Kelly A. Pike

The chicken B‐cell receptor complex and its role in avian B‐cell development

Summary: The bursa of Fabricius is critical to normal B‐lymphocyte development in birds. During embryonic life, B‐cell precursors migrate to the bursal rudiment and those which have undergone productive V(D)J recombination colonize lymphoid follicles and undergo immunoglobulin V gene diversification by gene conversion. The chicken surface IgM complex appears structurally and functionally equivalent to its mammalian counterpart, with homologs to CD79a and CD79b. Expression of a truncated Ig chain is sufficient to drive the early stages of B‐cell development in the embryo bursa. Bursal cells expressing the truncated receptor complex proliferate in bursal follicles, and those which contain V gene rearrangements undergo V gene diversification by gene conversion. The bursa is a gut‐associated organ and antigen is focused to bursal lymphoid follicles after hatch. While expression of the truncated chain is sufficient to support B‐cell development in the embryo, B cells expressing this receptor are rapidly eliminated after hatch. We suggest the possibility that B‐cell development in the bursa after hatch is driven by encounter with antigen leading to redistribution of B cells within the lymphoid follicle, B‐cell proliferation and V gene repertoire development by gene conversion.

The Cytoplasmic Domain of Igα Is Necessary and Sufficient to Support Efficient Early B Cell Development

The B cell receptor complex (BcR) is essential for normal B lymphocyte function, and surface BcR expression is a crucial checkpoint in B cell development. However, functional requirements for chains of the BcR during development remain controversial. We have used retroviral gene transfer to introduce components of the BcR into chicken B cell precursors during embryonic development. A chimeric heterodimer, in which the cytoplasmic domains of chicken Igα and Igβ are expressed by fusion with the extracellular and transmembrane domains of murine CD8α and CD8β, respectively, targeted the cytoplasmic domains of the BcR to the cell surface in the absence of extracellular BcR domains. Expression of this chimeric heterodimer supported all early stages of embryo B cell development: bursal colonization, clonal expansion, and induction of repertoire diversification by gene conversion. Expression of the cytoplasmic domain of Igα, in the absence of the cytoplasmic domain of Igβ, was not only necessary, but sufficient to support B cell development as efficiently as the endogenous BcR. In contrast, expression of the cytoplasmic domain of Igβ in the absence of the cytoplasmic domain of Igα failed to support B cell development. The ability of the cytoplasmic domain of Igα to support early B cell development required a functional Igα immunoreceptor tyrosine-based activation motif. These results support a model in which expression of surface IgM following productive V(D)J recombination in developing B cell precursors serves to chaperone the cytoplasmic domain of Igα to the B cell surface, thereby initiating subsequent stages of development.

The avian B-cell receptor complex: distinct roles of Igα and Igβ in B-cell development

Summary:  The bursa of Fabricius has evolved in birds as a gut‐associated site of B‐cell lymphopoiesis that is segregated from the development of other hematopoietic lineages. Despite differences in the developmental progression of chicken as compared to murine B‐cell lymphopoiesis, cell‐surface immunoglobulin (sIg) expression has been conserved in birds as an essential checkpoint in B‐cell development. B‐cell precursors that express an sIg complex that includes the evolutionarily conserved Igα/β heterodimer colonize lymphoid follicles in the bursa, whereas B‐cell precursors that fail to express sIg due to non‐productive V(D)J recombination are eliminated. Productive retroviral gene transfer has allowed us to introduce chimeric receptor constructs into developing B‐cell precursors in vivo. Chimeric proteins comprising the extracellular and transmembrane regions of murine CD8α fused to the cytoplasmic domain of chicken Igα efficiently supported B‐cell development in precursors that lacked endogenous sIg expression. By contrast, expression of an equivalent chimeric receptor containing the cytoplasmic domain of Igβ actively inhibited B‐cell development. Consequently, the cytoplasmic domains of Igα and Igβ play functionally distinct roles in chicken B‐cell development.

Protein Tyrosine Phosphatase 1B Is a Regulator of the Interleukin-10–Induced Transcriptional Program in Macrophages

The specificity of cytokine target genes may be fine-tuned by protein tyrosine phosphatases. Tailoring Cytokine Responses Although the proinflammatory cytokine interleukin-6 (IL-6) and the anti-inflammatory cytokine IL-10 activate the same transcription factor [signal transducer and activator of transcription 3 (STAT3)], which requires tyrosine phosphorylation, they stimulate the expression of distinct genes. Pike et al. found that mouse macrophages deficient in the protein tyrosine phosphatase 1B (PTP1B) exhibited enhanced activation of STAT3 in response to IL-10, but not IL-6, as well as increased expression of genes required for anti-inflammatory responses. Furthermore, unchecked IL-10 signaling led to the expression of a distinct set of inflammation-associated genes, suggesting that phosphatases may regulate both the identity and extent of expression of cytokine target genes. Both pro- and anti-inflammatory cytokines activate the Janus kinase (JAK)–signal transducer and activator of transcription (STAT) pathway; however, they elicit distinct transcriptional programs. Posttranslational modifications of STAT proteins, such as tyrosine phosphorylation, are critical to ensure the differential expression of STAT target genes. Although JAK-STAT signaling is dependent on reversible tyrosine phosphorylation, whether phosphatases contribute to the specificity of STAT-dependent gene expression is unclear. We examined the role of protein tyrosine phosphatase 1B (PTP1B) in regulating the interleukin-10 (IL-10)–dependent, STAT3-mediated anti-inflammatory response. We found that IL-10–dependent STAT3 phosphorylation and anti-inflammatory gene expression were enhanced in macrophages from PTP1B–/– mice compared to those in macrophages from wild-type mice. Consistent with this finding, the IL-10–dependent suppression of lipopolysaccharide-induced macrophage activation was increased in PTP1B–/– macrophages compared to that in wild-type macrophages, as was the IL-10–dependent increase in the cell surface expression of the anti-inflammatory cytokine receptor IL-4Rα. Furthermore, RNA sequencing revealed the expression of genes encoding proinflammatory factors in IL-10–treated PTP1B–/– macrophages, which correlated with increased phosphorylation of STAT1, which is not normally highly activated in response to IL-10. These findings identify PTP1B as a central regulator of IL-10R–STAT3 and IL-10R–STAT1 signaling, and demonstrate that phosphatases can tailor the quantitative and qualitative properties of cytokine-induced transcriptional responses.

Cell surface immunoglobulin receptors in B cell development.

Expression of surface immunoglobulin (sIg) related receptors has been conserved in phylogenetically distinct species as a critical checkpoint in B cell development. The sIg receptor comprises extracellular IgM heavy and light chains, with the potential for ligand binding, complexed to the Igalpha/Igbeta heterodimer that is responsible for signal transduction through sIg. Experimental systems, from both avian and murine models of B cell development, have been designed to identify the function of individual receptor components in B cell development. In this review, we assess the regulatory functions of different components of the sIg receptor complex during early development in experimental systems from evolutionarily distinct species.

BLNK Binds Active H-Ras to Promote B Cell Receptor-mediated Capping and ERK Activation

Cross-linked B cell receptor (BCR) aggregates on the cell surface, then assembles into the “cap” where Ras is co-localized, and transduces various intracellular signals including Ras-ERK activation. BCR signals induce proliferation, differentiation, or apoptosis of B cells depending on their maturational stage. The adaptor protein BLNK binds various signaling proteins and Igα, a signaling subunit of the BCR complex, and plays an important role in the BCR signal transduction. BLNK was shown to be required for activation of ERK, but not of Ras, after BCR cross-linking, raising a question how BLNK facilitates ERK activation. Here we demonstrate that BLNK binds the active form of H-Ras, and their binding is facilitated by BCR cross-linking. We have identified a 10-amino acid Ras-binding domain within BLNK that is necessary for restoration of BCR-mediated ERK activation in BLNK-deficient B cells and for anti-apoptotic signaling. The Ras-binding domain fused with a CD8α-Igα chimeric receptor could induce prolonged ERK phosphorylation, transcriptional activation of Elk1, as well as the capping of the receptor in BLNK-deficient B cells. These results indicate that BLNK recruits active H-Ras to the BCR complex, which is essential for sustained surface expression of BCR in the form of the cap and for the signal leading to functional ERK activation.

Dual Requirement for the Igα Immunoreceptor Tyrosine-Based Activation Motif (ITAM) and a Conserved Non-Igα ITAM Tyrosine in Supporting Igαβ-Mediated B Cell Development

Surface Ig (sIg) expression is a critical checkpoint during avian B cell development. Only cells that express sIg colonize bursal follicles, clonally expand, and undergo Ig diversification by gene conversion. Expression of a heterodimer, in which the extracellular and transmembrane domains of murine CD8α or CD8β are fused to the cytoplasmic domains of chicken Igα (chIgα) or Igβ, respectively (murine CD8α (mCD8α):chIgα + mCD8β:chIgβ), or an mCD8α:chIgα homodimer supported bursal B cell development as efficiently as endogenous sIg. In this study we demonstrate that B cell development, in the absence of chIgβ, requires both the Igα ITAM and a conserved non-ITAM Igα tyrosine (Y3) that has been associated with binding to B cell linker protein (BLNK). When associated with the cytoplasmic domain of Igβ, the Igα ITAM is not required for the induction of strong calcium mobilization or BLNK phosphorylation, but is still necessary to support B cell development. In contrast, mutation of the Igα Y3 severely compromised calcium mobilization when expressed as either a homodimer or a heterodimer with the cytoplasmic domain of Igβ. However, coexpression of the cytoplasmic domain of Igβ partially complemented the Igα Y3 mutation, rescuing higher levels of BLNK phosphorylation and, more strikingly, supporting B cell development.

Immature T-cell clustering and efficient differentiation require the polarity protein Scribble

T-cell polarization is required for cell migration and cell–cell interactions, cellular behaviors crucial for lymphocyte differentiation. Despite expression of the epithelial polarity network in T cells, neither its contribution to thymocyte polarity nor its requirement during development is known. We report here that depletion of the polarity protein Scribble in hematopoietic progenitor cells results in inefficient T-cell development characterized by a partial developmental block during the early double-negative (DN) stage of differentiation. Scribble-depleted hematopoietic progenitor cells exhibit a delayed transition into late CD44lo/−CD25+ DN3 cells, evidenced by the accumulation of early CD44intCD25+ DN3 cells. As a consequence, a limited cellular expansion and a reduced frequency of intracellular T-cell receptor β–positive DN3 cells are observed among Scribble-deficient differentiating T cells. Moreover, whereas purified Scribble-depleted DN2 and DN3 cells do not exhibit compromised spontaneous motility, T-cell clustering and prolonged homotypic interactions among such cells are reduced. This deficiency correlates with a lack of polarization of the integrin LFA-1 during T-cell migration or on the initiation of T-cell–T-cell interactions. Scribble is therefore a critical contributor to the clustering of immature T cells, an event shown here to be necessary for efficient developmental progression.

TC-PTP and PTP1B: Regulating JAK–STAT signaling, controlling lymphoid malignancies

Lymphoid malignancies are characterized by an accumulation of genetic lesions that act co-operatively to perturb signaling pathways and alter gene expression programs. The Janus kinases (JAK)-signal transducers and activators of transcription (STATs) pathway is one such pathway that is frequently mutated in leukemia and lymphoma. In response to cytokines and growth factors, a cascade of reversible tyrosine phosphorylation events propagates the JAK-STAT pathway from the cell surface to the nucleus. Activated STAT family members then play a fundamental role in establishing the transcriptional landscape of the cell. In leukemia and lymphoma, somatic mutations have been identified in JAK and STAT family members, as well as, negative regulators of the pathway. Most recently, inactivating mutations in the protein tyrosine phosphatase (PTP) genes PTPN1 (PTP1B) and PTPN2 (TC-PTP) were sequenced in B cell lymphoma and T cell acute lymphoblastic leukemia (T-ALL) respectively. The loss of PTP1B and TC-PTP phosphatase activity is associated with an increase in cytokine sensitivity, elevated JAK-STAT signaling, and changes in gene expression. As inactivation mutations in PTPN1 and PTPN2 are restricted to distinct subsets of leukemia and lymphoma, a future challenge will be to identify in which cellular contexts do they contributing to the initiation or maintenance of leukemogenesis or lymphomagenesis. As well, the molecular mechanisms by which PTP1B and TC-PTP loss co-operates with other genetic aberrations will need to be elucidated to design more effective therapeutic strategies.

Regulating naïve and memory CD8 T cell homeostasis--a role for protein tyrosine phosphatases.

A complex network of signalling events coordinate the differentiation, activation and maintenance of T lymphocytes. Tyrosine phosphorylation and dephosphorylation by protein tyrosine kinases and protein tyrosine phosphatases (PTPs) respectively, are critical for the activation and propagation of these signalling cascades. Intriguingly, the removal of tyrosyl phosphate moieties from phosphorylated proteins by phosphatases can contribute to both the positive and negative regulation of signalling events. The complex and diverse roles of individual PTP family members in immune cells is evident by the range of immune disorders caused by PTP deficiencies. Central to several such immune disorders is the disturbance of T cell homeostasis, as characterized by aberrant cell growth, survival and activation. The survival and homeostatic proliferation of naïve and memory CD8 T cells is primarily regulated by signalling events downstream of the T cell receptor complex and common γ chain cytokine receptors, events frequently targeted by PTP activity. We review the primary PTPs involved in CD8 T cell homeostasis, focusing on the signalling nodes that they target. In addition, because the mechanisms that co‐ordinate PTP activity are only partially understood, we discuss currently proposed models of regulation and highlight unanswered questions.