Catherine V. Bare

CCL25 increases thymopoiesis after androgen withdrawal

Although studies have demonstrated that androgen withdrawal increases thymic size, molecular mechanisms underlying this expansion remain largely unknown. We show that decreased androgen signaling leads to enhanced immigration of bone marrow T-cell precursors, as manifested by both an early increase of early thymic progenitors (ETP) and improved uptake of adoptively transferred quantified precursors into congenic castrated hosts. We provide evidence that the ETP niche is enhanced after androgen withdrawal by proliferation of UEA(+) thymic epithelial cells (TEC) and increased TEC production of CCL25, a ligand critical for ETP entry. Moreover, the greatest increase in CCL25 production is by UEA(+) TEC, linking function of this subset with the increase in ETP immigration. Furthermore, blockade of CCL25 abrogated the effects of castration by impairing ETP entry, retarding immature thymocyte development, limiting increase of thymic size, and impairing increase of thymopoiesis. Taken together, these findings describe a cohesive mechanism underlying increased thymic productivity after androgen withdrawal.

Differential inducibility of the transcriptional repressor ICER and its role in modulation of Fas ligand expression in T and NK lymphocytes.

The engagement of antigen receptor can initiate apoptosis of T lymphocytes through the induced expression of Fas ligand (FasL). Forskolin, an activator of the cAMP/PKA pathway, results in antagonism of Fas‐dependent, activation‐induced cell death (AICD) by suppressed expression of the FasL. We report that forskolin‐mediated induction of inducible cAMP early repressor (ICER) correlates with transcriptional attenuation of FasL expression in the AICD model 2B4 T cell hybridoma. ICER is inducible in human peripheral blood CD3+ T cells, but in CD19+ B cells, its induction is less responsive to forskolin treatment. Increased expression of ICER correlates with decreased FasL expression in both T and NK cells. ICER binds specifically to the proximal DNA binding siteof the nuclear factor of activated T cells (NFAT) in the FasL promoter and in the presence of the minimal NFAT DNA‐binding domain, the proximal NFAT motif allows ICER and NFAT to form an NFAT/ICER ternary complex in vitro. Moreover, in the activated 2B4 T cell hybridoma, the proximal NFAT motif participates in the down‐regulation of the FasL promoter mediated by ICER. These findings provide further insight into the mechanism involved in cAMP‐mediated transcriptional attenuation of FasL expression in T and NK lymphocytes.

Sensitive GC-MS/MS Method to Measure Deuterium Labeled Deoxyadenosine in DNA from Limited Mouse Cell Populations

A rapid and sensitive gas chromatography-tandem mass spectrometry (GC-MS/MS) method was developed to quantitatively measure low levels of DNA base deoxyadenosine (dA) and its isotopologues (e.g., dA M+1) from limited mouse cell populations. Mice undergoing allogeneic hematopoietic transplantation (AHSCT) received deuterated water at biologically relevant time intervals post AHSCT, allowing labeling of DNA upon cell division, which was detected as the dA M+1 isotopologue. Targeted mouse cell populations were isolated from lymphoid organs and purified by multiparameter fluorescence activated cell sorting. Cell lysis, DNA extraction, and hydrolysis were accomplished using available commercial procedures. The novel analytical method utilized a hydrophilic-lipophilic balanced sample preparation, rapid online hot GC inlet gas phase sample derivatization, fast GC low thermal mass technology, and a recently marketed GC-MS/MS system. Calibration standards containing dA and fortified with relevant levels of dA M+1 (0.25-20%) and dA M+5 (internal standard) were used for sample quantitation. The method employed a quadratic fit for calibration of dA M+1 (0.25-20%) and dA, demonstrated excellent accuracy and precision, and had limits of detection of 100 fg on-column for the dA isotopologues. The method was validated and required only 20 000 cells to characterize population dynamics of cells involved in the biology of chronic graft-versus-host disease, the main cause of late morbidity and nonrelapse-mortality following AHSCT. The high sensitivity and specificity of the method makes it useful for investigating in vivo kinetics on limited and important cell populations (e.g., T regulatory cells) from disease conditions or in disease models that are immune-mediated, such as diabetes, human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS), arthritis, inflammatory bowel disease, and multiple sclerosis.

Comparing DNA enrichment of proliferating cells following administration of different stable isotopes of heavy water

Deuterated water (2H2O) is a label commonly used for safe quantitative measurement of deuterium enrichment into DNA of proliferating cells. More recently, it has been used for labeling proteins and other biomolecules. Our in vitro - in vivo research reports important stable isotopic labeling enrichment differences into the DNA nucleosides and their isotopologues (e.g. deoxyadenosine (dA) M + 1, dA M + 2, dA M + 3), as well as tumor cell proliferation effects for various forms of commercially available stable heavy water (2H2O, H218O, and 2H218O). Using an in vitro mouse thymus tumor cell line, we determined that H218O provides superior DNA labeling enrichment quantitation, as measured by GC-positive chemical ionization (PCI)-MS/MS. In addition, at higher but physiologically relevant doses, both 2H218O and 2H2O down modulated mouse thymus tumor cell proliferation, whereas H218O water had no observable effects on cell proliferation. The in vivo labeling studies, where normal mouse bone marrow cells (i.e. high turnover) were evaluated post labeling, demonstrated DNA enrichments concordant with measurements from the in vitro studies. Our research also reports a headspace-GC-NCI-MS method, which rapidly and quantitatively measures stable heavy water levels in total body water.

FLT3 ligand regulates thymic precursor cells and hematopoietic stem cells through interactions with CXCR4 and the marrow niche

Impaired immune reconstitution after hematopoietic stem cell transplantation (HSCT) is attributed in part to impaired thymopoiesis. Recent data suggest that precursor input may be a point of regulation for the thymus. We hypothesized that administration of FLT3 ligand (FLT3L) would enhance thymopoiesis after adoptive transfer of aged, FLT3L-treated bone marrow (BM) to aged, Lupron-treated hosts by increasing murine HSC (Lin[minus]Sca1+c-Kit+ [LSK] cells) trafficking and survival. In murine models of aged and young hosts, we show that FLT3L enhances thymopoiesis in aged, Lupron-treated hosts through increased survival and export of LSK cells via CXCR4 regulation. In addition, we elucidate an underlying mechanism of FLT3L action on BM LSK cells-FLT3L drives LSK cells into the stromal niche using Hoescht (Ho) dye perimortem. In summary, we show that FLT3L administration leads to: (1) increased LSK cells and early thymocyte progenitor precursors that can enhance thymopoiesis after transplantation and androgen withdrawal, (2) mobilization of LSK cells through downregulation of CXCR4, (3) enhanced BM stem cell survival associated with Bcl-2 upregulation, and (4) BM stem cell enrichment through increased trafficking to the BM niche. Therefore, we show a mechanism by which FLT3L activity on hematopoeitic and thymic progenitor cells may contribute to thymic recovery. These data have potential clinical relevance to enhance thymic reconstitution after cytoreductive therapy.

In vivo kinetics and nonradioactive imaging of rapidly proliferating cells in graft-versus-host disease

Hematopoietic stem cell transplantation (HSCT) offers a cure for cancers that are refractory to chemotherapy and radiation. Most HSCT recipients develop chronic graft-versus-host disease (cGVHD), a systemic alloimmune attack on host organs. Diagnosis is based on clinical signs and symptoms, as biopsies are risky. T cells are central to the biology of cGVHD. We found that a low Treg/CD4+ T effector memory (Tem) ratio in circulation, lymphoid, and target organs identified early and established mouse cGVHD. Using deuterated water labeling to measure multicompartment in vivo kinetics of these subsets, we show robust Tem and Treg proliferation in lymphoid and target organs, while Tregs undergo apoptosis in target organs. Since deuterium enrichment into DNA serves as a proxy for cell proliferation, we developed a whole-body clinically relevant deuterium MRI approach to nonradioactively detect cGVHD and potentially allow imaging of other diseases characterized by rapidly proliferating cells.

IMPAIRED IMMUNE RECONSTITUTION POST SEQUENTIAL HIGH-DOSE CHEMOTHERAPY AND PERIPHERAL BLOOD PROGENITOR CELL (PBPC) INFUSION. † 962

IMPAIRED IMMUNE RECONSTITUTION POST SEQUENTIAL HIGH-DOSE CHEMOTHERAPY AND PERIPHERAL BLOOD PROGENITOR CELL (PBPC) INFUSION. † 962