Expression and function of the αVβ5 integrin during human B lymphopoiesis

The integrin αVβ5 is a receptor for sCD23 molecule and the αVβ5-CD23 interaction sustains proliferation of the pre-B cell line SMS-SB. This thesis describes further investigation into the role of αVβ5 integrin during human B cell development. B cell development in the bone marrow involves stepwise maturation of progenitor cells through different defined stages. A tight regulation of proliferation and differentiation mediates the progression of progenitor cells through this developmental pathway. A variety of signals from soluble molecules and adhesive interactions regulate this balance of proliferation and differentiation. The main aim of this work was to assess the importance of the integrin αVβ5 during B cell development by defining its expression and function during specific stages of B cell development in the bone marrow. The αVβ5 integrin was expressed by B cell precursors in the bone marrow, by different pre-B cell lines and the αVβ5-CD23 interaction sustained proliferation of some pre-B cell lines. The pre-B cell lines SMS-SB, RS4;11 and 697 showed a significant proliferative response to αVβ5 stimulation by sCD23, a sCD23-derived long peptide and anti-αVβ5 MAb 15F11. Transitional and more mature B cell lines down-regulated αVβ5 expression and did not show a proliferative response. Both αVβ5 and αVβ3 integrin could be detected on normal bone marrow B cell precursor populations, though αVβ5 was the more highly expressed integrin. In preliminary functional experiments, stimulation of CD19+/κ- cells with sCD23 induced cell proliferation whereas equivalent treatment of CD19+/κ+ cells did not. The data are consistent with the interpretation that αVβ5 integrin is expressed in B cell precursors but that its ability to sustain growth of these cells wanes as the cells mature towards a membrane immunoglobulin-positive state. The αVβ5-mediated proliferation was enhanced by the chemokine SDF-1 and PDGF but not by the cytokines IL-3, IL-4, IL-7 or IL-11. This effect was apparently restricted to earlier B cell precursors and was independent of levels of expression of both αVβ5 and CXCR4. Stimulation of SMS-SB cells by αVβ5 ligands provoked ERK phosphorylation and co-stimulation with SDF-1 promoted a more rapid and sustained ERK activation. PDGF induced a similar effect on αVβ5-mediated activation of ERKphosphorylation. These data suggest that ligation of αVβ5 by soluble, adhesion-independent stimuli activates ERK phosphorylation and this pathway can be modulated by inputs from G-protein-coupled and tyrosine kinase receptors. The murine pro-B cell line BAF03 also displayed αVβ5-mediated proliferation in response to human CD23. Preliminary experiments showed that human CD23 sustained growth of murine bone marrow B cell precursors. Therefore, these data suggest that murine B cells can also use αVβ5 integrin to sustain their growth and the studies described here in human cell lines could be translated into in vivo murine models. Further work is needed to confirm the proliferative response due to the αVβ5-CD23 interaction in normal B cell precursors in the bone marrow and to define the exact stage of development where this interaction is critical. In addition to its expression in the bone marrow B cell precursors, previous work has also demonstrated the expression of αVβ5 integrin in B cells from patients with ALL. Therefore, the αVβ5-CD23 interaction could have important implications not only in proliferation of normal B cell precursors but also in proliferation of neoplastic B cells. These data identify the αVβ5-CD23 interaction as a potentially important interaction during early B cell development, as αVβ5 expression and function is stage-specific, regulated by other molecules and can be demonstrated in both human and murine cell lines

Expression and function of the αVβ5 integrin during human B lymphopoiesis

The integrin αVβ5 is a receptor for sCD23 molecule and the αVβ5-CD23 interaction sustains proliferation of the pre-B cell line SMS-SB. This thesis describes further investigation into the role of αVβ5 integrin during human B cell development. B cell development in the bone marrow involves stepwise maturation of progenitor cells through different defined stages. A tight regulation of proliferation and differentiation mediates the progression of progenitor cells through this developmental pathway. A variety of signals from soluble molecules and adhesive interactions regulate this balance of proliferation and differentiation. The main aim of this work was to assess the importance of the integrin αVβ5 during B cell development by defining its expression and function during specific stages of B cell development in the bone marrow. The αVβ5 integrin was expressed by B cell precursors in the bone marrow, by different pre-B cell lines and the αVβ5-CD23 interaction sustained proliferation of some pre-B cell lines. The pre-B cell lines SMS-SB, RS4;11 and 697 showed a significant proliferative response to αVβ5 stimulation by sCD23, a sCD23-derived long peptide and anti-αVβ5 MAb 15F11. Transitional and more mature B cell lines down-regulated αVβ5 expression and did not show a proliferative response. Both αVβ5 and αVβ3 integrin could be detected on normal bone marrow B cell precursor populations, though αVβ5 was the more highly expressed integrin. In preliminary functional experiments, stimulation of CD19+/κ- cells with sCD23 induced cell proliferation whereas equivalent treatment of CD19+/κ+ cells did not. The data are consistent with the interpretation that αVβ5 integrin is expressed in B cell precursors but that its ability to sustain growth of these cells wanes as the cells mature towards a membrane immunoglobulin-positive state. The αVβ5-mediated proliferation was enhanced by the chemokine SDF-1 and PDGF but not by the cytokines IL-3, IL-4, IL-7 or IL-11. This effect was apparently restricted to earlier B cell precursors and was independent of levels of expression of both αVβ5 and CXCR4. Stimulation of SMS-SB cells by αVβ5 ligands provoked ERK phosphorylation and co-stimulation with SDF-1 promoted a more rapid and sustained ERK activation. PDGF induced a similar effect on αVβ5-mediated activation of ERKphosphorylation. These data suggest that ligation of αVβ5 by soluble, adhesion-independent stimuli activates ERK phosphorylation and this pathway can be modulated by inputs from G-protein-coupled and tyrosine kinase receptors. The murine pro-B cell line BAF03 also displayed αVβ5-mediated proliferation in response to human CD23. Preliminary experiments showed that human CD23 sustained growth of murine bone marrow B cell precursors. Therefore, these data suggest that murine B cells can also use αVβ5 integrin to sustain their growth and the studies described here in human cell lines could be translated into in vivo murine models. Further work is needed to confirm the proliferative response due to the αVβ5-CD23 interaction in normal B cell precursors in the bone marrow and to define the exact stage of development where this interaction is critical. In addition to its expression in the bone marrow B cell precursors, previous work has also demonstrated the expression of αVβ5 integrin in B cells from patients with ALL. Therefore, the αVβ5-CD23 interaction could have important implications not only in proliferation of normal B cell precursors but also in proliferation of neoplastic B cells. These data identify the αVβ5-CD23 interaction as a potentially important interaction during early B cell development, as αVβ5 expression and function is stage-specific, regulated by other molecules and can be demonstrated in both human and murine cell lines.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Inflammation-induced effector CD4+ T cell interstitial migration is alpha-v integrin dependent

Leukocytes must traverse inflamed tissues to effectively control local infection. Although motility in dense tissues appears to be integrin-independent actin-myosin based, during inflammation changes to the extracellular matrix (ECM) may necessitate distinct motility requirements. Indeed, we found that T cell interstitial motility was critically dependent on RGD-binding integrins in the inflamed dermis. Inflammation-induced deposition of fibronectin was functionally linked to increased αv integrin expression on effector CD4+ T cells. Using intravital multi-photon imaging, we found that CD4+ T cell motility was dependent on αv expression. Selective αv blockade or knockdown arrested TH1 motility in the inflamed tissue and attenuated local effector function. These data show a context-dependent specificity of lymphocyte movement in inflamed tissues that is essential for protective immunity

Preferential Expression of Integrin αvβ8 Promotes Generation of Regulatory T Cells by Mouse CD103⁺Dendritic Cells

BACKGROUND and AIMS: Immune responses in the intestine are controlled by regulatory T cells (Tregs), which prevent inflammation in response to commensal bacteria. A specific population of intestinal dendritic cells (DCs), marked by expression of CD103, generate Tregs more efficiently than other DC populations through mechanisms that involve retinoic acid and transforming growth factor (TGF)-β. However, it is not clear how CD103(+) DCs are specialized for this function. We investigated the ability of CD103(+) DCs to promote Treg generation through activation of TGF-β and the role of integrins with the αv subunit in this process. METHODS: Naïve T cells were cultured with purified DCs from mesenteric lymph nodes (MLNs) or intestines of wild-type and αv conditional knockout mice to assess generation of Tregs. Antigens were administered orally to mice, and antigen-specific generation of Tregs was measured in intestinal tissues. Expression of the integrin αv subunit was measured in purified subpopulations of DCs by quantitative polymerase chain reaction and immunoblot analyses. RESULTS: In vitro, CD103(+) DCs generated more Tregs in the presence of latent TGF-β than other MLN DCs. Efficient generation of Tregs required expression of the integrin αv subunit by DCs; mice that lacked αv in immune cells did not convert naïve T cells to intestinal Tregs in response to oral antigen. CD103(+) DCs derived from the MLNs selectively expressed high levels of integrin αvβ8 compared with other populations of DCs. CONCLUSIONS: Expression of αvβ8 is required for CD103(+) DCs to become specialized and activate latent TGF-β and generate Tregs during the induction of tolerance to intestinal antigens in mice

The Role of Autophagy-Related Proteins in Candida albicans Infections

Autophagy plays an important role in maintaining cell homeostasis by providing nutrients during periods of starvation and removing damaged organelles from the cytoplasm. A marker in the autophagic process is the reversible conjugation of LC3, a membrane scaffolding protein, to double membrane autophagosomes. Recently, a role for LC3 in the elimination of pathogenic bacteria and fungi, including Candida albicans (C. albicans), was demonstrated, but these organisms reside in single membrane phagosomes. This process is distinct from autophagy and is termed LC3-associated phagocytosis (LAP). This review will detail the hallmarks of LAP that distinguish it from classical autophagy and review the role of autophagy proteins in host response to C. albicans and other pathogenic fungi

Autophagy Induced by Toll-like Receptor Ligands Regulates Antigen Extraction and Presentation by B Cells

The engagement of B cells with surface-tethered antigens triggers the formation of an immune synapse (IS), where the local secretion of lysosomes can facilitate antigen uptake. Lysosomes intersect with other intracellular processes, such as Toll-like Receptor (TLR) signaling and autophagy coordinating immune responses. However, the crosstalk between these processes and antigen presentation remains unclear. Here, we show that TLR stimulation induces autophagy in B cells and decreases their capacity to extract and present immobilized antigens. We reveal that TLR stimulation restricts lysosome repositioning to the IS by triggering autophagy-dependent degradation of GEF-H1, a Rho GTP exchange factor required for stable lysosome recruitment at the synaptic membrane. GEF-H1 degradation is not observed in B cells that lack αV integrins and are deficient in TLR-induced autophagy. Accordingly, these cells show efficient antigen extraction in the presence of TLR stimulation, confirming the role of TLR-induced autophagy in limiting antigen extraction. Overall, our results suggest that resources associated with autophagy regulate TLR and BCR-dependent functions, which can finetune antigen uptake by B cells. This work helps to understand the mechanisms by which B cells are activated by surface-tethered antigens in contexts of subjacent inflammation before antigen recognition, such as sepsis

αv Integrin expression by DCs is required for Th17 cell differentiation and development of experimental autoimmune encephalomyelitis in mice

Th17 cells are a distinct lineage of T helper cells that protect the body from bacterial and fungal infection. However, Th17 cells also contribute to inflammatory and autoimmune disorders such as multiple sclerosis. Th17 cell generation requires exposure of naive T cells to the cytokine TGF-β in combination with proinflammatory cytokines. Here we show that differentiation of Th17 cells is also critically dependent on αv integrins. In mice, lack of integrin αv in the immune system resulted in loss of Th17 cells in the intestine and lymphoid tissues. It also led to protection from experimental autoimmune encephalomyelitis (EAE). Further analysis indicated that αv integrins on DCs activated latent TGF-β during T cell stimulation and thereby promoted differentiation of Th17 cells. Furthermore, pharmacologic inhibition of αv integrins using cyclic RGD peptides blocked TGF-β activation and Th17 cell generation in vitro and protected mice from EAE. These data demonstrate that activation of TGF-β by αv-expressing myeloid cells may be a critical step in the generation of Th17 cells and suggest that αv integrins could be therapeutic targets in autoimmune disease

αv Integrin expression by DCs is required for Th17 cell differentiation and development of experimental autoimmune encephalomyelitis in mice

Th17 cells are a distinct lineage of T helper cells that protect the body from bacterial and fungal infection. However, Th17 cells also contribute to inflammatory and autoimmune disorders such as multiple sclerosis. Th17 cell generation requires exposure of naive T cells to the cytokine TGF-β in combination with proinflammatory cytokines. Here we show that differentiation of Th17 cells is also critically dependent on αv integrins. In mice, lack of integrin αv in the immune system resulted in loss of Th17 cells in the intestine and lymphoid tissues. It also led to protection from experimental autoimmune encephalomyelitis (EAE). Further analysis indicated that αv integrins on DCs activated latent TGF-β during T cell stimulation and thereby promoted differentiation of Th17 cells. Furthermore, pharmacologic inhibition of αv integrins using cyclic RGD peptides blocked TGF-β activation and Th17 cell generation in vitro and protected mice from EAE. These data demonstrate that activation of TGF-β by αv-expressing myeloid cells may be a critical step in the generation of Th17 cells and suggest that αv integrins could be therapeutic targets in autoimmune disease