Homing characteristics of donor T cells after experimental allogeneic bone marrow transplantation and posttransplantation therapy for multiple myeloma.

Relapse and graft-versus-host disease remain major problems associated with allogeneic bone marrow (BM) transplantation (allo-BMT) and posttransplantation therapy in patients with multiple myeloma (MM) and other hematologic malignancies. A possible strategy for selectively enhancing the graft-versus-myeloma response and possibly reducing graft-versus-host disease is to increase the migration of alloreactive T cells toward the MM-containing BM. In the present study, we characterized the BM-homing behavior of donor-derived effector T cells in a novel allo-BMT model for the treatment of MM. We observed that posttransplantation immunotherapy consisting of donor lymphocyte infusion (DLI) and vaccination with minor histocompatibility antigen-loaded dendritic cells (DCs) was associated with prolonged survival compared with allo-BMT with no further treatment. Moreover, CD8(+) effector T cells expressing inflammatory homing receptors, including high levels of CD44, LFA-1, and inflammatory chemokine receptors, were recruited to MM-bearing BM. This was paralleled by strongly increased expression of IFN-γ and IFN-γ-inducible chemokines, including CXCL9, CXCL10, and CXCL16, especially in mice treated with DLI plus minor histocompatibility antigen-loaded DC vaccination. Remarkably, expression of the homeostatic chemokine CXCL12 was reduced. Furthermore, IFN-γ and TNF-α induced BM endothelial cells to express high levels of the inflammatory chemokines and reduced or unaltered levels of CXCL12. Finally, presentation of CXCL9 by multiple BM endothelial cell-expressed heparan sulfate proteoglycans triggered transendothelial migration of effector T cells. Taken together, our data demonstrate that both post-transplantation DLI plus miHA-loaded DC vaccination and MM growth result in an increased expression of inflammatory homing receptors on donor T cells, decreased levels of the homeostatic BM-homing chemokine CXCL12, and strong induction of inflammatory chemokines in the BM. Thus, along with increasing the population of alloreactive T cells, post-transplantation immunotherapy also might contribute to a more effective graft-versus-tumor response by switching homeostatic T cell migration to inflammation-driven migration.

The tetraspanin CD37 orchestrates the α(4)ß(1) integrin-Akt signaling axis and supports long-lived plasma cell survival.

Signaling by the serine and threonine kinase Akt (also known as protein kinase B), a pathway that is common to all eukaryotic cells, is central to cell survival, proliferation, and gene induction. We sought to elucidate the mechanisms underlying regulation of the kinase activity of Akt in the immune system. We found that the four-transmembrane protein CD37 was essential for B cell survival and long-lived protective immunity. CD37-deficient (Cd37(-/-)) mice had reduced numbers of immunoglobulin G (IgG)-secreting plasma cells in lymphoid organs compared to those in wild-type mice, which we attributed to increased apoptosis of plasma cells in the germinal centers of the spleen, areas in which B cells proliferate and are selected. CD37 was required for the survival of IgG-secreting plasma cells in response to binding of vascular cell adhesion molecule 1 to the α(4)ß(1) integrin. Impaired α(4)ß(1) integrin-dependent Akt signaling in Cd37(-/-) IgG-secreting plasma cells was the underlying cause responsible for impaired cell survival. CD37 was required for the mobility and clustering of α(4)ß(1) integrins in the plasma membrane, thus regulating the membrane distribution of α(4)ß(1) integrin necessary for activation of the Akt survival pathway in the immune system.

The lymphoid chemokine CCL21 triggers LFA-1 adhesive properties on human dendritic cells.

Dendritic cells (DCs) are the most potent APCs, involved in the induction of immunity and tolerance. Recently we showed that during differentiation of human DCs from monocyte precursors, Lymphocyte function-associated antigen-1 (LFA-1)-binding capacity is lost, although integrin expression levels were maintained constant, suggesting a different regulation mechanism of this integrin on different cell types. However, the exact role of LFA-1 in DC adhesion and migration remains obscure. Chemokines are potent regulators of integrin function, influencing migratory and adhesive properties of leukocytes. Here, we show that upon vaccination of cancer patients with human DCs, cells that have migrated in vivo into the lymph nodes upregulated the active form of LFA-1. We further show that exposure of human DCs to the lymphoid chemokine CCL21 specifically restores the high-affinity form of LFA-1 and induces binding to its ligand ICAM-1 under low shear stress. Our data indicate that on DCs LFA-1 may function as an inducible anchor during lymphatic transmigration or within the lymph nodes. A thorough understanding of the adhesive events during the DC life cycle will help to improve the outcome of DC-based antitumor clinical trials.

Chemokine induction by all-trans retinoic acid and arsenic trioxide in acute promyelocytic leukemia: triggering the differentiation syndrome.

In acute promyelocytic leukemia (APL), differentiation therapy with all-trans retinoic acid (ATRA) and/or arsenic trioxide can induce a differentiation syndrome (DS) with massive pulmonary infiltration of differentiating leukemic cells. Because chemokines are implicated in migration and extravasation of leukemic cells, chemokines might play a role in DS. ATRA stimulation of the APL cell line NB4 induced expression of multiple CC-chemokines (CCLs) and their receptors (> 19-fold), resulting in increased chemokine levels and chemotaxis. Induction of CCL2 and CCL24 was directly mediated by ligand-activated retinoic acid receptors. In primary leukemia cells derived from APL patients at diagnosis, ATRA induced chemokine production as well. Furthermore, in plasma of an APL patient with DS, we observed chemokine induction, suggesting that chemokines might be important in DS. Dexamethasone, which efficiently reduces pulmonary chemokine production, did not inhibit chemokine induction in APL cells. Finally, chemokine production was also induced by arsenic trioxide as single agent or in combination with ATRA. We propose that differentiation therapy may induce chemokine production in the lung and in APL cells, which both trigger migration of leukemic cells. Because dexamethasone does not efficiently reduce leukemic chemokine production, pulmonary infiltration of leukemic cells may induce an uncontrollable hyperinflammatory reaction in the lung.

Modulation of cell motility by spatial repositioning of enzymatic ATP/ADP exchange capacity.

ATP is the "principal energy currency" in metabolism and the most versatile small molecular regulator of cellular activities. Although already much is known about the role of ATP in fundamental processes of living systems, data about its compartmentalization are rather scarce, and we still have only very limited understanding of whether patterns in the distribution of intracellular ATP concentration ("ATP inhomogeneity") do exist and have a regulatory role. Here we report on the analysis of coupling of local ATP supply to regulation of actomyosin behavior, a widespread and dynamic process with conspicuous high ATP dependence, which is central to cell shape changes and cell motility. As an experimental model, we use embryonic fibroblasts from knock-out mice without major ATP-ADP exchange enzymes, in which we (re)introduce the ATP/ADP exchange enzyme adenylate kinase-1 (AK1) and deliberately manipulate its spatial positioning by coupling to different artificial location tags. By transfection-complementation of AK1 variants and comparison with yellow fluorescent protein controls, we found that motility and spreading were enhanced in cells with AK1 with a focal contact guidance tag. Intermediary enhancement was observed in cells with membrane-targeted or cytosolic AK1. Use of a heterodimer-inducing approach for transient translocation of AK1 to focal contacts under conditions of constant global AK1 activity in the cell corroborated these results. Based on our findings with these model systems, we propose that local ATP supply in the cell periphery and "on site" fuelling of the actomyosin machinery, when maintained via enzymes involved in phosphoryl transfer, are codetermining factors in the control of cell motility.

Heparan sulfate on activated glomerular endothelial cells and exogenous heparinoids influence the rolling and adhesion of leucocytes.

BACKGROUND: Proliferative glomerulonephritides are characterized by the influx of leucocytes. Heparan sulfate (HS) plays an important role in the recruitment, rolling and firm adhesion of leucocytes to activated endothelium. Recently, we have shown the importance of HS on activated mouse glomerular endothelial cells (mGEnC-1) for the firm adhesion of leucocytes in a static adhesion assay. In the present study, we evaluated the role of HS on glomerular endothelial cells and the effect of adding heparinoids on the leucocyte-glomerular endothelium interaction under dynamic flow conditions. METHODS: The number of rolling and firmly adhering leucocytes, and the rolling velocity of leucocytes was determined on a monolayer of unactivated or TNF-alpha-activated mGEnC-1 under dynamic flow conditions using physiological relevant shear stress rates in a flow chamber system. Furthermore, the effects of removal of HS on TNF-alpha-activated mGEnC-1 by heparinase III treatment, and of different concentrations of heparin, tinzaparin and HS, on the rolling and adhesion of leucocytes were evaluated. RESULTS: At the calculated physiological shear stress rate of 0.8 dynes/cm2 the number of rolling and firmly adhering leucocytes to mGEnC-1 increased 2-fold after activation with TNF-alpha, whereas the rolling velocity of the leucocytes decreased 2-fold. Addition of heparin, tinzaparin or HS, and the removal of HS on mGEnC-1 reduced the number of leucocytes rolling and adhering to activated mGEnC-1 about 2-3-fold, while the rolling velocity increased more than 2-fold. CONCLUSIONS: HS on activated glomerular endothelial cells is important for the interaction with leucocytes under flow conditions, while exogenous heparinoids interfere with this interaction. These results suggest that supplementary treatment of proliferative glomerulonephritides with heparinoids is an interesting option to pursue.