Dulce Soler

Unique subpopulations of CD56+ NK and NK-T peripheral blood lymphocytes identified by chemokine receptor expression repertoire.

CD56, an adhesion molecule closely related to neual cell adhesion molecule, is an immunophenotypic marker for several unique populations of PBLs. Although CD56+ cells derive from multiple lymphocyte lineages, they share a role in immunosurveillance and antitumor responses. We have studied the chemokine receptor expression patterns and functional migratory responses of three distinct CD56+ populations from human peripheral blood. NK-T cells were found to differ greatly from NK cells, and CD16+ NK cells from CD16− NK cells. CD16+ NK cells were the predominant population responding to IL-8 and fractalkine, whereas NK-T cells were the predominant population responding to the CCR5 ligand macrophage-inflammatory protein-1β. CD16− NK cells were the only CD56+ population that uniformly expressed trafficking molecules necessary for homing into secondary lymphoid organs through high endothelial venule. These findings describe a diverse population of cells that may have trafficking patterns entirely different from each other, and from other lymphocyte types.

CCR7 Expression and Memory T Cell Diversity in Humans

CCR7, along with L-selectin and LFA-1, mediates homing of T cells to secondary lymphoid organs via high endothelial venules (HEV). CCR7 has also been implicated in microenvironmental positioning of lymphocytes within secondary lymphoid organs and in return of lymphocytes and dendritic cells to the lymph after passage through nonlymphoid tissues. We have generated mAbs to human CCR7, whose specificities correlate with functional migration of lymphocyte subsets to known CCR7 ligands. We find that CCR7 is expressed on the vast majority of peripheral blood T cells, including most cells that express adhesion molecules (cutaneous lymphocyte Ag α4β7 integrin) required for homing to nonlymphoid tissues. A subset of CD27(neg) memory CD4 T cells from human peripheral blood is greatly enriched in the CCR7(neg) population, as well as L-selectin(neg) cells, suggesting that these cells are incapable of homing to secondary lymphoid organs. Accordingly, CD27(neg) T cells are rare within tonsil, a representative secondary lymphoid organ. All resting T cells within secondary lymphoid organs express high levels of CCR7, but many activated cells lack CCR7. CCR7 loss in activated CD4 cells accompanies CXC chemokine receptor (CXCR)5 gain, suggesting that the reciprocal expression of these two receptors may contribute to differential positioning of resting vs activated cells within the organ. Lymphocytes isolated from nonlymphoid tissues (such as skin, lung, or intestine) contain many CD27(neg) cells lacking CCR7. The ratio of CD27(neg)/CCR7(neg) cells to CD27(pos)/CCR7(pos) cells varies from tissue to tissue, and may correlate with the number of cells actively engaged in Ag recognition within a given tissue.

CCR10 expression is a common feature of circulating and mucosal epithelial tissue IgA Ab-secreting cells

The dissemination of IgA-dependent immunity between mucosal sites has important implications for mucosal immunoprotection and vaccine development. Epithelial cells in diverse gastrointestinal and nonintestinal mucosal tissues express the chemokine MEC/CCL28. Here we demonstrate that CCR10, a receptor for MEC, is selectively expressed by IgA Ab-secreting cells (large s/cIgA(+)CD38(hi)CD19(int/-)CD20(-)), including circulating IgA(+) plasmablasts and almost all IgA(+) plasma cells in the salivary gland, small intestine, large intestine, appendix, and tonsils. Few T cells in any mucosal tissue examined express CCR10. Moreover, tonsil IgA plasmablasts migrate to MEC, consistent with the selectivity of CCR10 expression. In contrast, CCR9, whose ligand TECK/CCL25 is predominantly restricted to the small intestine and thymus, is expressed by a fraction of IgA Ab-secreting cells and almost all T cells in the small intestine, but by only a small percentage of plasma cells and plasmablasts in other sites. These results point to a unifying role for CCR10 and its mucosal epithelial ligand MEC in the migration of circulating IgA plasmablasts and, together with other tissue-specific homing mechanisms, provides a mechanistic basis for the specific dissemination of IgA Ab-secreting cells after local immunization.

A novel chemokine ligand for CCR10 and CCR3 expressed by epithelial cells in mucosal tissues.

Mucosae-associated epithelial chemokine (MEC) is a novel chemokine whose mRNA is most abundant in salivary gland, with strong expression in other mucosal sites, including colon, trachea, and mammary gland. MEC is constitutively expressed by epithelial cells; MEC mRNA is detected in cultured bronchial and mammary gland epithelial cell lines and in epithelia isolated from salivary gland and colon using laser capture microdissection, but not in the endothelial, hemolymphoid, or fibroblastic cell lines tested. Although MEC is poorly expressed in skin, its closest homologue is the keratinocyte-expressed cutaneous T cell-attracting chemokine (CTACK; CCL27), and MEC supports chemotaxis of transfected lymphoid cells expressing CCR10, a known CTACK receptor. In contrast to CTACK, however, MEC also supports migration through CCR3. Consistent with this, MEC attracts eosinophils in addition to memory lymphocyte subsets. These results suggest an important role for MEC in the physiology of extracutaneous epithelial tissues, including diverse mucosal organs.

Expression Cloning of the STRL33/BONZO/TYMSTR Ligand Reveals Elements of CC, CXC, and CX3C Chemokines

STRL33/BONZO/TYMSTR is an orphan chemokine and HIV/SIV coreceptor receptor that is expressed on activated T lymphocytes. We describe an expression cloning strategy whereby we isolated a novel chemokine, which we name CXCL16. CXCL16 is an α (CXC) chemokine but also has characteristics of CC chemokines and a structure similar to fractalkine (neurotactin) in having a transmembrane region and a chemokine domain suspended by a mucin-like stalk. A recombinant version of CXCL16 fails to mediate chemotaxis to all known chemokine receptor transfectants tested but does mediate robust chemotaxis, high affinity binding, and calcium mobilization to Bonzo receptor transfectants, indicating that this is a unique receptor ligand interaction. In vitro polarized T cell subsets including Th1, Th2, and Tr1 cells express functional Bonzo, suggesting expression of this receptor in chronic inflammation, which we further verified by demonstration of CXCL16-mediated migration of tonsil-derived CD4+ T lymphocytes. CXCL16 is expressed on the surface of APCs including subsets of CD19+ B cells and CD14+ monocyte/macrophages, and functional CXCL16 is also shed from macrophages. The combination of unique structural features of both Bonzo and CXCL16 suggest that this interaction may represent a new class of ligands for this receptor family. Additionally, this chemokine might play a unique dual role of attracting activated lymphocyte subsets during inflammation as well as facilitating immune responses via cell-cell contact.

The Binding Specificity and Selective Antagonism of Vedolizumab, an Anti-α4β7 Integrin Therapeutic Antibody in Development for Inflammatory Bowel Diseases

Vedolizumab is a humanized monoclonal antibody that targets the α4β7 integrin exclusively, and modulates inflammation in the gastrointestinal tract without inducing the systemic immunosuppression that characterizes anti-α4 chain monoclonal antibodies, such as natalizumab. This unique pharmacologic profile is largely attributable to four determinants. The first determinant is the restriction of the expression of the α4β7 integrin to subsets of leukocytes. Vedolizumab does not bind to the majority of memory CD4+ T lymphocytes (60%), neutrophils, and most monocytes. The highest level of vedolizumab binding is to a subset (∼25%) of human peripheral blood memory CD4+ T lymphocytes that include gut-homing interleukin 17 T-helper lymphocytes. Vedolizumab also binds to eosinophils at high levels, and to naive T-helper lymphocytes, naive and memory cytotoxic T lymphocytes, B lymphocytes, natural killer cells, and basophils at lower levels; vedolizumab binds to memory CD4+ T and B lymphocytes with subnanomolar potency (EC50 = 0.3–0.4 nM). The second determinant is binding specificity; vedolizumab binds exclusively to the α4β7 integrin, and not to the α4β1 and αEβ7 integrins. The third determinant is selective antagonism; vedolizumab selectively inhibits adhesion of α4β7-expressing cells to mucosal addressin cell adhesion molecule 1 (median inhibition concentration [IC50] = 0.02–0.06 μg/ml) and fibronectin (IC50 = 0.02 μg/ml), but not vascular cell adhesion molecule 1. The fourth determinant is the gastrointestinal-specific tropism of the α4β7 integrin function. These pharmacologic properties of vedolizumab, in conjunction with the gastrointestinal tropism of α4β7 integrin function, may ultimately confer an improved risk-to-benefit profile for patients with inflammatory bowel diseases.

Mast cell–expressed orphan receptor CCRL2 binds chemerin and is required for optimal induction of IgE-mediated passive cutaneous anaphylaxis

Mast cells contribute importantly to both protective and pathological IgE-dependent immune responses. We show that the mast cell–expressed orphan serpentine receptor mCCRL2 is not required for expression of IgE-mediated mast cell–dependent passive cutaneous anaphylaxis but can enhance the tissue swelling and leukocyte infiltrates associated with such reactions in mice. We further identify chemerin as a natural nonsignaling protein ligand for both human and mouse CCRL2. In contrast to other “silent” or professional chemokine interreceptors, chemerin binding does not trigger ligand internalization. Rather, CCRL2 is able to bind the chemoattractant and increase local concentrations of bioactive chemerin, thus providing a link between CCRL2 expression and inflammation via the cell-signaling chemerin receptor CMKLR1.

Beneficial effects of targeting CCR5 in allograft recipients.

BACKGROUND The chemokine receptor, CCR5, and its three high-affinity ligands, macrophage inflammatory protein- (MIP) 1alpha, MIP-1beta, and regulated on activation normal T cell expressed and secreted (RANTES), are expressed by infiltrating mononuclear cells during the rejection of clinical and experimental organ allografts, although the significance of these molecules in the pathogenesis of rejection has not been established. METHODS We studied intragraft events in four allograft models. First, we studied cardiac transplants in fully MHC-mismatched mice that were deficient in CCR5 or two of its ligands, MIP-1alpha or RANTES. Second we tested the effects of a neutralizing rat anti-mCCR5 monoclonal antibody on allograft survival. Third we assessed whether a subtherapeutic course of cyclosporine would potentiate enhance survival in CCR5-deficient recipients. Finally, we tested the effect of targeting CCR5 in a class II-mismatched model. RESULTS Whereas mice deficient in expression of MIP-1alpha or RANTES reject fully MHC-mismatched cardiac allografts normally, CCR5-/- mice, or CCR5+/+ mice treated with a neutralizing mAb to mCCR5, show enhanced allograft survival. MHC class II-disparate mismatched are permanently accepted in CCR5-/- but not CCR5+/+ recipients. Finally, the beneficial effects of targeting of CCR5 are markedly synergistic with the effects of cyclosporine, resulting in permanent engraftment without development of chronic rejection. CONCLUSIONS We conclude that CCR5 plays a key role in the mechanisms of host T cell and macrophage recruitment and allograft rejection, such that targeting of CCR5 clinically may be of therapeutic significance.

Differential chemokine responses and homing patterns of murine TCR alpha beta NKT cell subsets.

NKT cells play important roles in the regulation of diverse immune responses. Therefore, chemokine receptor expression and chemotactic responses of murine TCRαβ NKT cells were examined to define their homing potential. Most NKT cells stained for the chemokine receptor CXCR3, while >90% of Vα14i-positive and ∼50% of Vα14i-negative NKT cells expressed CXCR6 via an enhanced green fluorescent protein reporter construct. CXCR4 expression was higher on Vα14i-negative than Vα14i-positive NKT cells. In spleen only, subsets of Vα14i-positive and -negative NKT cells also expressed CXCR5. NKT cell subsets migrated in response to ligands for the inflammatory chemokine receptors CXCR3 (monokine induced by IFN-γ/CXC ligand (CXCL)9) and CXCR6 (CXCL16), and regulatory chemokine receptors CCR7 (secondary lymphoid-tissue chemokine (SLC)/CC ligand (CCL)21), CXCR4 (stromal cell-derived factor-1/CXCL12), and CXCR5 (B cell-attracting chemokine-1/CXCL13); but not to ligands for other chemokine receptors. Two NKT cell subsets migrated in response to the lymphoid homing chemokine SLC/CCL21: CD4− Vα14i-negative NKT cells that were L-selectinhigh and enriched for expression of Ly49G2 (consistent with the phenotype of most NKT cells found in peripheral lymph nodes); and immature Vα14i-positive cells lacking NK1.1 and L-selectin. Mature NK1.1+ Vα14i-positive NKT cells did not migrate to SLC/CCL21. BCA-1/CXCL13, which mediates homing to B cell zones, elicited migration of Vα14i-positive and -negative NKT cells in the spleen. These cells were primarily CD4+ or CD4−CD8− and were enriched for Ly49C/I, but not Ly49G2. Low levels of chemotaxis to CXCL16 were only detected in Vα14i-positive NKT cell subsets. Our results identify subsets of NKT cells with distinct homing and localization patterns, suggesting that these populations play specialized roles in immunological processes in vivo.

Lack of chemokine receptor CCR1 enhances Th1 responses and glomerular injury during nephrotoxic nephritis

During the development of nephrotoxic nephritis (NTN) in the mouse, we find that a variety of chemokines and chemokine receptors are induced: CCR1 (RANTES, MIP-1alpha), CCR2 (MCP-1), CCR5 (RANTES, MIP-1alpha, MIP-1beta), CXCR2 (MIP-2), and CXCR3 (IP-10). Their timing of expression indicated that CXCR2 and CCR1 are probably important in the neutrophil-dependent heterologous phase of the disease, whereas CCR1, CCR2, CCR5, and CXCR3 accompany the subsequent mononuclear cell infiltration characteristic of autologous disease. We therefore assessed the role of CCR1 in NTN using CCR1(-/-) mice. We found that neutrophil accumulation in CCR1(-/-) mice was comparable to that in wild-type animals but that renal recruitment of CD4(+) and CD8(+) T cells and macrophages increased significantly. Moreover, CCR1(-/-) mice developed more severe glomerulonephritis than did controls, with greater proteinuria and blood urea nitrogen, as well as a higher frequency of crescent formation. In addition, CCR1(-/-) mice showed enhanced Th1 immune responses, including titers of antigen-specific IgG2a antibody, delayed-type hypersensitivity responses, and production of IFN-gamma and TNF-alpha. Lastly, using recombinant proteins and transfected cells that overexpressed CCR1, we demonstrated that MIP-1alpha, but not RANTES, bound CCR1 and induced cell chemotaxis. Thus, rather than simply promoting leukocyte recruitment during NTN, CCR1 expression profoundly alters the effector phase of glomerulonephritis. Therapeutic targeting of chemokine receptors may, on occasion, exacerbate underlying disease.