System for delivery into XCR1 positive cell and uses thereof

Abstract

The present invention relates to a delivery system suitable for delivering a substance into a XCR1 positive professional antigen-presenting cell, one or more nucleic acids coding for the same, a vector comprising the nucleic acid(s), a medicament comprising the delivery system or the one or more nucleic acid(s) and an adjuvant comprising XCL1 or a functionally active fragment thereof.

Claims

1. A method for inducing, in a subject, a memory immune response against a substance, comprising administering to the subject a delivery system, or nucleic acids encoding said delivery system, suitable for delivering the substance into a chemokine (C motif) receptor 1 (XCR1) positive professional antigen-presenting cell, the delivery system comprising i) a chemokine (C motif) ligand 1 (XCL1); ii) the substance to be delivered, wherein the substance is a peptide of a viral, bacterial, or fungal pathogenic protein or a tumor antigen, the substance being bound to the XCL1; and iii) a danger signal adjuvant, wherein said administering of the delivery system to the subject induces a memory immune response to the substance in the subject.

2. The method of claim 1, wherein the method prevents or treats a tumor and/or an infection.

3. The method of claim 1, wherein the memory immune response is a Th1 response.

4. The method of claim 3, wherein the Th1 response is a Th1 cytotoxic response.

5. The method of claim 1, wherein the delivery system is composed of one (poly)peptide.

6. The method of claim 1, wherein the danger signal adjuvant is not bound to the other components of the delivery system.

Description

FIGURES

(1) FIG. 1 shows the observed number of XCR1 copies after quantitative PCR of polyA-mRNA of diverse murine splenic cell populations, normalized to the expression in 10 000 cells. Only CD11c.sup.+CD8.sup.+ DC express significant amounts of XCR1 mRNA.

(2) FIG. 2A-FIG. 2B show activation of XCR1-bearing DC by XCL1. CD8.sup.+CD11c.sup.+ (A) or CD8.sup.+CD11c.sup.+ (B) dendritic cells (DC) were immobilized on poly-L-lysine-coated glass coverslips and loaded with fura-2/AM (2 M). Cells were imaged in a monochromator-assisted digital video imaging system and challenged with 100 nM ATAC at 60 s. Data represent intracellular Ca.sub.2+ concentrations ([Ca.sup.2++].sub.i) in 27-33 single cells (thin lines) measured in 3 independent experiments. Thick lines: mean [Ca.sub.2+].sub.i signal averaged over all cells measured. XCL1 induces a [Ca.sub.2+].sub.i signal in CD8.sup.+CD11c.sup.+(A) but not in CD8.sup.+CD11c.sup.+(B) dendritic cells.

(3) FIG. 3A-FIG. 3B show the percentage of migrated splenic CD8.sup.+ DC and CD8.sup.DC in an in vitro transwell chemotaxis assay in the presence of 1-1000 ng/ml XCL1 and 500 ng/ml CCL21. Only CD8.sup.+ DC migrate in response to XCL1.

(4) FIG. 4A-FIG. 4B show the percentage of migrated lymph node CD8.sup.+ DC and CD8.sup. DC in an in vitro transwell chemotaxis assay in the presence of 100 ng/ml XCL1 and 500 ng/ml CCL21. Only CD8.sup.+ DC migrate in response to XCL1.

(5) FIG. 5A-FIG. 5C show the migration behaviour of splenic B cells, T cells and NK cells in an in vitro transwell chemotaxis assay in the presence of 1-1000 ng/ml XCL1 or 200 ng/ml CXCL12, 100 ng/ml CCL21 or 200 ng/ml CXCL9, respectively. None of the cell populations migrate in response to XCL1.

(6) FIG. 6 shows maps of the endogenous ATAC locus containing three exons (numbered black boxes, top), the targeting vector ATAC.sub.mut/pTV-0 (middle) and the expected structure of the targeted locus (bottom). Restriction sites: X, XbaI; Sc, SacI; E.sub.1, EcoRI. Selection markers: neo, neomycin resistance; tk, thymidin kinase from herpes simplex virus. The sizes of the expected XbaI restriction fragments of the endogenous and targeted ATAC locus are indicated (16 kb and 22.5 kb, respectively).

(7) FIG. 7A-FIG. 7I show the gating strategy for the analysis of splenic CD11c.sup.+CD8.sup.+ DC by flow cytometry. The stained cell surface markers are indicated on the axes. The CD11c.sup.+MHC-11.sup.+ cells represented around 4% of splenic nucleated cells, after dead cells (DAPI.sup.+, 7D) and CD19.sup.+ cells (7E) were gated out. These CD11c.sup.+MHC-11.sup.+ cells were further subdivided into CD11b.sup.+ and CD8.sup.+ (dendritic) cells (7G). The fluorescence signal (CFSE) is shown for CD11c.sup.+CD8.sup.+ (7H) and CD11c.sup.+CD11b.sup.+ (7I) (dendritic) cells.

(8) FIG. 8A-FIG. 8B show the percentage of splenic CSFE.sup.+DC after injection of CSFE-labeled cell lines. Data obtained with CD8.sup.+ DC are shown in A, data obtained with CD8.sup. DC are shown in B. XCL1 significantly improves cell (antigen) uptake into CD8.sup.+ DC.

(9) FIG. 9 shows the percentage of OT-I cells in spleens of recipient mice on day 3 after injection of PBS, DEC-205-OVA or DEC-205-OVA/-CD40. A higher percentage is seen in wild type mice (black circles) compared to ATAC-KO-mice (white circles).

(10) FIG. 10 shows the percentage of IFN--expressing OT-I cells isolated from spleens of recipient mice on day 3 and restimulated in vitro. A higher percentage of IFN--secreting OT-I cells is seen in wild type mice (black circles) compared to ATAC-KO-mice (white circles), indicating the adjuvant effect of XCL1 on the differentiation of T cells.

(11) FIG. 11 shows a Western Blot of immunoprecipitates (i.p.) of human XCR1 protein with mAb 6F8.

(12) lane 1: marker

(13) lane 2: i.p. with mAb 6F8 from transfectant 5 c-myc/hATACR/P3X

(14) lane 3: i.p. with mAb 6F8 from P3X wild-type line

(15) lane 4: i.p. with mAb 6F8 from transfectant 3 c-myc/hATACR/P3X

(16) lane 5: i.p. with mAb 6F8 from transfectant hATACR/300-19

(17) lane 6: i.p. with mAb 6F8 from 300-19 wild-type line

(18) FIG. 12 shows a Coomassie stained SDS-PAGE loaded with different preparations of recombinant murine XCL1.

(19) lane 1: marker

(20) lane 2: metal-affininity purified XCL1-SUMO fusion protein

(21) lane 3: XCL1-SUMO fusion protein after digestion with SUMO protease

(22) lane 4: purified XCL1

(23) FIG. 13 shows the OVA-specific cytotoxicity of OT-I T cells and ATAC-KO OT-I T cells after adoptive transfer into C57BL/6 or ATAC-KO mice, respectively. OVA/300-19 cells were used for immunization on day 1 after transfer, and the in vivo cytotoxicity assay was performed on day 6.

(24) FIG. 14 shows expression of XCR1 in splenic DC.

EXAMPLES

Example 1: Exclusive Detection of XCR1 mRNA in CD8.SUP.+ DC

(25) Spleens from C57BL/6 mice were digested in RPMI1640 containing 2% (v/v) FBS (low endotoxin; PAA, Pasching, Austria), 500 g/ml collagenase D, and 20 g/ml DNase I (both from Roche Diagnostics GmbH, Penzberg, Germany) for 25 min in a shaking water bath at 37 C. The suspension was adjusted to 10 mM EDTA and incubated for 5 additional minutes. Cells were passed through a 70-vim-mesh (BD Biosciences, San Jose, Calif., USA) and rinsed with MACS-PBS (PBS, 2 mM EDTA, 0.5% (w/v) BSA low endotoxin). After sedimentation with 380g at 4 C. the cells were suspended in MACS-PBS.

(26) For the magnetic isolation of B cells, T cells, NK cells, granulocytes or macrophages, the cells of digested spleen were depleted of DC (dendritic cells) by negative selection with anti-CD11c-microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany). B cells were purified by positive selection with anti-CD19-microbeads, total T cells with anti-CD90-microbeads, NK cells with anti-DX5-microbeads, granulocytes with anti-Ly6G-microbeads, macrophages with biotin-conjugated mAb F4/80 (ATCC, Manassas, Va., USA) and anti-biotin microbeads (Miltenyi Biotec, supra), all according to the manufacturer's instructions (Miltenyi Biotec, supra). For isolation of DC, cells of digested spleen were underlayed with 1.069 g/ml Nycodenz solution (Axis-Shield, Oslo, Norway) and centrifuged for 20 min with 800g at 4 C. Low density cells were harvested from the interphase and washed once with MACS-PBS. Total DC were purified by magnetic cell sorting with anti-CD11c-microbeads according to the manufacturer's instructions (Miltenyi Biotec, supra). Briefly, cells were preincubated for 5 min at 4 C. with MACS-PBS containing 200 g/ml anti-FcRII/III (mAb 2.4G2; ATCC, supra) and 500 g/ml purified rat IgG (Nordic, Tilburg, The Netherlands) to prevent unspecific binding. CD11c-microbeads were added for additional 15 min, and washed twice with MACS-PBS. Cells were loaded onto a LS column (Miltenyi Biotec, supra) fitted in a MidiMACS Seperator magnet (Miltenyi Biotec, supra) and washed 3-times; CD11c-positive cells were retained on the column and eluted after removing the column from the magnetic field by adding 5 ml of MACS-PBS. CD11c.sup.+ splenic cells were stained in FACS-PBS (PBS, 2.5% (v/v) FBS, 0.1% (w/v) NaN3) containing 200 g/ml anti-FcR11/III (mAb 2.4G2), 500 g/ml purified rat IgG (both as blocking reagents), with anti-CD8 (mAb 53-6.72; ATCC, supra), anti-CD11b (mAb 5C6; ATCC, supra), anti-CD11c (mAb N418; ATCC, supra), and anti-MHC class II (mAb M5/114.15.2; ATCC, supra) for 20 min at 4 C. After washing, the cells were sorted on an Aria Cell Sorter (BD Bioscience) into CD11c.sup.+CD8.sup. and CD11c.sup.+CD8.sup.+ DC subpopulations to a purity>95%.

(27) Total RNA was prepared using the High Pure RNA Isolation Kit (Roche Diagnostics GmbH, supra) according to the protocol. In brief, cells (10.sup.5-10.sup.7) were collected by centrifugation and suspended in 200 l PBS and mixed with 400 l Lysis/Binding buffer. The lysate was applied onto the filter tube and centrifuged for 15 s with 8000g. The filter was washed once with 500 l Wash Buffer I and incubated for 15 min with DNase I to remove remaining DNA. After washing with 500 l of Wash Buffer I and twice with Wash Buffer II, the RNA was eluted twice with 50 l Elution Buffer. RNA concentration and purity of the combined eluate was determined on the Agilent 2100 bioanalyzer (Agilent Technologies, Waldbronn, Germany) and by photometrical reading.

(28) Small scale mRNA from 10.sup.5-10.sup.7 cells was isolated with the MACS mRNA Isolation Kit (Miltenyi Biotec, supra). The cell sediment was lysed in 1 ml of Lysis/Binding Buffer and centrifuged with 13000g for 3 min. After the addition of 50 l Oligo-(dT)-microbeads, the lysate was loaded onto a MACS column fitted into a MACS separation magnet. The column was rinsed twice with 200 l of Lysis/Binding Buffer and 4-times with Wash Buffer. Traces of remaining DNA were removed by digestion with 5 U DNase I (Promega, Madison, Wis., USA) for 1 min. Washing steps were repeated to remove digested DNA and DNase. Preheated Elution Buffer (120 l, 70 C.) was used to elute the purified mRNA. Quality control was performed as described above.

(29) Total RNA or mRNA were reverse-transcribed into cDNA with the Reverse Transcription System according to the manufacturer's instructions (Promega, Madison, Wis., USA). In short, 0.1-1 g total RNA or 1-10 ng poly(A).sup.+ mRNA was denatured at 70 C. for 10 min and immediately chilled thereafter. Reverse-transcription was performed with Oligo(dT)15 primers and AMV reverse transcriptase for 15 min at RT, followed by an incubation at 42 C. Reaction was stopped by a 5 min heating step at 95 C. followed by incubation at 4 C. for 5 min. The cDNA was then analyzed by quantitative PCR for their content on XCR1 copies and 2-microglobulin was used as an internal standard. For amplification of murine XCR1, 400 nM forward primer (5-TGCCTGTGTTGATCTCAGCAC-3; SEQ ID NO: 11), 200 nM reverse primer (5-CGGTGGATGGTCATGATGG-3; SEQ ID NO: 12), and 150 nM hybridization probe (5-FAM-CATCAGCCTCTACAGCAGCATCTTCTTCCT-TAMRA-3) were used. Murine the 2-microglobulin was amplified using 300 nM forward primer (5-CGCTCGGTGACCCTAGTCTTT-3; SEQ ID NO: 13), 300 nM reverse primer (5-TTCAGTATGTTCGGCTTCCCA-3; SEQ ID NO: 14), and 150 nM hybridization probe (5-FAM-CGGCTTGTATGCTATCCAGAAAACCCCTCA-TAMRA-3). In order to generate a standard for mRNA/cDNA copy quantification, the specific XCR1 gene fragments was amplified and cloned into pZErO vector using the Zero Background cloning kit (Invitrogen, Groningen, The Netherlands). For qPCR, primers were mixed with 10 l ABsolute QPCR Mix including ROX (ABgene, Epsom, UK) and 1/10th of the cDNA in a 20 l PCR-reaction. PCR was performed and quantified on the ABI Prism 7000 or 7700 Sequence Detection Systems (Applied Biosystems, Foster City, Calif., USA) with initial enzyme activation for 15 min at 95 C. followed by 50 cycles (95 C., 15 s; 60 C., 1 min). For quantification, several dilutions of the cloned gene fragment ranging from 10.sup.0 to 10.sup.8 copies were run in parallel to generate a standard curve. The results are shown in the following table 1.

(30) TABLE-US-00004 TABLE 1 Quantification of number of mRNA copies Cell type number of mRNA copy/10000 cells splenocytes 912 T cells 15 B cells 16 NK cells 0 granulocytes 5 macrophages 41 CD11c.sup.+CD8.sup.+DC 925 CD11c.sup.+CD8.sup. DC 148717

Example 2: Selective Activation of CD8.SUP.+.DC by XCL1

(31) CD8.sup.+ and CD8.sup. DC, freshly sorted to a purity>95% by flow sorting as described in Example 1, were supplemented with 2 M fura-2/AM (Molecular Probes, Brattleboro) and allowed to settle on poly-L-lysine-coated glass coverslips at 37 C. and 5% CO.sub.2 for 30 min in a humidified atmosphere. Adherent cells were superfused with a HEPES-buffered solution containing (in mM) 128 NaCl, 6 KCl, 1 MgCl.sub.2, 1 CaCl.sub.2, 5.5 glucose, 10 HEPES, 0.2% (w/v) BSA, and mounted onto the stage of an inverted microscope (Axiovert 100, Zeiss, Jena, Germany). During application of XCL1 (100 nM of synthetic murine XCL1 (Dictagene, Lausanne, Switzerland)), fura-2 was sequentially excited with monochromatic light of 340 nm, 358 nm, 380 nm and 480 nm, and fluorescence emission was detected through a 512 nm long pass filter with a cooled CCD-camera (TILL-Photonics, Grfelfing, Germany). Weakly interfering signals of FITC-labeled antibodies bound to CD8.sup.+ DC were eliminated, and [Ca.sup.2+].sub.i was calculated after spectral unmixing (Lenz J. Cell Biol. 2002, 179:291-301). Data represent intracellular Ca.sup.2+ concentrations ([Ca.sup.2+].sub.i) in 45-56 single cells (black lines) measured in 3 independent experiments. Thick black lines: mean [Ca.sup.2+].sub.i signal averaged over all cells measured. The results demonstrate that XCL1 induces a strong Ca.sup.2+-signal in CD8.sup.+DC (FIG. 2, A), but not CD8.sup.DC (FIG. 2, B). The results thus demonstrate the capacity of XCL1 to specifically activate CD8.sup.+DC and XCL1 thus acts as an adjuvant for XCR1-bearing APC.

Example 3: XCL1 Induces Chemotaxis of CD8.SUP.+.DC, but not of CDd8.SUP..DC, B Cells, T Cells, or NK Cells

(32) CD11c.sup.+ cells were highly enriched from C57BL/6 splenocytes by magnetic separation using CD11c-microbeads according to the manufacturer's protocol (Miltenyi Biotec, Bergisch Gladbach, Germany). CD11c.sup.+ cells (0.5-110.sup.6) were suspended in 100 medium and transferred to a 6.5 mm Transwell Permeable Support containing a 5-m pore polycarbonate membrane (Corning Costar Co., Acton, Mass., USA). The Transwell Permeable Support was inserted into 24 well plate (Corning Costar Co., supra) filled with 600 l medium containing either serial dilutions of chemically synthesized XCL1/ATAC (Dictagene, Lausanne, Switzerland) or with 500 ng/ml CCL21 (chemokine (CC motif) ligand 21; R&D Systems, Minneapolis, Minn., USA), the latter used as a positive control; all experiments were performed in duplicates. Cells were incubated for 120-150 minutes at 37 C. in a cell incubator. The lower side of the membrane was gently rinsed and the cells in the lower chamber were analyzed by flow cytometry for the expression of CD8 (53-6.72-FITC; ATCC, supra), CD11b (5C6-PE; ATCC, supra) and CD11c (N418-Cy5; ATCC, supra). Cell suspensions from each well were analyzed for a defined time (5 min) and the absolute number of live cells (DAPI-negative) was determined. The percentage of migrated cells was calculated by dividing the number of cells in the lower chamber by the number of input cells [number migrated cells/number input cells100]. A representative experiment is shown in FIG. 3. In response to XCL1, CD8.sup.+ DC display the characteristic bell curve of chemotactic migration with no migration at a concentration of 1 ng/ml, a maximum migration at 100 ng/ml and a declining response at 1000 ng/ml. CD8.sup. DC did not respond to XCL1 but migrated in the presence of CCL21.

(33) DC from peripheral lymph nodes were isolated by collagenase digestion of the tissues, followed by positive magnetic sorting with CD11c-microbeads as described above. The chemotaxis assay was performed in Costar Transwell Chambers as above, using XCL1 at a concentration of 100 ng/ml and CCL21 in a concentration of 500 ng/ml. Cells by analyzed by flow cytometry, and the percentage of migrated cells was calculated as above. Again, only CD8.sup.+ DC migrated in response to XCL1, while CD8.sup. DC responded only to CCL21 (FIG. 4).

(34) To investigate the chemotactic response of other splenic cell populations, T cells were isolated by positive magnetic selection from C57BL/6 splenocytes with anti-CD90 conjugated beads, NK cells with anti-49b conjugated beads, and B cells with a combination of biotinylated anti-CD19 antibody (clone 1D3) and anti-Biotin conjugated beads, according to the manufacturer's instructions (see also Example 1). The chemotaxis assays were performed as above using serial dilutions of XCL1/ATAC. The positive control for B cells was CXCL12 (chemokine (CXC motif) ligand 12) at 200 ng/ml, CCL21 (chemokine (CC motif) ligand 21) for T cells at 100 ng/ml, and CXCL9 ((chemokine (CXC motif) ligand 9) for NK cells at 200 ng/ml (all from R&D Systems, Minneapolis, Minn., USA). B cells, T cells, or NK cells failed to respond to XCL1/ATAC with chemotaxis, while the respective positive controls induced significant cell migration in these cell populations (FIG. 5). These experiments demonstrated that XCL1 induces chemotaxis in CD8.sup.+DC, but not in CD8.sup.DC, T cells, B cells, or NK cells. These experiments thus demonstrated that XCL1 acts as a specific adjuvant for XCR1-bearing APC.

Example 4: XCL1-Facilitated Cell/Antigen Uptake into CDd8.SUP.+ Dendritic Cells

(35) Mice deficient for XCL1 (ATAC-KO) were generated by disruption of the murine ATAC gene in embryonic stem cells by homologous recombination using a targeting vector in which exons two and three of the ATAC gene were replaced by the inverted neomycin gene (FIG. 6). Correctly targeted embryonic stem cells, as identified by Southern blotting, were used for the generation of chimeric mice. After germ-line transmission of the mutant allele and breeding of heterozygous ATAC deficient mice inter se, homozygous ATAC-deficient mice were born at expected Mendelian frequency in the F.sub.2-generation and backcrossed to the C57BL/6 background for 10 generations. The murine pre-B cell line 300-19 (Alt et al., 1981, Cell 27, 381-90) was transfected by electroporation with the BCMGS.sub.neo vector (Karasuyama et al., 1989, J Exp Med 169, 13-25) into which the complete coding region of murine XCL1 (GenBank Acc. No.: NM_008510) was cloned by standard methods. After subcloning in G418-containing selection medium, a cell line (referred to as muATAC/300-19) stably secreting murine XCL1/ATAC was obtained, as determined by intracellular flow cytometry (Dorner et al., 2002, Proc. Natl. Acad. Sci. USA 99, 6181-86). Wild-type 300-19 (wt/300-19) cells and muATAC/300-19 cells were fluorescence-labeled by incubation with 10 M 5,6-carboxyfluorescein succinimidyl ester (CFSE, Molecular Probes) for 10 min at 37 C., washed, and injected (1010.sup.6 cells each) intravenously into female XCL1-deficient C57BL/6 (ATAC-KO) mice; control mice were injected with PBS only. After 12 h, mice were sacrificed, the spleens removed and the splenocytes isolated according to standard methods. Splenocytes were stained for CD3, CD4, CD8, CD11b, CD11c, CD19, MHC II, and NK1.1 by standard methods and the CSFE signal was correlated to cell surface markers by analysis on the LSR II (BD Biosciences) flow cytometer (result is shown in FIG. 7) using FlowJo (Tree Star Inc., Ashland, Oreg., USA) for evaluation of the data. The results demonstrated that already 300-19 wild-type cells were taken up by CD8.sup.+DC in the spleen (FIG. 8A). However, the XCL1-transfected 300-19 cells (muATAC/300-19) were taken up to a clearly higher degree (increase of around 50%) (FIG. 8A). These results demonstrated that XCL1 facilitates antigen uptake by CD11c.sup.+CD8.sup.+DC. No cell uptake was observed by splenic CD11c.sup.+CD8.sup. DC (FIG. 8B).

Example 5: Expression of ATAC by CD4.SUP.+ T Cells During Induction of Tolerance or Immunity In Vivo

(36) Splenic cells containing 5-710.sup.6 KJ1-26.sup.+ transgenic D011.10 CD4.sup.+ T cells (Murphy et al., 1990, Science 250, 1720-3) were adoptively transferred into syngeneic BALB/c mice. These transgenic DO11.10 CD4.sup.+ T cells are specific for chicken ovalbumin (OVA) peptide 323-339 (ISQAVHAAHAEINEAGR). Recipient mice were immunized with 100 g OVA, or 100 g OVA+ the adjuvant LPS (10 g) into footpads. Alternatively, recipient mice were immunized with 2 mg OVA injected intravenously. OVA-specific KJ1-26.sup.+ CD4.sup.+ T cells were recovered from the recipients after 14 h, 24 h, or 48 h by flow cytometry cell sorting (purity>97%), either from the draining popliteal lymph nodes (in the case of footpad OVA injection), or from all peripheral lymph nodes (in the case of intravenous OVA injection). Total RNA was isolated from the recovered transgenic T cells and subjected to gene expression analysis using a custom TaqMan Low Density Array (Applied Biosystems). The data obtained are listed in Table 2.

(37) The Ct-values (a parameter obtained when using quantitative PCR) increased in all experimental setups at 14, 24, and 48 h approximately by the value of 5, when compared to the 0 h time point control. This increase represents an approximately 30 fold increase in XCL1 mRNA expression upon in vivo activation of the transgenic T cells in all experimental conditions. These data indicate that XCL1 is expressed and utilized by the immune system, both at immunogenic as well as tolerogenic conditions. These data thus indicate that XCL1 can be used for delivery of a substance both to achieve immunity/memory (in the presence of a danger signal) or to achieve tolerance (in the absence of a danger signal).

(38) TABLE-US-00005 TABLE 2 OVA s.c. OVA + LPS s.c. OVA i.v. Avg Ct Ct Avg Ct Ct Avg Ct Ct time 18S RNA XCL1 18S RNA XCL1 18S RNA XCL1 0 h 7.55 33.94 7.55 33.94 7.55 33.94 14 h 8.59 29.02 10.04 33.91 8.25 27.64 24 h 7.20 28.99 9.53 n.d. 7.82 28.63 48 h 5.96 28.64 6.03 32.04 6.20 30.85

Example 6: XCL1-Mediated, Improved Antigen Recognition by CD8.SUP.+ T Cells Interacting with CD8^{+.DC In Vivo}

(39) ATAC-KO mice (see Example 4) were backcrossed 10 to the C57BL/6 background and then backcrossed to OT-I transgenic mice (OT-I ATAC-KO). OT-I transgenic mice express a transgenic T-cell receptor specific for the SIINFEKL peptide (SEQ ID NO: 15) an 8 amino acid epitope of ovalbumin) derived from chicken ovalbumin (OVA) (Hogquist et al., 1994, Cell 76, 17-27). Total splenocytes containing 210.sup.60T-I T cells were adoptively transferred into syngeneic C57BL/6 recipient mice by intravenous (i.v.) injection. In parallel, total splenocytes containing 210.sup.60T-I ATAC-KO T cells were adoptively transferred into syngeneic C57BL/6 ATAC-KO recipient mice. In all cases, female donor and recipient mice were used. Twenty four hours after cell transfer, recipient mice were challenged with 100 ng OVA conjugated to an anti-DEC205 antibody (DEC-205-OVA) to achieve a preferential delivery of antigen to CD8.sup.+DC, as described previously (Bonifaz et al., 2002, J. Exp. Med. 196, 1627-38).

(40) DEC-205-OVA was generated by incubating 1 mg anti-DEC-205 mAb NLDC-145 (obtained from Georg Kraal, Amsterdam) with 2 mg SMCC-activated OVA according to the manufacturer's protocol (Pierce Chemical Co.). Protein G precipitation of the reagent was performed to remove unconjugated OVA, and the amount of conjugated OVA per mg antibody was carefully determined by analyzing Coomassie-stained non-reducing SDS-gels. DEC-205-OVA was applied i.v. in a volume of 200 l; control mice received PBS. Some mice were injected with DEC-205-OVA alone, which, in the absence of a danger signal, has tolerogenic effects (Bonifaz et al., 2002, J. Exp. Med. 196, 1627-38). Other mice were injected with DEC-205-OVA in combination with 6 g of anti-CD40 antibody FGK (obtained from Ton Rolink, Basel), in which the anti-CD40 mAb which provides danger signals to DC ((Bonifaz et al., 2002, J. Exp. Med. 196, 1627-38). Three days after DEC-205-OVA injection, mice were sacrificed and the splenocytes were stained for CD3, CD8, CD90.1, and MHC II expression by standard methods, and analyzed on a LSR II flow cytometer using FlowJo software in order to determine the presence of OT-I CD8.sup.+ T cells. In addition, splenocytes from the sacrificed mice were incubated in vitro with 50 ng/ml of peptide SIINFEKL in the presence of 5 g/ml Brefeldin A for 5 h. After this period, OT-I T cells and OT-I ATAC-KO T cells were analyzed for secretion of IFN- by intracellular flow cytometry according to standard methods. The results demonstrated that in the absence of XCL1, the interaction of CD8.sup.+T-I T cells with CD8.sup.+DC, either under tolerogenic (no anti-CD40 mAb) or immunogenic (addition of anti-CD40 mAb) conditions, leads to reduced activation and expansion of T cells (FIG. 9). At the same time, the absence of XCL1 leads, either under tolerogenic or immunogenic conditions, to reduced differentiation of CD8.sup.+ T cells into IFN- secreting effector T cells (FIG. 10). Both results demonstrate the activating and adjuvant effects of XCL1 on CD8.sup.+DC interacting with CD8.sup.+ T cells.

Example 7: Generation of Monoclonal Antibodies Against the Human XCR1 (hXCR1)

(41) Female BALB/c mice were immunized with a peptide representing the first 31 N-terminal amino acids of hXCR1 (MESSGNPEST TFFYYDLQSQ PCENQAWVFA T; SEQ ID NO: 18). The N-terminus of the peptide was coupled to keyhole limpet hemocyanin using glutaraldehyde (31-N-hXCR1-KLH; synthesis by P. Henklein, Charit, Berlin). Initial immunization was performed with 31-N-hXCR1-KLH (30 g applied intraperitoneally and 30 g subcutaneuosly) in complete Freund's adjuvant. Mice were boosted twice after 3-4 week intervals with 50 g 31-N-hXCR1KLH in incomplete Freund's adjuvant applied intraperitoneally. Six weeks after the second boost, mice were injected with the 31-N-hXCR1 peptide bound to bovine serum albumin (31-N-hXCR1-BSA) intravenously (50 g) in saline. Three days later the mice were sacrificed and spleen cells were fused with the myeloma line P3X63Ag8.653 according to standard protocols for monoclonal antibody generation. Screening of the hybridoma supernatants was performed using the uncoupled 31-N-hXCR1 peptide adsorbed to 96-well plates in a standard ELISA assay. One hybridoma (6F8) gave a strong and consistent signal in the ELISA assay; the hybridoma was therefore subcloned and the 6F6 antibody used for further characterization of hXCR1. To this end, several hXCR1 transfectants were generated by cloning the entire coding region of hXCR1/hATACR (GenBank Acc. No.: L36149) into the vector BCMGS.sub.neo (supra) in such a fashion that it was either at the 3 or 5 end tagged with a c-myc epitope EQKLISEEDL (SEQ ID NO: 19). Subsequently, the murine myeloma line P3X63Ag8.653 was transfected by electroporation with either version of the vector and the two transfected cell lines 5 c-myc/hATACR/P3X and 3 c-myc/hATACR/P3X were established after subcloning in G418-containing selection medium. Included in the studies was also the murine cell line transfected with hXCR1 obtained from Dr. Bernhard Moser, Bern, Switzerland (hATACR/300-19). Supernatants of the mAb 6F8 were used to immunoprecipitate the hXCR1 protein from various cell lines (FIG. 11). To this end, lysates from the transfectants 5 c-myc/hATACR/P3X, 3 c-myc/hATACR/P3X, and hATACR/300-19, and the respective wild-type lines were generated from 5-1010.sup.6 cells each according to standard methods (lysis buffer: 50 mM Tris/HCl (pH 8), 150 mM NaCl, 1 mM EDTA, +1% (v/v) Nonident P-40, 1 mM PMSF, 10 M leupeptin A, 1 M pepstatin, 10 g/ml aprotinin). These lysates, after preclearing, were incubated with mAb 6F8 supernatant (5-10 ml), and immunoprecipitated with protein G beads according to standard methods. The immunoprecipitate was denatured in SDS buffer, separated on a reducing 12% SDS-gel, and electroblotted on a Immobilon P membrane (Millipore) according to standard methods. The blot was stained with a polyclonal rabbit-anti-hXCR1 serum (generated against a peptide representing the N-terminus of hXCR1, MESSGNPEST TFFYYDLQSQ PCENQAWVFA T, SEQ ID NO: 18, using a standard protocol) diluted 1:2500 in blocking buffer and developed using biotin-coupled goat-anti-rabbit-IgG (1:5000 in blocking buffer), avidin-alkaline phosphatase and the Western Light/CDP-Star detection system (Tropix). The detection of the light signal was with XOMatAR-film (Kodak). The rabbit anti-hXCR1 serum had been generated by immunizing rabbits 3 with 250 g of the 31-N-hXCR1 peptide in complete Freund's adjuvant over a period 11 weeks.

Example 8: Generation of Recombinant Murine XCL1 in its Biologically Active Form

(42) Native murine XCL1 is generated in vivo by proteolytic removal of a signal peptide, resulting in a protein with N-terminal valine (Dorner et al., 1997, J. Biol. Chem. 272, 8817-23). To generate a corresponding recombinant murine XCL1 starting with N-terminal valine, amino acids 22-114 of full-length murine ATAC were fused to the C-terminus of a histidine-tagged SUMO-protein, using standard DNA recombinant technology and the expression vector pET SUMO (Invitrogen, Groningen, The Netherlands). The fusion protein was expressed in E. coli using standard protocols and purified by immobilized metal affinity chromatography (Ni-NTA Superflow, Qiagen, Hilden, Germany) according to the manufacturer's protocol. Site-specific cleavage of the fusion protein was achieved by incubation with SUMO protease (Invitrogen) for 3 h at 37 C. A second immobilized metal affinity chromatography step was performed to remove the histidine-tagged SUMO fusion part. Using this protocol, a biologically active form of recombinant murine XCL1 protein was generated with high yield and purity (FIG. 12).

Example 9: Enhanced Cytoxicity by WT OT-I in Comparison to ATAC-KO OT-I

(43) Transgenic CD8.sup.+ T cells specific for OVA peptide were purified from splenocytes of OT-I or ATAC-KO OT-1 mice by magnetic depletion of other splenic cell populations using antibodies against CD4, CD11b, CD11c, NK1.1, and B220. OT-I or OT-I ATAC-KO T cells (310.sup.5) were adoptively transferred into syngeneic C57BL/6 or ATAC-KO mice, respectively. Both groups of mice were immunized 24 h later with 310.sup.6 300-19 cells transfected with OVA (OVA/300-19). OVA/300-19 cells were generated by electroporation of wild-type 300-19 cells with the BCMGS.sub.neO vector (Karasuyama et al., 1989, J Exp Med 169, 13-25) into which a truncated coding region of OVA (corresponding to amino acids 138-386; GenBank Acc. No.: NM_205152) was cloned by standard methods. At day 6 after immunization with OVA/300-19 cells, an in vivo cytotoxicity assay was performed as previously described (Romano et al., 2004, J. Immunol. 172, 6913-6921). Shortly, splenocytes of C57BL/6 mice were isolated and incubated for 1 h at 37 C., either in medium alone or in the presence of 10 M of the specific OVA peptide SIINFEKL. After washing, peptide-pulsed cells were labeled with 10 M 5,6-carboxyfluorescein diacetate succimidyl ester (CSFE, Molecular Probes, Oregon, USA), while unpulsed cells were labeled with 1 M CSFE. Equal amounts of CSFE-low and CSFE-high/SIINFEKL splenocytes (1010.sup.6 cells each) were injected into the OVA/300-19 immunized mice and the relative abundance of surviving CSFE-low and CSFE-high/SIINFEKL splenocytes was determined by flow cytometry 18 h later. OVA-specific cytotoxicity was calculated as described (Hernandez et al., 2007, J. Immunol. 178, 2844-2852). Injection of OVA/300-19 cells induced 324% OVA-specific cytotoxicity in the presence of OT-I T cells, but only 1410% cytotoxicity in the presence of ATAC-KO OT-I T cells (FIG. 13). Control immunization of mice with wild-type 300-19 cells did not induce cytotoxicity by transferred OT-I T cells. This experiment demonstrates that ATAC acts as an adjuvant in the induction of CD8.sup.+ T cell cytotoxicity.

Example 10: XCR1 Expression In Vivo is Limited to a Supopulation of DC

(44) Organ tissues from B6.129P2-Xcr1.sup.tm1Dgen/J mice (The Jackson Laboratory, Maine, USA), in which the ATAC gene has been replaced by a lacZ-reporter gene (knock-in), were analyzed for in situ B-galactosidase activity. To this end, pieces of organs were immersed in 0.1% glutaraldehyde and 4% paraformaldehyde in PBS for 4 h at 4 C., incubated in 10% sucrose/PBS at 4 C. overnight, and snap frozen. Cryosections of the tissues were re-fixed in 0.1% glutaraldehyde and 4% paraformaldehyde in PBS for 10 min at RT, washed 3 with cold PBS (pH 7.4) for 5 min, incubated with X-Gal staining solution (Sanes et al., 1986, EMBO J. 5, 3133-3142) overnight at 37 C., washed 3 in PBS, and counterstained by Neutral Red.

(45) Expression of lacZ (and thus the XCR1 gene) was observed in the spleen, thymus, lymph nodes, lung, liver, testis, ovary, placenta, Payer's patches, small intestine, and large intestine.) In the spleen, the signals obtained corresponded to the distribution pattern of CD8.sup.+ DC. In the other organs the (usually low) abundance, the morphology, and the tissue distribution of the signals were fully compatible with the concept of an XCR1 expression limited to a subpopulation of DC.

Example 11: Expression of XCR1 in Murine Splenocytes Analyzed by Flow Cytometry

(46) Splenocytes from B6.129P2-Xcr1.sup.tm1Dgen/J mice mice were isolated and stained for CD3, CD4, CD8, CD19, CD11c, MHC II and NK.1.1 by standard methods. Expression of the lacZ reporter gene, assayed with fluorescein di--D-galacto pyranoside (FDG, Invitrogen) according to the manufacturer's protocol, was detected in 7%-10% of CD4.sup.CD8.sup. DC and in 75%-90% of CD8.sup.+ DC, but not in CD4.sup.+ DC (FIG. 14). All other splenic populations were negative. These results demonstrate that XCR1 is, within the immune system, only expressed in a subpopulation of DC, which in the spleen mostly carries the CD8 cell surface marker.