No abstract

The chemokine stromal cell-derived factor-1 (SDF-1/ CXCL12) and its G-protein-coupled receptor (GPCR) CXCR4 play fundamental roles in many physiological processes, and CXCR4 is a drug target for various diseases such as cancer metastasis and human immunodeficiency virus, type 1, infection. However, almost no structural information about the SDF-1-CXCR4 interaction is available, mainly because of the difficulties in expression, purification, and crystallization of CXCR4. In this study, an extensive investigation of the preparation of CXCR4 and optimization of the experimental conditions enables NMR analyses of the interaction between the full-length CXCR4 and SDF-1. We demonstrated that the binding of an extended surface on the SDF-1 ␤-sheet, 50-s loop, and N-loop to the CXCR4 extracellular region and that of the SDF-1 N terminus to the CXCR4 transmembrane region, which is critical for G-protein signaling, take place independently by methyl-utilizing transferred cross-saturation experiments along with the usage of the CXCR4-selective antagonist AMD3100. Furthermore, based upon the data, we conclude that the highly dynamic SDF-1 N terminus in the 1st step bound state plays a crucial role in efficiently searching the deeply buried binding pocket in the CXCR4 transmembrane region by the "fly-casting" mechanism. This is the first structural analyses of the interaction between a full-length GPCR and its chemokine, and our methodology would be applicable to other GPCR-ligand systems, for which the structural studies are still challenging.Chemokines are a number of small (8 -10 kDa) secreted proteins that direct cell migration in immune systems by activating their receptors expressed on the cell surface (1, 2). The chemokine, stromal cell-derived factor-1 (SDF-1, 2 also known as CXCL12) (3, 4), and its receptor, CXCR4 (5-7), play many essential physiological roles, such as homeostatic regulation of leukocyte traffic, hematopoiesis, and embryonic development (8 -11). The interaction between SDF-1 and CXCR4 also controls cancer metastasis (12, 13), and CXCR4 is a co-receptor for T-tropic strains of human immunodeficiency virus, type 1 (5, 14).The most abundant splice variant of SDF-1 (SDF-1␣) is composed of 68 amino acids, and its NMR (15, 16) and crystal structures (17, 18) demonstrated that SDF-1␣ assumes a typical chemokine fold as follows: an unstructured N terminus (Lys 1 -Tyr 7 ) followed by a long flexible loop (N-loop), a three-stranded anti-parallel ␤-sheet, and an ␣-helix. The mutational analyses revealed that although the SDF-1␣ N terminus is critical for the CXCR4-mediated signaling (15), both the N terminus and the N-loop residues are implicated in the receptor binding (15,18,19). In addition, recent mutational analysis suggested that the residues on the SDF-1␣ ␤-sheet are also important for receptor binding (20).CXCR4, composed of 352 amino acids, belongs to the class A G-protein-coupled receptor (GPCR) family, with the seven transmembrane (TM) helices. Whereas GPCR activation is mediated by the conformation...

Acute graft-versus-host disease (a-GVHD) is initiated primarily by immunologically competent cytotoxic T cells (CTLs) that express anti-host specificities. However, the host lymphoid compartment in which these precursor CTLs are initially stimulated remains unclear. Here we show that gut Peyer's patches (PPs) are required to activate anti-host CTL responses in a well characterized murine acute graft-versus-host reaction (a-GVHR) model, involving transfer of parent lymphocytes into F1 hybrid recipients. The a-GVHR was prevented when recruitment of donor T cells into PP was interrupted either by disrupting the gene encoding chemokine receptor CCR5 or by blocking integrin alpha(4)beta(7)-MAdCAM-1 (mucosal vascular addressin) interactions. Mice deficient for PPs failed to develop a-GVHD in two models of disease induction. Thus, blockade of CTL generation in PPs might offer new strategies for circumventing a-GVHD.

Ligation of the chemokine receptor CCR2 on monocytes and macrophages with its ligand CCL2 results in activation of the cascade consisting of phosphatidylinositol-3-OH kinase (PI(3)K), the small G protein Rac and lamellipodium protrusion. We show here that a unique clathrin heavy-chain repeat homology protein, FROUNT, directly bound activated CCR2 and formed clusters at the cell front during chemotaxis. Overexpression of FROUNT amplified the chemokine-elicited PI(3)K-Rac-lamellipodium protrusion cascade and subsequent chemotaxis. Blocking FROUNT function by using a truncated mutant or antisense strategy substantially diminished signaling via CCR2. In a mouse peritonitis model, suppression of endogenous FROUNT markedly prevented macrophage infiltration. Thus, FROUNT links activated CCR2 to the PI(3)K-Rac-lamellipodium protrusion cascade and could be a therapeutic target in chronic inflammatory immune diseases associated with macrophage infiltration.

Depletion of CD4þ cells in tumor-bearing mice has strong antitumor effects. However, the mechanisms underlying these effects and the therapeutic benefits of CD4 þ cell depletion relative to other immunotherapies have not been fully evaluated. Here, we investigated the antitumor effects of an anti-CD4-depleting mAb as a monotherapy or in combination with immune checkpoint mAbs. In B16F10, Colon 26, or Lewis lung carcinoma subcutaneous tumor models, administration of the anti-CD4 mAb alone had strong antitumor effects that were superior to those elicited by CD25 þ Treg depletion or other immune checkpoint mAbs, and which were completely reversed by CD8 þ cell depletion. CD4 þ cell depletion led to the proliferation of tumor-specific CD8 þ T cells in the draining lymph node and increased infiltration of PD-1into the tumor, with a shift toward type I immunity within the tumor. Combination treatment with the anti-CD4 mAb and immune checkpoint mAbs, particularly anti-PD-1 or anti-PD-L1 mAbs, synergistically suppressed tumor growth and greatly prolonged survival. To our knowledge, this work represents the first report of robust synergy between anti-CD4 and anti-PD-1 or anti-PD-L1 mAb therapies.