Anti-XCL1 (aa 22-114) polyclonal antibody

Chemokines adopt a conserved tertiary structure stabilized by two disulfide bridges and direct the migration of leukocytes. Lymphotactin (Ltn) is a unique chemokine in that it contains only one disulfide and exhibits large-scale structural heterogeneity. Under physiological solution conditions (37 C and 150 mM NaCl), Ltn is in equilibrium between the canonical chemokine fold (Ltn10) and a distinct four-stranded, beta-sheet (Ltn40). Consequently, it has not been possible to address the biological significance of each structural species independently. To stabilize the Ltn10 structure in a manner independent of specific solution conditions, Ltn variants containing a second disulfide bridge were designed. Placement of the new cysteines was based on a sequence alignment of Ltn with either the first (Ltn-CC1) or third disulfide (Ltn-CC3) in the CC chemokine, HCC-2. NMR data demonstrate that both CC1 and CC3 retain the Ltn10 chemokine structure and no longer exhibit structural rearrangement. The ability of each mutant to activate the Ltn receptor, XCR1, has been tested using an intracellular Ca2+ flux assay. These data support the conclusion that the chemokine fold of Ltn10 is responsible for receptor activation. We also examined the role of amino- and carboxyl-terminal residues in Ltn-mediated receptor activation. In contrast to previous reports, we find that the 25 residues comprising the novel C-terminal extension do not participate in receptor activation, while the native N-terminus is absolutely required for Ltn function.

Targeting antigen to surface receptors on dendritic cells (DCs) can improve antibody response against subunit vaccines. We have previously observed that human XCL1-fusion vaccines target murine Xcr1(+) DCs without actively inducing endocytosis of the antigen, resulting in enhanced antibody responses in mice. However, the use of foreign chemokines for targeting is undesirable when translating this observation to human or veterinary medicine due to potential cross-reactive responses against the endogenous chemokine. Here we have identified a mutant version of murine Xcl1, labeled Xcl1(Delta 1) owing to removal of a conserved valine in position 1 of the mature chemokine, that retains specific binding to Xcr1(+) DCs without inducing endocytosis of the receptor. DNA immunization with Xcl1(Delta 1) conjugated to influenza hemagglutinin (HA) induced improved antibody responses, with higher end point titers of IgG compared to WT Xcl1-HA. The Xcl1(Delta 1) fusion vaccine also resulted in an increased number of HA reactive germinal center B cells with higher avidity toward the antigen, and serum transfer experiments show that Xcl1(Delta 1)-HA induced antibody responses provided better protection against influenza infection as compared to WT Xcl1-HA. In summary, our observations indicate that targeting antigen to Xcr1(+) DCs in an endocytosis deficient manner enhances antibody responses. This effect was obtained by introducing a single mutation to Xcl1, suggesting our strategy may easily be translated to human or veterinary vaccine settings.