enhancing their DNA double-strand break (DNA DSB) repair capacity. Since the thymus is also a hypoxic environment, we investigated the potential effects of hypoxia on the DDR of DN2 thymocytes. Finally, we demonstrate for the first time that de novo DN2 thymocytes are able to rapidly repair DNA DSBs following thymic irradiation in vivo.

Vitamin B12 deficiency and impaired expression of amnionless during aging.

BACKGROUND: Physical frailty and loss of mobility in elderly individuals lead to reduced independence, quality of life, and increased mortality. Vitamin B12 deficiency has been linked to several age-related chronic diseases, including in the musculo-skeletal system, where vitamin B12 deficiency is generally believed to be linked to poor nutritional intake. In the present study, we asked whether aging and frailty associate with altered vitamin B12 homeostasis in humans and investigated the underlying molecular mechanisms using preclinical models. METHODS: We analysed a subset of the Singapore Longitudinal Aging Study and stratified 238 participants based on age and Fried frailty criteria. Levels of methyl-malonic acid (MMA), a marker for vitamin B12 deficiency, and amnionless, the vitamin B12 co-receptor that anchors the vitamin B12 transport complex to the membrane of epithelial cells, were measured in plasma. In addition, vitamin B12 levels and the molecular mechanisms of vitamin B12 uptake and excretion were analysed in ileum, kidney, liver, and blood using a rat model of natural aging where nutritional intake is fully controlled. RESULTS: We demonstrate that aging and frailty are associated with a higher prevalence of functional vitamin B12 deficiency that can be detected by increased levels of MMA in blood (ρ = 0.25; P = 0.00013). The decline in circulating vitamin B12 levels is recapitulated in a rat model of natural aging where food composition and intake are stable. At the molecular level, these perturbations involve altered expression of amnionless in the ileum and kidney. Interestingly, we demonstrate that amnionless can be detected in serum where its levels increase during aging in both rodents and human (P = 3.3e-07 and 9.2e-07, respectively). Blood amnionless levels negatively correlate with vitamin B12 in rats (r2  = 0.305; P = 0.0042) and positively correlate with the vitamin B12 deficiency marker MMA in humans (ρ = 0.22; P = 0.00068). CONCLUSIONS: Our results demonstrate that aging and frailty cause intrinsic vitamin B12 deficiencies, which can occur independently of nutritional intake. Mechanistically, vitamin B12 deficiency involves the physio-pathological decline of both the intestinal uptake and the renal reabsorption system for vitamin B12. Finally, amnionless is a novel biomarker which can detect perturbed vitamin B12 bioavailability during aging and physical frailty.

Health relevance of the modification of low grade inflammation in ageing (inflammageing) and the role of nutrition.

Ageing of the global population has become a public health concern with an important socio-economic dimension. Ageing is characterized by an increase in the concentration of inflammatory markers in the bloodstream, a phenomenon that has been termed "inflammageing". The inflammatory response is beneficial as an acute, transient reaction to harmful conditions, facilitating the defense, repair, turnover and adaptation of many tissues. However, chronic and low grade inflammation is likely to be detrimental for many tissues and for normal functions. We provide an overview of low grade inflammation (LGI) and determine the potential drivers and the effects of the "inflamed" phenotype observed in the elderly. We discuss the role of gut microbiota and immune system crosstalk and the gut-brain axis. Then, we focus on major health complications associated with LGI in the elderly, including mental health and wellbeing, metabolic abnormalities and infections. Finally, we discuss the possibility of manipulating LGI in the elderly by nutritional interventions. We provide an overview of the evidence that exists in the elderly for omega-3 fatty acid, probiotic, prebiotic, antioxidant and polyphenol interventions as a means to influence LGI. We conclude that slowing, controlling or reversing LGI is likely to be an important way to prevent, or reduce the severity of, age-related functional decline and the onset of conditions affecting health and well-being; that there is evidence to support specific dietary interventions as a strategy to control LGI; and that a continued research focus on this field is warranted.

Pro-B cells propagated in stromal cell-free cultures reconstitute functional B-cell compartments in immunodeficient mice.

Up to now long-term in vitro growth of pro-B cells was thought to require stromal cells. However, here we show that fetal liver (FL) and bone marrow (BM) derived pro-B cells can be propagated long-term in stromal cell-free cultures supplemented with IL-7, stem cell factor and FLT3 ligand. Within a week, most cells expressed surface CD19, CD79A, λ5, and VpreB antigens and had rearranged immunoglobulin D-J heavy chain genes. Both FL and BM pro-B cells reconstituted the B-cell compartments of immuno-incompetent Rag2-deficient mice, with FL pro-B cells generating follicular, marginal zone (MZB) and B1a B cells, and BM pro-B cells giving rise mainly to MZB cells. Reconstituted Rag2-deficient mice generated significant levels of IgM and IgG antibodies to a type II T-independent antigen; mice reconstituted with FL pro-B cells generated surprisingly high IgG1 titers. Finally, we show for the first time that mice reconstituted with mixtures of pro-B and pro-T cells propagated in stromal cell-free in vitro cultures mounted a T-cell-dependent antibody response. This novel stromal cell-free culture system facilitates our understanding of B-cell development and might be applied clinically.

Metabolic phenotyping of an adoptive transfer mouse model of experimental colitis and impact of dietary fish oil intake.

Inflammatory bowel diseases are acute and chronic disabling inflammatory disorders with multiple complex etiologies that are not well-defined. Chronic intestinal inflammation has been linked to an energy-deficient state of gut epithelium with alterations in oxidative metabolism. Plasma-, urine-, stool-, and liver-specific metabonomic analyses are reported in a naïve T cell adoptive transfer (AT) experimental model of colitis, which evaluated the impact of long-chain n-3 polyunsaturated fatty acid (PUFA)-enriched diet. Metabolic profiles of AT animals and their controls under chow diet or fish oil supplementation were compared to describe the (i) consequences of inflammatory processes and (ii) the differential impact of n-3 fatty acids. Inflammation was associated with higher glycoprotein levels (related to acute-phase response) and remodeling of PUFAs. Low triglyceride levels and enhanced PUFA levels in the liver suggest activation of lipolytic pathways that could lead to the observed increase of phospholipids in the liver (including plasmalogens and sphingomyelins). In parallel, the increase in stool excretion of most amino acids may indicate a protein-losing enteropathy. Fecal content of glutamine was lower in AT mice, a feature exacerbated under fish oil intervention that may reflect a functional relationship between intestinal inflammatory status and glutamine metabolism. The decrease in Krebs cycle intermediates in urine (succinate, α-ketoglutarate) also suggests a reduction in the glutaminolytic pathway at a systemic level. Our data indicate that inflammatory status is related to this overall loss of energy homeostasis.

A stromal cell free culture system generates mouse pro-T cells that can reconstitute T-cell compartments in vivo.

T-cell lymphopenia following BM transplantation or diseases such as AIDS result in immunodeficiency. Novel approaches to ameliorate this situation are urgently required. Herein, we describe a novel stromal cell free culture system in which Lineage(-) Sca1(+)c-kit(+) BM hematopoietic progenitors very efficiently differentiate into pro-T cells. This culture system consists of plate-bound Delta-like 4 Notch ligand and the cytokines SCF and IL-7. The pro-T cells developing in these cultures express CD25, CD117, and partially CD44; express cytoplasmic CD3ε; and have their TCRß locus partially D-J rearranged. They could be expanded for over 3 months and used to reconstitute the T-cell compartments of sublethally irradiated T-cell-deficient CD3ε(-/-) mice or lethally irradiated WT mice. Pro-T cells generated in this system could partially correct the T-cell lymphopenia of pre-Tα(-/-) mice. However, reconstituted CD3ε(-/-) mice suffered from a wasting disease that was prevented by co-injection of purified CD4(+) CD25(high) WT Treg cells. In a T-cell-sufficient or T-lymphopenic setting, the development of disease was not observed. Thus, this in vitro culture system represents a powerful tool to generate large numbers of pro-T cells for transplantation and possibly with clinical applications.

Effects of increase in fish oil intake on intestinal eicosanoids and inflammation in a mouse model of colitis.

BACKGROUND: Inflammatory bowel diseases (IBD) are chronic intestinal inflammatory diseases affecting about 1% of western populations. New eating behaviors might contribute to the global emergence of IBD. Although the immunoregulatory effects of omega-3 fatty acids have been well characterized in vitro, their role in IBD is controversial. METHODS: The aim of this study was to assess the impact of increased fish oil intake on colonic gene expression, eicosanoid metabolism and development of colitis in a mouse model of IBD. Rag-2 deficient mice were fed fish oil (FO) enriched in omega-3 fatty acids i.e. EPA and DHA or control diet for 4 weeks before colitis induction by adoptive transfer of naïve T cells and maintained in the same diet for 4 additional weeks. Onset of colitis was monitored by colonoscopy and further confirmed by immunological examinations. Whole genome expression profiling was made and eicosanoids were measured by HPLC-MS/MS in colonic samples. RESULTS: A significant reduction of colonic proinflammatory eicosanoids in FO fed mice compared to control was observed. However, neither alteration of colonic gene expression signature nor reduction in IBD scores was observed under FO diet. CONCLUSION: Thus, increased intake of dietary FO did not prevent experimental colitis.

Metabolic phenotyping of the Crohn's disease-like IBD etiopathology in the TNF(ΔARE/WT) mouse model.

The underlying biochemical consequences of inflammatory bowel disease (IBD) on the systemic and gastrointestinal metabolism have not yet been fully elucidated but could help to better understand the disease pathogenesis and to identify tissue-specific markers associated with the different disease stages. Here, we applied a metabonomic approach to monitor metabolic events associated with the gradual development of Crohn's disease (CD)-like ileitis in the TNF(ΔARE/WT) mouse model. Metabolic profiles of different intestinal compartments from the age of 4 up to 24 weeks were generated by combining proton nuclear magnetic resonance ((1)H NMR) spectroscopy and liquid chromatography-mass spectrometry (LC-MS). From 8 weeks onward, mice developed CD similar to the immune and tissue-related phenotype of human CD with ileal involvement, including ileal histological abnormalities, reduced fat mass and body weight, as well as hallmarks of malabsorption with higher energy wasting. The metabonomic approach highlighted shifts in the intestinal lipid metabolism concomitant to the histological onset of inflammation. Moreover, the advanced disease status was characterized by a significantly altered metabolism of cholesterol, triglycerides, phospholipids, plasmalogens, and sphingomyelins in the inflamed tissue (ileum) and the adjacent intestinal parts (proximal colon). These results describe different biological processes associated with the disease onset, including modifications of the general cell membrane composition, alteration of energy homeostasis, and finally the generation of inflammatory lipid mediators. Taken together, this provides novel insights into IBD-related alterations of specific lipid-dependant processes during inflammatory states.

Time-resolved quantitative proteome analysis of in vivo intestinal development.

Postnatal intestinal development is a very dynamic process characterized by substantial morphological changes that coincide with functional adaption to the nutritional change from a diet rich in fat (milk) to a diet rich in carbohydrates on from weaning. Time-resolved studies of intestinal development have so far been limited to investigation at the transcription level or to single or few proteins at a time. In the present study, we elucidate proteomic changes of primary intestinal epithelial cells from jejunum during early suckling (1-7 days of age), middle suckling (7-14 days), and weaning period (14-35 days) in mice, using a label-free proteomics approach. We show differential expression of 520 proteins during intestinal development and a pronounced change of the proteome during the middle suckling period and weaning. Proteins involved in several metabolic processes were found differentially expressed along the development. The temporal expression profiles of enzymes of the glycolysis were found to correlate with the increase in carbohydrate uptake at weaning, whereas the abundance changes of proteins involved in fatty acid metabolism as well as lactose metabolism indicated a nondiet driven preparation for the nutritional change at weaning. Further, we report the developmental abundance changes of proteins playing a vital role in the neonatal acquisition of passive immunity. In addition, different isoforms of several proteins were quantified, which may contribute to a better understanding of the roles of the specific isoforms in the small intestine. In summary, we provide a first, time-resolved proteome profile of intestinal epithelial cells along postnatal intestinal development.

Auto-reconstitution of the T-cell compartment by radioresistant hematopoietic cells following lethal irradiation and bone marrow transplantation.

OBJECTIVE: In lethally irradiated bone marrow chimeras, part of the reconstituted T-cell compartment is derived from the irradiated host, but the detailed origin and functional activity of host-derived T cells has not been thoroughly analyzed. Herein, we determine the origin and function of radioresistant host-derived T cells. MATERIALS AND METHODS: Lethally irradiated thymectomized or nonthymectomized C57BL/6 host mice were reconstituted with syngeneic bone marrow, itself incapable of generating T cells. Using fetal thymic organ cultures, bulk and limiting dilution assays on OP9-DL1 stromal cells, unambiguous cohorts of thymus-derived and peripheral T-cell-derived T cells were phenotypically characterized by flow cytometry and functionally characterized by their ability to participate in a T-cell-dependent antibody response. RESULTS: Both thymus-derived and peripheral T-cell-derived host T cells are functional and can reconstitute 35% of the normal T-cell pool. By comparing thymectomized vs nonthymectomized hosts, host-derived T cells were shown to comprise a major (70%) subpopulation of de novo generated, thymus-derived, polyclonal, naïve cells, and a minor subpopulation of surviving, peripheral, oligoclonal, memory-like cells. Unlike euthymic recipients, mice whose T cells were derived from surviving peripheral T cells were frequently incapable of mounting a T-cell-dependent antibody response. Host-derived thymocytes regenerated in an interleukin-7-dependent fashion from conventional DN2 thymocytes and their differentiation recapitulated normal thymic ontogeny. CONCLUSION: We characterized, for the first time, functional radioresistant DN2-phenotype thymic T-cell precursors, the T-cell progeny of which might provide a first line of defense against infections during the lymphopenic phase post-bone marrow transplantation.

Tolerance checkpoints in B-cell development: Johnny B good.

B-cell development up to the immature B-cell stage takes place in the bone marrow, while final maturation into mature B cells occurs in the spleen. During differentiation, the precursor and immature B cells have to pass several checkpoints, including those in which they are censored for being auto-reactive, and therefore being potentially dangerous. Numerous studies have shown that the immature B-cell stage in the bone marrow and the transitional B-cell stages in the spleen comprise distinct checkpoints at which auto-reactivity is censored. Recently, evidence has been provided that the large pre-BII stage in the bone marrow, at which the pre-BCR is expressed, is yet another B-cell tolerance checkpoint. Here, we review these findings and speculate on directions for possible further experimentation.

Crucial role for BAFF-BAFF-R signaling in the survival and maintenance of mature B cells.

Defects in the expression of either BAFF (B cell activating factor) or BAFF-R impairs B cell development beyond the immature, transitional type-1 stage and thus, prevents the formation of follicular and marginal zone B cells, whereas B-1 B cells remain unaffected. The expression of BAFF-R on all mature B cells might suggest a role for BAFF-R signaling also for their in vivo maintenance. Here, we show that, 14 days following a single injection of an anti-BAFF-R mAb that prevents BAFF binding, both follicular and marginal zone B cell numbers are drastically reduced, whereas B-1 cells are not affected. Injection of control, isotype-matched but non-blocking anti-BAFF-R mAbs does not result in B cell depletion. We also show that this depletion is neither due to antibody-dependent cellular cytotoxicity nor to complement-mediated lysis. Moreover, prevention of BAFF binding leads to a decrease in the size of the B cell follicles, an impairment of a T cell dependent humoral immune response and a reduction in the formation of memory B cells. Collectively, these results establish a central role for BAFF-BAFF-R signaling in the in vivo survival and maintenance of both follicular and marginal zone B cell pools.

Expansion of peripheral naturally occurring T regulatory cells by Fms-like tyrosine kinase 3 ligand treatment.

Fms-like tyrosine kinase 3 ligand (FLT3L) plays a major role in dendritic cell (DC) biology. Deficiency of FLT3L causes a dramatic decrease in DC numbers, whereas increasing its availability (by repetitive injections for 7-10 days) leads to a 10-fold increase in DC numbers. In this study, we show that FLT3L treatment indirectly leads to an expansion of peripheral naturally occurring T regulatory cells (NTregs). The FLT3L-induced increase in NTregs was still observed in thymectomized mice, ruling out the role of the thymus in this mechanism. Instead, the increased number of NTregs was due to proliferation of preexisting NTregs, most likely due to favored interactions with increased number of DCs. In vitro, we show that DCs induce regulatory T-cell (Treg) proliferation by direct cell contact and in an interleukin-2-dependent, T-cell receptor-independent manner. FLT3L could prevent death induced by acute graft-versus-host disease (GVHD). This study demonstrates unique aspects in the regulation of Treg homeostasis by DCs, which were unappreciated until now. It also reinforces the relevance of FLT3L treatment in GVHD by its ability to increase both the number of tolerizing DCs and NTregs.

Peripheral T cells in the thymus: have they just lost their way or do they do something?

In young adult mice, the thymus produces about a million newly formed T cells every day that colonize peripheral lymphoid tissues. Mostly regarded as a primary lymphoid organ only, the relationship between the thymus and peripheral lymphoid organs is considered unidirectional. However, this perception has been challenged by reports showing that peripheral lymphocytes, mostly T cells, can migrate back into the thymus. The presence of recirculating T cells in the thymus is rather incongruous and raises the question: is the presence of 'peripheral' T cells in the thymus superfluous or do these cells fulfill some relevant physiologic functions? There is now evidence that cells of the hematopoietic lineage, including T cells, can play an active role during thymocyte selection, a role generally considered the exclusive property of thymic epithelial cells and dendritic cells. Although, on a per cell basis, peripheral T cells in the thymus may be less efficient than thymus epithelial cells or dendritic cells at thymocyte positive and negative selection, they may nevertheless contribute to selection by influencing the selectable TCR repertoire and post-selection T cell functionality. Here, peripheral lymphocytes re-entering the thymus may be envisioned as Trojan horses as these cells may introduce antigens necessary for both positive and negative selection of T cells.

The B lineage potential of thymus settling progenitors is critically dependent on mouse age.

The nature and lineage potential, particularly that for B cells, of thymus settling progenitors (TSP) in the adult mouse has been the subject of considerable debate. Lack of B cell potential would suggest pre-thymic, whereas its presence would suggest intra-thymic loss of B cell potential. Using limiting dilution analysis (LDA) in vitro and transfer experiments in vivo, we show that the B cell potential of TSP is critically dependent on mouse age, reaching a maximum of about 1 in 20 cells at birth, decreasing 50-fold in adult mice. Cells with a TSP phenotype can be found in the neonatal blood. Furthermore, using LDA, we show that Notch ligand signaling of TSP results in the loss of B cell potential with a half-life of approximately 12 h. Taken together, these results indicate that loss of B cell potential by TSP is an intra-thymic event and highlight the developmental pressure acting on the immune system to rapidly colonize primary lymphoid organs with functional progenitors. This critical time coincides with birth in the mouse. In the adult mouse, we estimate than only about 5 TSP cells/day would be required to maintain steady-state thymopoiesis.

Anti-tumor immunotherapy via antigen delivery from a live attenuated genetically engineered Pseudomonas aeruginosa type III secretion system-based vector.

Immunotherapy requiring an efficient T lymphocyte response is initiated by antigen delivery to antigen-presenting cells. Several studies have assessed the efficiency of various antigen loading procedures, including microbial vectors. Here a live strain of Pseudomonas aeruginosa was engineered to translocate a recombinant antigenic protein into mammalian cells via the type III secretion system, a bacterial device translocating effector proteins into host cells. Optimization of the vector included virulence attenuation and determination of the N-terminal sequence allowing translocation of fused antigens into cells. In vitro delivery of an ovalbumin fragment by the bacterial vector into dendritic cells induced the activation of ovalbumin-specific CD8(+) T lymphocytes. Mice injected with the ovalbumin-delivering vector developed ovalbumin-specific CD8(+) T lymphocytes and were resistant to a subsequent challenge with an ovalbumin-expressing melanoma. Moreover, in a curative assay, injection of the vaccine vector 5 and 12 days after tumor implantation led to a complete cure in five of six animals. These results highlight the utility of type III secretion system-based vectors for anti-tumor immunotherapy.

Peripheral T cell lymphopenia and concomitant enrichment in naturally arising regulatory T cells: the case of the pre-Talpha gene-deleted mouse.

In pre-Talpha (pTalpha) gene-deleted mice, the positively selectable CD4+ CD8+ double-positive thymocyte pool is only 1% that in wild-type mice. Consequently, their peripheral T cell compartment is severely lymphopenic with a concomitant increase in proportion of CD25+ FoxP3+ regulatory T cells. Using mixed bone marrow chimeras, where thymic output was 1% normal, the pTalpha(-/-) peripheral T cell phenotype could be reproduced with normal cells. In the pTalpha(-/-) thymus and peripheral lymphoid organs, FoxP3+ CD4+ cells were enriched. Parabiosis experiments showed that many pTalpha(-/-) CD4+ single-positive thymocytes represented recirculating peripheral T cells. Therefore, the enrichment of FoxP3+ CD4+ single-positive thymocytes was not solely due to increased thymic production. Thus, the pTalpha(-/-) mouse serves as a model system with which to study the consequences of chronic decreased thymic T cell production on the physiology of the peripheral T cell compartment.