Cytokine Signaling and Hematopoietic Homeostasis Are Disrupted in Lnk-deficient Mice

The adaptor protein Lnk, and the closely related proteins APS and SH2B, form a subfamily of SH2 domain-containing proteins implicated in growth factor, cytokine, and immunoreceptor signaling. To elucidate the physiological function of Lnk, we derived Lnk-deficient mice. Lnk−/− mice are viable, but display marked changes in the hematopoietic compartment, including splenomegaly and abnormal lymphoid and myeloid homeostasis. The in vitro proliferative capacity and absolute numbers of hematopoietic progenitors from Lnk−/− mice are greatly increased, in part due to hypersensitivity to several cytokines. Moreover, an increased synergy between stem cell factor and either interleukin (IL)-3 or IL-7 was observed in Lnk−/− cells. Furthermore, Lnk inactivation causes abnormal modulation of IL-3 and stem cell factor–mediated signaling pathways. Consistent with these results, we also show that Lnk is highly expressed in multipotent cells and committed precursors in the erythroid, megakaryocyte, and myeloid lineages. These data implicate Lnk as playing an important role in hematopoiesis and in the regulation of growth factor and cytokine receptor–mediated signaling

Identification of a Novel Developmental Stage Marking Lineage Commitment of Progenitor Thymocytes

Bipotent progenitors for T and natural killer (NK) lymphocytes are thought to exist among early precursor thymocytes. The identification and functional properties of such a progenitor population remain undefined. We report the identification of a novel developmental stage during fetal thymic ontogeny that delineates a population of T/NK-committed progenitors (NK1.1+/CD117+/CD44+/CD25−). Thymocytes at this stage in development are phenotypically and functionally distinguishable from the pool of multipotent lymphoid-restricted (B, T, and NK) precursor thymocytes. Exposure of multipotent precursor thymocytes or fetal liver– derived hematopoietic progenitors to thymic stroma induces differentiation to the bipotent developmental stage. Continued exposure to a thymic microenvironment results in predominant commitment to the T cell lineage, whereas coculture with a bone marrow–derived stromal cell line results in the generation of mature NK cells. Thus, the restriction point to T and NK lymphocyte destinies from a multipotent progenitor stage is marked by a thymus-induced differentiation step

Bone marrow basophils provide survival signals to immature B cells in vitro but are dispensable in vivo.

Immature B cells are the first B cell progenitors to express a fully formed B cell receptor and are therefore subject to extensive selection processes that act to mitigate the emergence of autoreactive clones. While it is well appreciated that most B cell generation in the bone marrow is highly dependent on access to molecules present in the local milieu, the existence of extrinsically provided factors that modulate immature B cell biology is ambiguous. Nonetheless, a population of CD49b+CD90lo cells has demonstrated in vitro potential to promote immature B cell survival. Using a mouse basophil reporter strain we confirmed the identity of these CD49b+CD90lo supportive cells as basophils. However, analysis of bone marrow B cell populations following lineage specific basophil depletion demonstrates that basophils do not have a significant role in vivo in modulating immature B cell biology during steady-state conditions

Basophils do not affect progenitor B cell numbers and λ light chain usage <i>in vivo</i>.

<p>(A-E) Basoph8 mice were crossed to the ROSA-DTA strain and bone marrow cell numbers and B cell populations were enumerated by flow cytometry. Age and sex matched ROSA-DTA mice were used as controls. (B) Total bone marrow cellularity was calculated by trypan blue staining and counting with a hemocytometer. (A, C) Representative bone marrow flow cytometry plots are shown. (D) B cell populations were calculated with pro-B and pre-B defined as CD19⁺CD93⁺IgM^-IgD^-, immature-B as CD19⁺CD93⁺IgM⁺IgD^-, transitional as CD19⁺CD93⁺IgM⁺IgD⁺, and mature as CD19⁺CD93^-IgM⁺IgD⁺. (E) Bone marrow was stained for λ light chain. Data shown (D, E) as median + quartile box-and-whisker plots and is pooled from three individual experiments using mice aged 10-16 weeks; n = 9 ROSA-DTA, n = 10 Basoph8 X DTA. All groups are non-significant.</p

<i>RAG1</i> expression is unchanged with basophil deletion.

<p>Real-time PCR analysis of <i>RAG1</i> in B cells from Basoph8 X ROSA-DTA or Basoph8 X B6 mice. B cells were purified by magnetic cell selection before RNA extraction. <i>RAG1</i> levels were first normalized to <i>Beta-actin</i> and then to mean <i>RAG1</i> levels in Basoph8 X B6 (control) samples. Data are from three independent experiments in which B cells from mice aged 12-20 weeks were pooled from one to three mice.</p

Basophils protect B cell progenitors in co-culture systems.

<p>(A) Bone marrow from 7-16 week old Basoph8 mice was stained for CD49b and CD90. CD49b⁺CD90^loYFP⁺ and CD49b^-CD90^loYFP^- cells were sorted by FACS and co-cultured overnight together with CD2⁺IgD^- FACS sorted B cell progenitors at a ratio of 1:25. In some culture wells anti-μ was added at 20 μg/mL to stimulate BCR crosslinking. Cell survival was measured by a Nicoletti assay. (B) Sorted CD2⁺IgM^-IgD^- B cell progenitors were co-cultured for two days with YFP⁺ cells. The absolute number of CD2⁺IgM⁺CD23^- and CD2⁺IgM⁺CD23⁺ B cells was then determined by flow cytometry. Data shown as mean ± standard deviation and are representative of three individual experiments; *<i>p</i><0.05; **<i>p</i><0.01; ***<i>p<</i>0.001.</p

Basophils do not influence immature B cell localization.

<p>Basoph8 X ROSA-DTA mice were intravenously injected with 1 μg anti-B220 conjugated to R-phycoerythin and euthanized two minutes later. The frequency of B220-PE staining B cells was determined by flow cytometry. Left panels show representative flow plots while the right graph shows data pooled from two independent experiments in a median + quartile box-and-whisker plot; n = 6 all groups mice were 8-16 weeks of age. All groups are non-significant.</p

Basophil share marrow localization patterns with B cells.

<p>Basoph8 mice were intravenously injected with 1 μg anti-FcεRIα-PE and euthanized two minutes later. FcεRIα-PE staining was then assessed by flow cytometry. Shown is a representative flow plot and quantification from three independent experiments.</p