Belimumab alters transitional B-cell subset proportions in patients with stable systemic lupus erythematosus.

OBJECTIVE: We evaluated the effects of the B-cell activating factor (BAFF)-targeting antibody Belimumab on human nonmemory B-cell pools. Human B-cell pools were identified using surface markers adapted from mouse studies that specifically assessed reductions in immature B cells due to BAFF depletion. Patients with systemic lupus erythematosus (SLE) have high levels of both BAFF and immature B cells. Mechanistic mouse studies provide a framework for understanding human responses to therapies that target B cells. METHODS: Peripheral blood mononuclear cells were isolated from healthy donors and SLE patients on Belimumab or standard-of-care therapy (SCT). Cells were stained for flow cytometry to identify B-cell subsets based on CD21/CD24. Differences in subset proportions were determined by one-way ANOVA and Tukey's post hoc test. RESULTS: Patients treated with Belimumab show alterations in the nonmemory B-cell pool characterized by a decrease in the Transitional 2 (T2) subset (p = 0.002), and an increase in the proportion of Transitional 1 (T1) cells (p = 0.005) as compared with healthy donors and SCT patients. The naïve B-cell compartment showed no significant differences between the groups (p = 0.293). CONCLUSION: Using a translational approach, we show that Belimumab-mediated BAFF depletion reduces the T2 subset in patients, similar to observations in mouse models with BAFF depletion.

Cutting edge: predominant expression of a novel Ikaros isoform in normal human hemopoiesis.

Murine studies implicate Ikaros proteins as regulators of hemopoiesis, particularly in the lymphoid lineages. High homology between murine and human Ikaros suggests that Ikaros expression in the two might be similar. However, initial human studies that focused on leukemia detected novel Ikaros transcripts in patient samples. Thus, novel Ikaros splice forms and DNA nonbinding isoforms were linked with malignancy. We undertook an extensive analysis of normal human Ikaros expression to determine whether novel mRNAs are expressed as proteins and the extent to which these splice variants are unique to leukemia. Here we show that both mRNA and protein for DNA nonbinding Ikaros isoforms and splice variants previously linked to leukemia are expressed in normal human cells. However, our studies identify a new Ikaros isoform not previously described in mouse or human. This isoform is the predominant Ikaros protein in normal human cells, but not in leukemia cell lines.

Identification of very early lymphoid precursors in bone marrow and their regulation by estrogen.

Estrogen is a negative regulator of lymphopoiesis and provides an experimental tool for probing relationships between lymphocyte precursors and stem cells. We found that expression of lymphocyte-associated genes and immunoglobulin (Ig) gene rearrangement occurred before CD45R acquisition. Lymphoid-restricted progenitors that were Lin(-)IL-7R alpha(+)c-kit(lo)TdT(+) (lineage marker(-), interleukin receptor 7 alpha(+), c-kit(lo) and terminal deoxynucleotidyl transferase(+)) were selectively depleted in estrogen-treated mice; within a less differentiated Lin-c-kit(hi) fraction, functional precursors of B and T, but not myeloid, cells were also selectively depleted. TdT and an Ig heavy chain transgene were detected within a hormone-regulated Lin(-)c-kit(hi)Sca-1(+)CD27(+)Flk-2(+)IL-7R alpha(-) subset of this multipotential progenitor population. Identification of these extremely early lymphoid precursors should facilitate investigation of the molecular mechanisms that control lineage-fate decisions in hematopoiesis.

Identification of a novel, human multilymphoid progenitor in cord blood.

The earliest stages of lymphoid commitment from human pluripotent hematopoietic stem cells have not been defined. A clonogenic subpopulation of CD34(+)CD38(-) cord blood cells were identified that expressed high levels of the CD7 antigen and possessed only lymphoid potential. CD34(+)CD38(-)CD7(+) (CD7(+)) cells uniformly coexpressed CD45RA and HLA-DR; c-kit and Thy-1 expression was absent to low. Clonal analysis demonstrated that single CD7(+) cells could generate B cells, natural killer cells, and dendritic cells but were devoid of myeloid or erythroid potential. In contrast, control CD34(+)CD38(-)CD7(-) (CD7(-)) cells generated both lymphoid and myelo-erythroid cells. The lymphoid potential (generation of lymphoid progeny in bulk and single cell cultures) of CD7(+) cells was equivalent to that of the pluripotent CD7(-) cells. RNA expression studies showed that CD7(+) cells expressed PU.1 and GATA-3, but did not express Pax-5, terminal deoxynucleotide transferase, or CD3epsilon. In contrast to the previously described murine common lymphoid progenitor, the alpha chain of the receptor for interleukin-7 was not detected by fluorescence-activated cell sorting analysis or RNA polymerase chain reaction in CD7(+) cells. These studies identify a clonogenic lymphoid progenitor with both B-cell and natural killer cell lineage potential with a molecular profile that suggests a developmental stage more primitive than previously identified lymphoid progenitors. The CD7(+) phenotype distinguishes primitive human lymphoid progenitors from pluripotent stem cells, thus allowing the study of regulation of early human lymphopoiesis and providing an alternative to pluripotent stem cells for genetic manipulation and transplantation. (Blood. 2001;97:3683-3690)

Early B-lymphocyte precursors and their regulation by sex steroids.

This review describes an improved characterization of early B-lymphocyte precursors in mice and the remarkable sensitivity of the same cells to hormones. The nuclear enzyme terminal deoxynucleotidyl transferase (TdT) was used as a marker to image and characterize bone marrow cells lacking all lineage-associated markers. Most early TdT+ precursors have a distinctive density of c-kit and express the interleukin-7Ralpha chain, as well as flt-3/flk2, but lack CD34. An understanding of those cell surface properties made it possible to obtain highly enriched, viable cells with the potential to give rise to CD19+ lymphocytes in culture. A series of other flow cytometry and culture experiments suggested a possible differentiation sequence for these early pro-B cells. This new model was used to advantage in our studies of sex steroids. It appears that early precursors represent a hormone-sensitive control point for determining numbers of new B lymphocytes that are produced within bone marrow. We also compare and contrast these findings with B lymphopoiesis in humans.

Functional assessment of precursors from murine bone marrow suggests a sequence of early B lineage differentiation events.

Most lineage marker-negative (Lin-)TdT+ cells from murine marrow lack CD34 but display c-kit at low density as well as IL-7Ralpha and Flk-2/Flt-3 receptors. Single cells with these characteristics generated CD45RA+CD19- as well as CD19+ lymphocytes in culture. CD45RA+CD19- marrow cells were resolved into three nonoverlapping subsets. One subset, lacking DX5 and Ly-6C antigens, yielded CD19+ cells in culture. Further analysis demonstrated CD24 on most Lin-TdT+ cells and all CD45R+CD19-DX5-Ly-6C- cells. Mac-1/CD11b was absent from these two subsets of B lineage precursors, while IL-7Ralpha was retained during subsequent differentiation to a CD19+ and stromal cell-independent stage. These findings contrast with previous descriptions of B lymphocyte precursors and suggest a sequence of early differentiation events.

Loss of c-kit accompanies B-lineage commitment and acquisition of CD45R by most murine B-lymphocyte precursors.

Using surface markers, we identified two bone marrow (BM) subsets enriched for TdT+ cells on the brink of CD45R acquisition. These two populations, Lin-c-kitLo and Lin-c-kit-, consisting of 35.4% and 7. 4%, respectively, TdT+ cells, generated B-lineage cells in overnight cultures. Approximately half of the c-kitLo B-lineage precursors were bipotential, yielding myeloid and lymphoid progeny, whereas most that were c-kit- gave rise only to lymphocytes. Analysis of B-lineage progression during a finite culture period showed that the most mature precursors were concentrated in the Lin-c-kit- population. Moreover, a majority of the earliest CD45R+ pro-B cells in BM, identified as CD45R+ CD43(+) BP-1(-) CD25(-) natural killer (NK)1.1(-) sIgM-, were also c-kit-. These c-kit- cells, like their c-kitLo counterparts, expressed TdT, proliferated in response to interleukin (IL)-7, and generated sIgM+ cells. These data suggest that TdT expression is initiated as c-kit downregulation begins in Lin- cells, with progressive loss of c-kit during B-lineage differentiation. CD45R expression is initiated during the transition from c-kitLo to c-kit- with many cells losing c-kit before acquiring CD45R. The ability to isolate highly enriched populations of viable CD45R- precursors will be instrumental in characterizing the earliest B-lineage cells.