Killing two birds with one drug: a new application for HIV-1 cell entry inhibitors in the treatment of metastatic cancer.

The chemokine receptors CCR5 and CXCR4 serve as co-receptors for the human immunodeficiency virus 1 (HIV-1) and thus, are important cellular components during HIV-1 cell entry. In recent years, a new biological role for chemokine receptors has emerged in assisting the spread of primary tumors to distant secondary sites within the human body (metastasis). This review highlights some of the HIV-1 cell entry inhibitors (antagonists), which are currently in development and/or under evaluation in clinical trials, and discusses the therapeutic use of these new antagonists for the treatment of certain forms of metastatic cancer

GAR22β regulates cell migration, sperm motility, and axoneme structure

© 2016 Gamper et al. Spatiotemporal cytoskeleton remodeling is pivotal for cell adhesion and migration. Here we investigated the function of Gas2-related protein on chromosome 22 (GAR22β), a poorly characterized protein that interacts with actin and microtubules. Primary and immortalized GAR22β-/- Sertoli cells moved faster than wild-type cells. In addition, GAR22β-/- cells showed a more prominent focal adhesion turnover. GAR22β overexpression or its reexpression in GAR22β-/- cells reduced cell motility and focal adhesion turnover. GAR22β-actin interaction was stronger than GAR22β-microtubule interaction, resulting in GAR22β localization and dynamics that mirrored those of the actin cytoskeleton. Mechanistically, GAR22β interacted with the regulator of microtubule dynamics end-binding protein 1 (EB1) via a novel noncanonical amino acid sequence, and this GAR22β-EB1 interaction was required for the ability of GAR22β to modulate cell motility. We found that GAR22β is highly expressed in mouse testes, and its absence resulted in reduced spermatozoa generation, lower actin levels in testes, and impaired motility and ultrastructural disorganization of spermatozoa. Collectively our findings identify GAR22β as a novel regulator of cell adhesion and migration and provide a foundation for understanding the molecular basis of diverse cytoskeleton-dependent processes

Relative Frequencies of Alloantigen-Specific Helper CD4 T Cells and B Cells Determine Mode of Antibody-Mediated Allograft Rejection.

Humoral alloimmunity is now recognized as a major determinant of transplant outcome. MHC glycoprotein is considered a typical T-dependent antigen, but the nature of the T cell alloresponse that underpins alloantibody generation remains poorly understood. Here, we examine how the relative frequencies of alloantigen-specific B cells and helper CD4 T cells influence the humoral alloimmune response and how this relates to antibody-mediated rejection (AMR). An MHC-mismatched murine model of cardiac AMR was developed, in which T cell help for alloantibody responses in T cell deficient (Tcrbd-/-) C57BL/6 recipients against donor H-2Kd MHC class I alloantigen was provided by adoptively transferred "TCR75" CD4 T cells that recognize processed H-2Kd allopeptide via the indirect-pathway. Transfer of large numbers (5 × 105) of TCR75 CD4 T cells was associated with rapid development of robust class-switched anti-H-2Kd humoral alloimmunity and BALB/c heart grafts were rejected promptly (MST 9 days). Grafts were not rejected in T and B cell deficient Rag2-/- recipients that were reconstituted with TCR75 CD4 T cells or in control (non-reconstituted) Tcrbd-/- recipients, suggesting that the transferred TCR75 CD4 T cells were mediating graft rejection principally by providing help for effector alloantibody responses. In support, acutely rejecting BALB/c heart grafts exhibited hallmark features of acute AMR, with widespread complement C4d deposition, whereas cellular rejection was not evident. In addition, passive transfer of immune serum from rejecting mice to Rag2-/- recipients resulted in eventual BALB/c heart allograft rejection (MST 20 days). Despite being long-lived, the alloantibody responses observed at rejection of the BALB/c heart grafts were predominantly generated by extrafollicular foci: splenic germinal center (GC) activity had not yet developed; IgG secreting cells were confined to the splenic red pulp and bridging channels; and, most convincingly, rapid graft rejection still occurred when recipients were reconstituted with similar numbers of Sh2d1a-/- TCR75 CD4 T cells that are genetically incapable of providing T follicular helper cell function for generating GC alloimmunity. Similarly, alloantibody responses generated in Tcrbd-/- recipients reconstituted with smaller number of wild-type TCR75 CD4 T cells (103), although long-lasting, did not have a discernible extrafollicular component, and grafts were rejected much more slowly (MST 50 days). By modeling antibody responses to Hen Egg Lysozyme protein, we confirm that a high ratio of antigen-specific helper T cells to B cells favors development of the extrafollicular response, whereas GC activity is favored by a relatively high ratio of B cells. In summary, a relative abundance of helper CD4 T cells favors development of strong extrafollicular alloantibody responses that mediate acute humoral rejection, without requirement for GC activity. This work is composed of two parts, of which this is Part I. Please read also Part II: Chhabra et al., 2019

Relative Frequencies of Alloantigen-Specific Helper CD4 T Cells and B Cells Determine Mode of Antibody-Mediated Allograft Rejection

Humoral alloimmunity is now recognized as a major determinant of transplant outcome. MHC glycoprotein is considered a typical T-dependent antigen, but the nature of the T cell alloresponse that underpins alloantibody generation remains poorly understood. Here, we examine how the relative frequencies of alloantigen-specific B cells and helper CD4 T cells influence the humoral alloimmune response and how this relates to antibody-mediated rejection (AMR). An MHC-mismatched murine model of cardiac AMR was developed, in which T cell help for alloantibody responses in T cell deficient (Tcrbd−/−) C57BL/6 recipients against donor H-2Kd MHC class I alloantigen was provided by adoptively transferred “TCR75” CD4 T cells that recognize processed H-2Kd allopeptide via the indirect-pathway. Transfer of large numbers (5 × 105) of TCR75 CD4 T cells was associated with rapid development of robust class-switched anti-H-2Kd humoral alloimmunity and BALB/c heart grafts were rejected promptly (MST 9 days). Grafts were not rejected in T and B cell deficient Rag2−/− recipients that were reconstituted with TCR75 CD4 T cells or in control (non-reconstituted) Tcrbd−/− recipients, suggesting that the transferred TCR75 CD4 T cells were mediating graft rejection principally by providing help for effector alloantibody responses. In support, acutely rejecting BALB/c heart grafts exhibited hallmark features of acute AMR, with widespread complement C4d deposition, whereas cellular rejection was not evident. In addition, passive transfer of immune serum from rejecting mice to Rag2−/− recipients resulted in eventual BALB/c heart allograft rejection (MST 20 days). Despite being long-lived, the alloantibody responses observed at rejection of the BALB/c heart grafts were predominantly generated by extrafollicular foci: splenic germinal center (GC) activity had not yet developed; IgG secreting cells were confined to the splenic red pulp and bridging channels; and, most convincingly, rapid graft rejection still occurred when recipients were reconstituted with similar numbers of Sh2d1a−/− TCR75 CD4 T cells that are genetically incapable of providing T follicular helper cell function for generating GC alloimmunity. Similarly, alloantibody responses generated in Tcrbd−/− recipients reconstituted with smaller number of wild-type TCR75 CD4 T cells (103), although long-lasting, did not have a discernible extrafollicular component, and grafts were rejected much more slowly (MST 50 days). By modeling antibody responses to Hen Egg Lysozyme protein, we confirm that a high ratio of antigen-specific helper T cells to B cells favors development of the extrafollicular response, whereas GC activity is favored by a relatively high ratio of B cells. In summary, a relative abundance of helper CD4 T cells favors development of strong extrafollicular alloantibody responses that mediate acute humoral rejection, without requirement for GC activity.This work is composed of two parts, of which this is Part I. Please read also Part II: Chhabra et al., 2019

Germinal Center Alloantibody Responses Mediate Progression of Chronic Allograft Injury

Different profiles of alloantibody responses are observed in the clinic, with those that persist, often despite targeted treatment, associated with poorer long-term transplant outcomes. Although such responses would suggest an underlying germinal center (GC) response, the relationship to cellular events within the allospecific B cell population is unclear. Here we examine the contribution of germinal center (GC) humoral alloimmunity to chronic antibody mediated rejection (AMR). A murine model of chronic AMR was developed in which T cell deficient (Tcrbd−/−) C57BL/6 recipients were challenged with MHC-mismatched BALB/c heart allografts and T cell help provided by reconstituting with 103 “TCR75” CD4 T cells that recognize self-restricted allopeptide derived from the H-2Kd MHC class I alloantigen. Reconstituted recipients developed Ig-switched anti-Kd alloantibody responses that were slow to develop, but long-lived, with confocal immunofluorescence and flow cytometric characterization of responding H-2Kd-allospecific B cells confirming persistent splenic GC activity. This was associated with T follicular helper (TFH) cell differentiation of the transferred TCR75 CD4 T cells. Heart grafts developed progressive allograft vasculopathy, and were rejected chronically (MST 50 days), with explanted allografts displaying features of humoral vascular rejection. Critically, late alloantibody responses were abolished, and heart grafts survived indefinitely, in recipients reconstituted with Sh2d1a−/− TCR75 CD4 T cells that were genetically incapable of providing TFH cell function. The GC response was associated with affinity maturation of the anti-Kd alloantibody response, and its contribution to progression of allograft vasculopathy related principally to secretion of alloantibody, rather than to enhanced alloreactive T cell priming, because grafts survived long-term when B cells could present alloantigen, but not secrete alloantibody. Similarly, sera sampled at late time points from chronically-rejecting recipients induced more vigorous donor endothelial responses in vitro than sera sampled earlier after transplantation. In summary, our results suggest that chronic AMR and progression of allograft vasculopathy is dependent upon allospecific GC activity, with critical help provided by TFH cells. Clinical strategies that target the TFH cell subset may hold therapeutic potential.This work is composed of two parts, of which this is Part II. Please read also Part I: Alsughayyir et al., 2019

Characterization of GAR22 function in the regulation of red blood cell differentiation and cell migration

Thyroid hormone receptors (TRs) are ligand-dependent transcription factors that have a major impact on erythroid cell development. In this study, I have investigated the influence of TR activity on red blood cell gene expression and identified TR target genes by employing a genome-wide approach with DNA microarray. Ligand-activated TR potently accelerated differentiation of SCF/Epo-dependent progenitors in vitro, concomitantly with inducing growth arrest. By comparing the gene expression profile of untreated and T3 treated SCF/Epo progenitor cells, I demonstrated that T3 potently regulated a subset of genes involved in erythroid differentiation, such as GATA-2, c-kit and Band 3. Furthermore, T3 regulated genes previously not implicated in differentiation of erythroid cells, including BTEB1 (basic transcription binding protein 1/ Krüppel-like factor 9) and the novel TR target gene GAR22 (growth arrest specific gene 2 on chromosome 22). The upregulation of GAR22 in terminally differentiated cells suggests that GAR22 might be involved in regulation of growth control and cell cycle. Accordingly, ectopic expression of GAR22 in SCF/Epo progenitor cells lengthened the cell cycle, but did not affect red cell gene expression. This finding is consistent with the proposed role of GAR22 as a tumor suppressor. GAR22 binds to microtubules and microfilaments, and this might contribute to the intense cytoskeletal remodeling that takes place during red blood cell differentiation. To investigate GAR22 binding to microfilaments and microtubules, I generated a panel of green fluorescent protein (GFP)-tagged GAR22 fusion proteins. For studying GAR22 dynamics, B16F1 mouse melanoma cells and NIH3T3 fibroblasts were chosen, since they have a flat morphology and are fairly motile, which makes these cells particularly well suitable for such study. GAR22 genes encodes for two splice variants, GAR22alpha and GAR22beta. Both GAR22 variants perfectly co-localized with actin in B16F1 and NIH3T3 cells. It was found that the dynamics of GAR22 proteins resembled that of actin suggesting that GAR22 is involved in the regulation of actin cytoskeleton remodeling and cell migration. Consistent with this hypothesis I found that overexpression of GAR22beta greatly impaired directional cell migration. To further characterize GAR22 function, I searched for GAR22 interacting proteins employing proteomics and mass spectrometry, and identified EB1 (end binding protein 1). GAR22beta interacts with EB1 through its carboxy-terminal domain. EB1 is a microtubule plus end binding protein that regulates stability and dynamics of microtubules. Thus, the GAR22-EB1 interaction might be involved in the regulation of microtubule dynamics and/ or function. This possibility is supported by the observation that overexpression of GAR22beta displaced EB1 from the plus tips of microtubules, causing the collapse of microtubule cytoskeleton. Moreover, overexpression of GAR22beta also impaired the reassembly of microtubules in nocoazole treated cells. Finally, the deletion of either the actin binding domain or the microtubule-binding domain within GAR22beta reduced the inhibitory phenotype of GAR22beta on cell migration. These findings uncovered a novel function of GAR22beta in regulating directional cell migration via the control of EB1 localization and microtubule dynamics. In this context through its dual binding to microfilaments and microtubules GAR22beta is proposed to support the functional interplay between actin and microtubule cytoskeletons. Thus, this GAR22beta activity is of fundamental importance for cell motility, and extends beyond its function in red cell differentiation

Characterization of GAR22 function in the regulation of red blood cell differentiation and cell migration

Thyroid hormone receptors (TRs) are ligand-dependent transcription factors that have a major impact on erythroid cell development. In this study, I have investigated the influence of TR activity on red blood cell gene expression and identified TR target genes by employing a genome-wide approach with DNA microarray. Ligand-activated TR potently accelerated differentiation of SCF/Epo-dependent progenitors in vitro, concomitantly with inducing growth arrest. By comparing the gene expression profile of untreated and T3 treated SCF/Epo progenitor cells, I demonstrated that T3 potently regulated a subset of genes involved in erythroid differentiation, such as GATA-2, c-kit and Band 3. Furthermore, T3 regulated genes previously not implicated in differentiation of erythroid cells, including BTEB1 (basic transcription binding protein 1/ Krüppel-like factor 9) and the novel TR target gene GAR22 (growth arrest specific gene 2 on chromosome 22). The upregulation of GAR22 in terminally differentiated cells suggests that GAR22 might be involved in regulation of growth control and cell cycle. Accordingly, ectopic expression of GAR22 in SCF/Epo progenitor cells lengthened the cell cycle, but did not affect red cell gene expression. This finding is consistent with the proposed role of GAR22 as a tumor suppressor. GAR22 binds to microtubules and microfilaments, and this might contribute to the intense cytoskeletal remodeling that takes place during red blood cell differentiation. To investigate GAR22 binding to microfilaments and microtubules, I generated a panel of green fluorescent protein (GFP)-tagged GAR22 fusion proteins. For studying GAR22 dynamics, B16F1 mouse melanoma cells and NIH3T3 fibroblasts were chosen, since they have a flat morphology and are fairly motile, which makes these cells particularly well suitable for such study. GAR22 genes encodes for two splice variants, GAR22alpha and GAR22beta. Both GAR22 variants perfectly co-localized with actin in B16F1 and NIH3T3 cells. It was found that the dynamics of GAR22 proteins resembled that of actin suggesting that GAR22 is involved in the regulation of actin cytoskeleton remodeling and cell migration. Consistent with this hypothesis I found that overexpression of GAR22beta greatly impaired directional cell migration. To further characterize GAR22 function, I searched for GAR22 interacting proteins employing proteomics and mass spectrometry, and identified EB1 (end binding protein 1). GAR22beta interacts with EB1 through its carboxy-terminal domain. EB1 is a microtubule plus end binding protein that regulates stability and dynamics of microtubules. Thus, the GAR22-EB1 interaction might be involved in the regulation of microtubule dynamics and/ or function. This possibility is supported by the observation that overexpression of GAR22beta displaced EB1 from the plus tips of microtubules, causing the collapse of microtubule cytoskeleton. Moreover, overexpression of GAR22beta also impaired the reassembly of microtubules in nocoazole treated cells. Finally, the deletion of either the actin binding domain or the microtubule-binding domain within GAR22beta reduced the inhibitory phenotype of GAR22beta on cell migration. These findings uncovered a novel function of GAR22beta in regulating directional cell migration via the control of EB1 localization and microtubule dynamics. In this context through its dual binding to microfilaments and microtubules GAR22beta is proposed to support the functional interplay between actin and microtubule cytoskeletons. Thus, this GAR22beta activity is of fundamental importance for cell motility, and extends beyond its function in red cell differentiation