InVivoMAb anti-mouse CXCL10 (IP-10)

The generation of an adaptive immune response against intracellular pathogens requires the recruitment of effector T cells to sites of infection. Here we show that the chemokine IP-10, a specific chemoattractant for activated T cells, controls this process in mice naturally infected with Toxoplasma gondii. Neutralization of IP-10 in infected mice inhibited the massive influx of T cells into tissues and impaired antigen-specific T cell effector functions. This resulted in >1000-fold increase in tissue parasite burden and a marked increase in mortality compared to control antibody-treated mice. These observations suggest that IP-10 may play a broader role in the localization and function of effector T cells at sites of Th1 inflammation.

The successful induction of T cell-mediated protective immunity against poorly immunogenic malignancies remains a major challenge for cancer immunotherapy. Here, we demonstrate that the induction of tumor-protective immunity by IL-12 in a murine neuroblastoma model depends entirely on the CXC chemokine IFN-gamma-inducible protein 10 (IP-10). This was established by in vivo depletion of IP-10 with mAbs in mice vaccinated against NXS2 neuroblastoma by gene therapy with a linearized, single-chain (sc) version of the heterodimeric cytokine IL-12 (scIL-12). The efficacy of IP-10 depletion was indicated by the effective abrogation of scIL-12-mediated antiangiogenesis and T cell chemotaxis in mice receiving s.c. injections of scIL-12-producing NXS2 cells. These findings were extended by data demonstrating that IP-10 is directly involved in the generation of a tumor-protective CD8+ T cell-mediated immune response during the early immunization phase. Four lines of evidence support this contention: First, A/J mice vaccinated with NXS2 scIL-12 and depleted of IP-10 by two different anti-IP-10 mAbs revealed an abrogation of systemic-protective immunity against disseminated metastases. Second, CD8+ T cell-mediated MHC class I Ag-restricted tumor cell lysis was inhibited in such mice. Third, intracellular IFN-gamma expressed by proliferating CD8+ T cells was substantially inhibited in IP-10-depleted, scIL-12 NXS2-vaccinated mice. Fourth, systemic tumor protective immunity was completely abrogated in mice depleted of IP-10 in the early immunization phase, but not if IP-10 was depleted only in the effector phase. These findings suggest that IP-10 plays a crucial role during the early immunization phase in the induction of immunity against neuroblastoma by scIL-12 gene therapy.

The ability of chemokines to induce the migration of cells expressing their cognate G-protein-coupled receptor is a characteristic property of chemokine function. To study this important function, in vitro chemotaxis assays are most often used, which, although useful, lack many components of the complex in vivo trafficking process. Reliable in vivo recruitment assays have been very difficult to establish. We describe a robust in vivo T-cell recruitment assay for adoptively transferred T lymphocytes in mice. Instillation of the CXCR3 chemokine ligands IP-10/CXCL10 or I-TAC/CXCL11 into the airways results in robust recruitment of transferred T lymphocytes. The assay thereby models the natural environment of chemokine function, as chemokines are expressed in the airways during inflammation, inducing selective leukocyte homing. This assay is particularly useful for the analysis of chemokine and chemokine receptor mutants in structure function studies and for testing the in vivo efficacy of inhibitory chemokine and chemokine receptor antibodies and small molecule antagonists.

Trafficking of leukocytes to sites of inflammation is an important step in the establishment of an immune response. Chemokines are critical regulators of leukocyte trafficking and are widely studied molecules for their important role in disease and for their potential as new therapeutic targets. The ability of chemokines to induce leukocyte recruitment has been mainly measured by in vitro chemotaxis assays, which lack many components of the complex biological process of leukocyte migration and therefore provide incomplete information about chemokine function in vivo. In vivo assays to study the activity of chemokines to induce leukocyte recruitment have been difficult to establish. We describe here the development of a robust in vivo recruitment assay for CD8(+) and CD4(+) T lymphocytes induced by the CXCR3 ligands IP-10 (CXCL10) and I-TAC (CXCL11). For this assay, in vitro activated T lymphocytes were adoptively transferred into the peritoneum of naïve mice. Homing of these transferred T lymphocytes into the airways was measured following intratracheal instillation of chemokines. High recruitment indices were achieved that were dependent on chemokine concentration and CXCR3 expression on the transferred lymphocytes. Recruitment was also inhibited by antibodies to the chemokine. The assay models the natural condition of chemokine-mediated lymphocyte migration into the airways as chemokines are expressed in the airways during inflammation. The nature of this model allows flexibility to study wildtype and mutant chemokines and chemokine receptors and the ability to evaluate chemokine antagonists and antibodies in vivo. This assay will therefore help elucidate a deeper understanding of the chemokine system in vivo.