Antigen-based vaccination and prevention of type 1 diabetes.

Insulin-dependent or type 1 diabetes (T1D) is a paradigm for prevention of autoimmune disease: Pancreatic ß-cell autoantigens are defined, at-risk individuals can be identified before the onset of symptoms, and autoimmune diabetes is preventable in rodent models. Intervention in asymptomatic individuals before or after the onset of subclinical islet autoimmunity places a premium on safety, a requirement met only by lifestyle-dietary approaches or autoantigen-based vaccination to induce protective immune tolerance. Insulin is the key driver of autoimmune ß-cell destruction in the nonobese diabetic (NOD) mouse model of T1D and is an early autoimmune target in children at risk for T1D. In the NOD mouse, mucosal administration of insulin induces regulatory T cells that protect against diabetes. The promise of autoantigen-specific vaccination in humans has yet to be realized, but recent trials of oral and nasal insulin vaccination in at-risk humans provide grounds for cautious optimism.

T cell regulation mediated by interaction of soluble CD52 with the inhibitory receptor Siglec-10.

Functionally diverse T cell populations interact to maintain homeostasis of the immune system. We found that human and mouse antigen-activated T cells with high expression of the lymphocyte surface marker CD52 suppressed other T cells. CD52(hi)CD4(+) T cells were distinct from CD4(+)CD25(+)Foxp3(+) regulatory T cells. Their suppression was mediated by soluble CD52 released by phospholipase C. Soluble CD52 bound to the inhibitory receptor Siglec-10 and impaired phosphorylation of the T cell receptor-associated kinases Lck and Zap70 and T cell activation. Humans with type 1 diabetes had a lower frequency and diminished function of CD52(hi)CD4(+) T cells responsive to the autoantigen GAD65. In diabetes-prone mice of the nonobese diabetic (NOD) strain, transfer of lymphocyte populations depleted of CD52(hi) cells resulted in a substantially accelerated onset of diabetes. Our studies identify a ligand-receptor mechanism of T cell regulation that may protect humans and mice from autoimmune disease.

Forward light scatter is a simple measure of T-cell activation and proliferation but is not universally suited for doublet discrimination.

Following activation by antigen, T cells enter the cell cycle in stages that can be defined with flow cytometric markers. We show that these markers include the increase of forward light scatter width (FSC-W) signal and the ratio of FSC area/peak. This change in light scatter precedes the first cell division and may reflect blast transformation. We show that the FSC-W parameter can be used, alone or in combination with other activation markers, to monitor the relative and absolute numbers of T cells responding to a proliferative stimulus. In contrast to dye dilution assays, the FSC-W method does not allow discrimination between consecutive cell divisions, but it has several advantages and could complement the dye dilution assay. Our findings also show that the routine elimination of doublets based on FSC signals may exclude proliferating T cells from the analysis.

Erythropoiesis from adult but not fetal blood-derived CD133+ stem cells depends strongly on interleukin-3.

We have shown previously that erythropoiesis in cultures from fetal blood depends less on interleukin-3 (IL3) than erythropoiesis from adult blood. This paper explores if this is due to different proportions of stem cell sub-populations with different IL3 requirement, or to different IL3 requirement on fetal and adult stem cells in general. The CD133 (AC133) antigen is found on half of all CD34+ cells with erythropoietic potential and is thought to mark a primitive stem cell sub-population. This work compares the IL3 requirements of CD133+ and CD133-CD34+ cells derived from fetal (12-20 weeks) and adult blood. Erythropoiesis was monitored using flow cytometry and colony counting. Erythropoiesis from adult CD133+ but not CD133-CD34+ cells was dramatically delayed (6-8 days) in the absence of IL3. In cultures from fetal blood, both CD133+ and CD133-CD34+ cells showed only a small dependency on IL3. The ratio of erythroid colony-forming CD133+ cells to erythroid colony-forming CD34+ cells was 0.5-0.6 in adult and 0.2-0.4 in fetal blood. The differential IL3 dependency of erythropoiesis from fetal and adult blood CD133+ and CD133-CD34+ stem cells may be relevant for the choice and preparation of stem cells for clinical purposes.

IL-3-dependent early erythropoiesis is stimulated by autocrine transforming growth factor beta.

Autocrine/paracrine transforming growth factor beta (TGF-beta) is an important regulator of stem cell quiescence and generally suppresses stem cell proliferation. However, we show here that during the first few days of an erythroid cell culture from adult blood stem cells, the presence of neutralizing antibodies against TGF-beta had a suppressive effect on subsequent erythropoiesis, indicating a stimulatory action of autocrine TGF-beta. The suppression occured in the form of a delay in erythroblast proliferation rather than a reduction in final erythroid colony numbers. The inhibitory effect of anti-TGF-beta occured in the presence of interleukin-3 (IL-3) but not in cultures with only stem cell factor and erythropoietin. Erythroblasts expressing gamma-globin (gamma+) were more strongly suppressed than erythroblasts expressing only beta-globin (gamma-beta+), so that stem cell treatment with anti-TGF-beta caused a decrease in the proportion of gamma+ cells. Anti-TGF-beta had an inhibitory effect on erythropoiesis only when administered during the first 4 days of culture, that is, before the onset of globin expression and dependence on erythropoietin. The decreasing effect of anti-TGF-beta with delayed addition coincided with a decreasing dependence on IL-3. CD133+ stem cells were more strongly suppressed by anti-TGF-beta than the complementary CD133-CD34+ stem cells, and the latter were also much less dependent on IL-3. The treatment of very early stem cell cultures with a pulse of added TGF-beta1 in the presence of IL-3 increased the subsequent proliferation of erythroblasts. Taken together, the data suggest that IL-3-driven early erythropoiesis from immature peripheral blood stem cells is stimulated by autocrine TGF-beta.

Reactivation of fetal hemoglobin in adult stem cell erythropoiesis by transforming growth factor-beta.

Treatment of adult blood-derived stem cells with transforming growth factor (TGF-beta) during the first 3-4 days in culture increases the proportions and absolute numbers of erythroid cells subsequently expressing fetal hemoglobin (F+ cells). The change in F+ cell proportions may be due to globin switching or to selective effects on the expansion of stem cell subpopulations with different globin expression programs. To distinguish between the two mechanisms, we compared the effects of TGF-beta on proliferation and globin expression with the effects of well-researched agents known to increase fetal hemoglobin (HbF) in sickle cell patients. Hydroxyurea suppressed F+ and F- erythroid cells equally and thus did not affect the F+ proportions. Aza-cytidine and sodium butyrate, known reactivators of gamma-globin expression, suppressed F+ and F- cells differentially and increased F+ cell proportions with a dependence on treatment timing similar to that of TGF-beta. In contrast to TGF-beta, these agents had no superimposed stimulatory effect. The data suggest that TGF-beta reactivates gamma-globin expression, combined with a sequential stimulation and suppression of erythropoiesis. The similarities between the actions of TGF-beta and therapeutic reactivators of fetal hemoglobin make it conceivable that TGF-beta may have the potential to increase HbF in patients with beta-hemoglobin disorders.

A simple and sensitive erythroblast scoring system to identify fetal cells in maternal blood.

OBJECTIVES: Nucleated red blood cells (NRBCs) of fetal origin appear to have distinguishable characteristics from that of maternal NRBCs in both nuclear morphology and properties of hemoglobin staining. However, these differences have yet to be quantified. Our aim was to develop an erythroblast scoring system using four distinct phenotypic parameters (nuclear roundness, nuclear morphology, gamma hemoglobin staining intensity, and peripheral brightness of the stained cytoplasm) to address this issue. METHODS: NRBCs were isolated from four maternal blood samples by density gradient separation, CD15/45 depletion, and gamma hemoglobin positive selection after elective termination of a trisomy 21 male fetus (47,XY,+21). All cells were deposited onto microscope slides and every NRBC was analyzed according to the scoring system. Each of the four individual parameters was given a value from 0 to 3 points and a combined score was obtained for each cell (range 0-12). Fluorescence in situ hybridization (FISH) using X- and Y-specific probes was performed to determine, on the basis of interphase karyotype, whether the cell was maternal or fetal. RESULTS: The majority of maternal NRBCs were found to have a combined score of 6 or less (103/117) and the majority of fetal NRBCs were found to have a score of 7 or greater (43/53). The proportion of cells that were identified correctly as fetal increased with each ascending category of combined score. For example, 5.7% of NRBCs with a combined score of 5 points or less were found to be fetal, whereas 19.2% of NRBCs with a combined score of 6 points were fetal. At combined scores of 11 and 12 points, 100% of NRBCs were found to be fetal. CONCLUSION: Fetal NRBCs have characteristic morphology and a gamma hemoglobin staining appearance that makes them distinguishable from maternal NRBCs. The scoring system presented here is a simple and sensitive method to distinguish fetal NRBCs from adult cells in maternal blood. This system may have clinical utility for noninvasive prenatal diagnosis as well as applications for basic research into the developmental biology of NRBCs. In addition, these defined parameters may serve as computational classifiers for the automated detection of fetal cells in maternal blood.

Fetal cell isolation from maternal blood cultures by flow cytometric hemoglobin profiles. Results of a preliminary clinical trial.

OBJECTIVE: We conducted a trial to test if the blood of pregnant women contains fetal clonogenic erythroid cells the progeny of which can be identified and isolated by a newly developed flow-sorting procedure. METHODS: We have previously demonstrated the identification of fetal nucleated red cells in cocultures of fetal and adult blood. The procedure is based on profiles of the correlated contents of fetal and adult hemoglobin (HbF and HbA, respectively), using antibodies specific for the different hemoglobin chains. In such profiles, fetal cells contain only HbF, while the vast majority of adult cells contain either only HbA or a combination of HbA and HbF. HbF+ HbA- cells are flow sorted and fetal cells identified by fluorescence in situ hybridization, using chromosome-specific probes. This technique provides a yield that approaches 100%, meaning that fetal cells will be found even if the culture contains only a single fetal erythroid colony among thousands of maternal colonies. Peripheral blood samples were obtained from 11 women carrying chromosomally normal male fetuses, from 5 women carrying trisomy 21 fetuses, and from 2 women carrying trisomy 18 fetuses. A further six samples came from women with an unknown fetal karyotype. As positive controls, we used blood samples drawn after termination procedures that tended to induce some fetomaternal hemorrhage. In parallel to the method being tested, we employed alternative techniques of fetal cell detection: one third of the mononuclear cell preparations from each maternal blood sample was not cultured but labeled with anti-HbF antibodies for flow sorting of F+ cells. Ten percent of the total harvested cell population of each culture was subjected to quantitative polymerase chain reaction analysis targeting a Y-chromosome-specific sequence. RESULTS: Most post-termination blood samples yielded fetal cells with high purity which demonstrates the validity of the method. However, no fetal cells were found in any of the maternal blood samples with normal or abnormal pregnancies, neither before nor after culture. CONCLUSION: We conclude that a cell culture approach targeting clonogenic erythroid cells offers no advantage over established methods of direct isolation.