Correction: Generation and Improvement of Effector Function of a Novel Broadly Reactive and Protective Monoclonal Antibody against Pneumococcal Surface Protein A of Streptococcus pneumoniae.

[This corrects the article DOI: 10.1371/journal.pone.0154616.].

Generation and Improvement of Effector Function of a Novel Broadly Reactive and Protective Monoclonal Antibody against Pneumococcal Surface Protein A of Streptococcus pneumoniae.

A proof-of-concept study evaluating the potential of Streptococcus pneumoniae Pneumococcal Surface Protein A (PspA) as a passive immunization target was conducted. We describe the generation and isolation of several broadly reactive mouse anti-PspA monoclonal antibodies (mAbs). MAb 140H1 displayed (i) 98% strain coverage, (ii) activity in complement deposition and opsonophagocytic killing (OPK) assays, which are thought to predict the in vivo efficacy of anti-pneumococcal mAbs, (iii) efficacy in mouse sepsis models both alone and in combination with standard-of-care antibiotics, and (iv) therapeutic activity in a mouse pneumonia model. Moreover, we demonstrate that antibody engineering can significantly enhance anti-PspA mAb effector function. We believe that PspA has promising potential as a target for the therapy of invasive pneumococcal disease by mAbs, which could be used alone or in conjunction with standard-of-care antibiotics.

Characterization and quantification of clonal heterogeneity among hematopoietic stem cells: a model-based approach.

Hematopoietic stem cells (HSCs) show pronounced heterogeneity in self-renewal and differentiation behavior, which is reflected in their repopulation kinetics. Here, a single-cell-based mathematical model of HSC organization is used to examine the basis of HSC heterogeneity. Our modeling results, which are based on the analysis of limiting dilution competitive repopulation experiments in mice, demonstrate that small quantitative but clonally fixed differences of cellular properties are necessary and sufficient to account for the observed functional heterogeneity. The model predicts, and experimental data validate, that competitive pressures will amplify small clonal differences into large changes in the number of differentiated progeny. We further predict that the repertoire of HSC clones will evolve over time. Last, our results suggest that larger differences in cellular properties have to be assumed to account for genetically determined differences in HSC behavior as observed in different inbred mice strains. The model provides comprehensive systemic and quantitative insights into the clonal heterogeneity among HSCs with potential applications in predicting the behavior of malignant and/or genetically modified cells.

A new mechanism for the aging of hematopoietic stem cells: aging changes the clonal composition of the stem cell compartment but not individual stem cells.

Whether hematopoietic stem cells (HSCs) change with aging has been controversial. Previously, we showed that the HSC compartment in young mice consists of distinct subsets, each with predetermined self-renewal and differentiation behavior. Three classes of HSCs can be distinguished based on their differentiation programs: lymphoid biased, balanced, and myeloid biased. We now show that aging causes a marked shift in the representation of these HSC subsets. A clonal analysis of repopulating HSCs demonstrates that lymphoid-biased HSCs are lost and long-lived myeloid-biased HSCs accumulate in the aged. Myeloid-biased HSCs from young and aged sources behave similarly in all aspects tested. This indicates that aging does not change individual HSCs. Rather, aging changes the clonal composition of the HSC compartment. We show further that genetic factors contribute to the age-related changes of the HSC subsets. In comparison with B6 mice, aged D2 mice show a more pronounced shift toward myeloid-biased HSCs with a corresponding reduction in the number of both T- and B-cell precursors. This suggests that low levels of lymphocytes in the blood can be a marker for HSC aging. The loss of lymphoid-biased HSCs may contribute to the impaired immune response to infectious diseases and cancers in the aged.

The hematopoietic stem compartment consists of a limited number of discrete stem cell subsets.

Hematopoietic stem cells (HSCs) display extensive heterogeneity in their behavior even when isolated as phenotypically homogeneous populations. It is not clear whether this heterogeneity reflects inherently diverse subsets of HSCs or a homogeneous population of HSCs diversified by their response to different external stimuli. To address this, we analyzed 97 individual HSCs in long-term transplantation assays. HSC clones were obtained from unseparated bone marrow (BM) through limiting dilution approaches. Following transplantation into individual hosts, donor-type cells in blood were measured bimonthly and the resulting repopulation kinetics were grouped according to overall shape. Only 16 types of repopulation kinetics were found among the HSC clones even though combinatorially 54 groups were possible. All HSC clones, regardless of their origin, could be assigned to this subset of groups, and the probability of finding new patterns is negligible. Thus, the full repertoire of repopulating HSCs was covered. These data indicate that the HSC compartment consists of a limited number of distinct HSC subsets, each with predictable behavior. Enrichment of HSCs (Lin- Rho- SP) changes the representation of HSC types by selecting for distinct subsets of HSCs. These data from the steady-state HSC repertoire could provide a basis for the diagnosis of perturbed patterns of HSCs potentially caused by disease or aging.

Myeloid-biased hematopoietic stem cells have extensive self-renewal capacity but generate diminished lymphoid progeny with impaired IL-7 responsiveness.

The adult hematopoietic stem cell (HSC) compartment contains a substantial population of lineage-biased (Lin-bi) HSCs. Lin-bi HSCs generate cells of all hematopoietic lineages, albeit with skewed ratios of lymphoid to myeloid cells. The biased ratios are stable through serial transplantation, demonstrating that lineage bias is an inherent function of the HSCs. To define the mechanisms that cause lineage bias, the developmental potential of myeloid-biased (My-bi) HSCs was characterized. In serial transplantation experiments, My-bi HSCs contributed significantly longer to repopulation than other types of HSCs. The long lifespan indicates that My-bi HSCs are important for the persistence of HSC function throughout life. My-bi HSCs produce normal levels of myeloid precursors but reduced levels of precursors for the T- and B- lymphocyte lineages. Gene array analysis suggested that the lymphoid progeny of My-bi HSCs express lowered levels of interleukin-7 (IL-7) receptor. Indeed, the progeny derived from My-bi HSCs failed to respond to IL-7 in vitro. Thus, My-bi HSCs are programmed for diminished lymphopoiesis through a mechanism that involves a blunted response of its progeny to the central lymphokine IL-7. The data demonstrate that epigenetic regulation on the level of the HSCs can directly affect the number, composition, and function of the mature progeny.

Limiting dilution analysis for estimating the frequency of hematopoietic stem cells: uncertainty and significance.

The ability to predict accurately the number of hematopoietic stem cells (HSCs) in a graft is important for the success of HSC transplantation. Limiting dilution analysis (LDA) in vitro and in vivo is widely used to enumerate HSCs. However, there have been few attempts to standardize this approach. Particularly, the role of statistical and experimental errors in the performance and evaluation of LDA has received little attention. Since these errors directly affect the interpretation, validity, and significance of the LDA results, we have here performed a systematic analysis of the contribution of different types of errors.Long-term culture-initiating cells (LTC-IC) in the bone marrow of C57BL/6 (B6) mice were measured. Experiments were designed to exclude systematically different types of experimental errors. Computer simulations were performed to estimate the statistical error. Analysis of 137 LTC-IC assays showed 2.8 +/- 1.06 LTC-IC per 10(5) cells in the bone marrow of B6 mice. The major components of the uncertainty were derived from variations introduced by performing the experiments at different time points and by the statistical error. Surprisingly, operator errors and mouse-to-mouse error, including age and sex of the animals, contributed little to the overall uncertainty. As expected, the errors were found to decrease when increasing numbers of replica were analyzed. A computer program was developed to assist with the optimal design of the assay. The analysis presented here provides rational strategies for standardizing the experimental design and for gauging the accuracy of LDA-based HSC measurements.

Deterministic regulation of hematopoietic stem cell self-renewal and differentiation.

Most current theories assume that self-renewal and differentiation of hematolymphoid stem cells (HSCs) is randomly regulated by intrinsic and environmental influences. A direct corollary of these tenets is that self-renewal will continuously generate functionally heterogeneous daughter HSCs. Decisions about self-renewal versus commitment are made by individual, single HSCs and, thus, require examination on the clonal level. We followed the behavior of individual, clonally derived HSCs through long-term, serial repopulation experiments. These studies showed that daughter HSCs derived from individual clones were remarkably similar to each other in the extent and kinetics of repopulation. Moreover, daughter HSCs within a clone showed equivalent contributions to the myeloid or lymphoid lineages. Lineage contribution could be followed because of the discovery of a new subset of HSCs that gave rise stably to skewed ratios of myeloid and lymphoid cells. Overall, the data argue that self-renewal does not contribute to the heterogeneity of the adult HSC compartment. Rather, all HSCs in a clone follow a predetermined fate, consistent with the generation-age hypothesis. By extension, this suggests that the self-renewal and differentiation behavior of HSCs in adult bone marrow is more predetermined than previously thought.