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Originally published online as doi:10.1189/jlb.0804480 on December 2, 2004

Published online before print December 2, 2004
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(Journal of Leukocyte Biology. 2005;77:303-310.)
© 2005 by Society for Leukocyte Biology

Human CD14 is an efficient target for recombinant immunoglobulin vaccine constructs that deliver T cell epitopes

Gro Tunheim*,1, Karoline W. Schjetne*, Agnete B. Fredriksen*, Inger Sandlie{dagger} and Bjarne Bogen*

* Institute of Immunology, University of Oslo and Rikshospitalet University Hospital, Norway; and
{dagger} Department of Molecular Biosciences, University of Oslo, Norway

1 Correspondence: Institute of Immunology, University of Oslo and Rikshospitalet University Hospital, N-0027 Oslo, Norway. E-mail: gro.tunheim{at}medisin.uio.no; bjarne.bogen{at}medisin.uio.no


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ABSTRACT
 
It has been shown in the mouse that recombinant immunoglobulin (Ig) molecules with T cell epitopes inserted into the constant domain (Troybodies) can target antigen-presenting cells (APC) for efficient delivery of T cell epitopes. Here, we have extended the Troybody concept to human applications. Moreover, we show that a receptor of innate immunity, CD14, which is a part of the lipopolysaccharide receptor complex on monocyte APC, is an efficient target. For construction of CD14-specific Troybodies, we used rearranged variable(diversity)joining regions cloned from the 3C10 mouse B cell hybridoma. As a model T cell epitope, amino acids 40–48 of mouse C{kappa}, presented on human leukocyte antigen-DR4, were inserted into a loop connecting ß-strands in CH1 of human {gamma}3. In the presence of monocytes, CD14-specific Troybodies were >100 times as efficient as a nontargeting control antibody (Ab) at stimulating C{kappa}40–48-specific/DR4-restricted T cells. Presentation was dependent on the conventional processing pathway for presentation on major histocompatibility complex (MHC) class II molecules. Enhanced presentation of the C{kappa} epitope was most likely a result of increased loading of MHC class II molecules, as the CD14-specific monoclonal Ab 3C10 did not induce maturation of the APC. The results show that CD14, a receptor of innate immunity, may be a promising target of recombinant Ig-based vaccines for elicitation of T cell responses in humans.

Key Words: antigen presentation • monocyte • CD4+ T cells • Ab • MHC class II


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INTRODUCTION
 
CD4+ T cells have a major immunoregulatory function as they help B cells and CD8+ T cells. In addition, they can themselves be cytotoxic, induce inflammation, and produce cytokines [1 ]. Thus, efficient vaccines should preferably elicit strong CD4+ T cell responses.

An ideal vaccine for induction of CD4+ T cell responses should induce peptide loading of major histocompatibility complex (MHC) class II molecules and maturation of antigen-presenting cells (APC) in the absence of additional adjuvants. To fulfill these two objectives, targeting vaccines to receptors of the innate immune system may be a useful strategy. Supporting this possibility, it has recently been shown that targeting antigen to DEC-205 [2 ], mannose receptor [3 , 4 ], dendritic cell-specific intracellular adhesion molecule-grabbing nonintegrin (DC-SIGN) [5 , 6 ], Toll-like receptor (TLR)2 [7 ], and chemokine receptors such as CC chemokine receptor 1 (CCR1), CCR2, and CXC chemokine receptor 4 [8 ] resulted in loading of MHC class II molecules and enhanced ability to induce CD4+ T cell responses. Thus, there appears to be a link between targeting receptors of host immunity and elicitation of adaptive CD4+ responses [7 ], which could be exploited for vaccination purposes.

We have investigated here if other endocytic receptors of the innate immune system represent good targets for enhancement of CD4+ T cell responses in humans and considered TLR4 and CD14 as interesting candidates. TLR4 is a member of the pattern recognition receptor (PRR) family of TLR [9 ]. TLR4 is a part of the lipopolysaccharide (LPS) receptor complex together with CD14 and MD-2. The TLR4 transmembrane molecule functions as the signaling component of the complex [9 , 10 ]. Mouse TLR4 appears to mediate inflammatory responses to ligands such as Taxol, respiratory syncytial virus, and heat shock protein 60 [9 ].

CD14, expressed on the surface of monocytes, is a glycophosphatidylinositol-linked protein, which is also part of the LPS receptor complex. CD14 binds LPS and as such, acts as a PRR [11 , 12 ]. In addition to binding LPS and other bacterial products such as peptidoglycan [13 , 14 ] and lipomannans [15 ], human CD14 has been suggested to mediate phagocytosis of bacteria and apoptotic cells [16 , 17 ].

Antibodies (Ab), which have been chemically [18 , 19 ] or genetically [20 , 21 ] coupled to antigen, can be used to target surface molecules on APC to enhance loading of antigenic peptide on MHC class II molecules and hence, stimulation of CD4+ T cells. In an extension of this approach, we have recently developed recombinant Ab-based vaccines, Troybodies, which have been equipped with variable (V) regions specific for surface molecules on APC and in addition, have T cell epitopes genetically introduced into loops connecting ß-strands in the CH1 domain [22 , 23 ]. Troybodies target APC and are endocytosed and processed. The liberated T cell epitopes are efficiently loaded onto MHC class II molecules for enhanced CD4+ T cell activation [22 23 24 ]. Previously, Troybodies have been targeted to various surface molecules on mouse APC such as immunoglobulin D (IgD) on B cells [22 ] and MHC class II (I–E) expressed on B cells, macrophages, as well as DCs [23 ]. These Troybodies have been equipped with a variety of model mouse T cell epitopes such as amino acids (aa) 91–101 {lambda}2315, 110–120 hemagglutinin, 323–339 ovalbumin, and 46–61 hen egg lysozyme [22 23 24 25 ]. Such Troybodies considerably enhanced CD4+ T cell responses in vitro and in vivo [22 , 23 , 25 ].

In a step toward a more realistic vaccine for use in humans, here, we have produced Troybodies specific for human CD14. More specifically, we have made chimeric mouse/human Troybodies, which have mouse V regions that target human CD14 and constant (C) regions from human {gamma}3 with a model T cell epitope inserted into a loop connecting ß-strands in the CH1 domain. Such CD14-specific Troybodies were >100 times more potent at stimulating human antigen-specific CD4+ T cells compared with control Ab.


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MATERIALS AND METHODS
 
Cell lines
The mouse B cell hybridoma 3C10 produces a mouse IgG2b{kappa} monoclonal Ab (mAb) with specificity for human CD14. 3C10, human embryo kidney (HEK) cells 293E, and NS0 were purchased from American Type Culture Collection (Manassas, VA). T18 is a human CD4+ T cell clone specific for aa 40–48 of mouse Ig C{kappa} and is restricted by human leukocyte antigen (HLA)-DR4 (DRA1, B1*0401) [6 ]. The T18 clone was kindly provided by Keith M. Thompson (University of Oslo, Norway). The {lambda}1-expressing murine myeloma cell line J558L was a kind gift from Sherie L. Morrison (University of California, Los Angeles). Peripheral blood mononuclear cells (PBMC) were purified from whole blood by LymphoPrep density gradient centrifugation (Nycomed, Oslo, Norway). To obtain monocytes, the PBMC were incubated for 1.5 h on plastic before washings, and monocytes were isolated as the adherent fraction. Monocyte-derived DCs were prepared by culturing adherent monocytes with granulocyte macrophage-colony stimulating factor (800 U/ml) and interleukin-4 (IL-4; 250 ng/ml; both from Peprotech, Rocky Hill, NJ) for 6 days, renewing cytokines every 2–3 days. Maturation of DCs was induced by 10 ng/ml LPS (Escherichia coli; Sigma-Aldrich, St. Louis, MO). Tissue-culture medium was RPMI 1640 (Life Technologies, Paisley, UK) supplemented with nonessential amino acids, sodium pyruvate, monothioglycerol, garamycin, glucose, L-glutamine, and 10% fetal calf serum. For the culture of human T cells, the medium was RPMI 1640 supplemented with 10% human serum from pooled blood donors.

Tumor necrosis factor {alpha} (TNF-{alpha}) measurement
Purified monocytes were cultured with titrated amounts of different mAb. LPS (20 ng/ml) or the TLR2 ligand Pam3Cys-HEL (46–61; 1 µg/ml) [7 ] was used as positive control. After 16 h, supernatant (SN) was collected, and the TNF-{alpha} concentrations were measured by sandwich enzyme-linked immunosorbent assay (ELISA; Human TNF-{alpha}/TNFSF1A DuoSet, R&D Systems Europe Ltd., Abingdon, UK).

Assay for APC maturation
Purified monocytes were cultured for 40 h with different mAb (10 µg/ml), LPS (20 ng/ml), or the TLR2 ligand Pam3Cys-HEL (46–61; 1 µg/ml) before the cells were harvested and stained for CD80, CD86, and MHC class II for flow cytometric analyses.

Measurements of free cytosolic Ca2+ in single T cells
Ca2+ mobilization measurements were performed essentially as described [7 , 26 ]. Briefly, 3C10 mAb (100 µg/ml) was added to monocytes in the presence or absence of brefeldin A (500 µM), chloroquine (10 µM), or leupeptin (5 µg/ml; Sigma-Aldrich). After incubation, Fura-2/AM (5 µM, Teflabs, Austin TX)-loaded, C{kappa}-specific CD4+ T cells (T18) were added onto the Ab-pulsed monocytes. The imaging and registration software were as described [26 ]. Statistical analyses were computed by using SPSS for Windows (Chicago, IL). Differences in the concentration of free cytosolic Ca2+ in the absence or presence of brefeldin A, chloroquine, or leupeptin were compared using Student’s t-test (significant level at P<0.01).

Construction of mutant IgG3 heavy (H)-chain genes encoding the C{kappa} T cell epitope
Amino acids 40–48 of the mouse C{kappa} epitope were inserted into the human C{gamma}3 gene already placed in the polylinker of pUC19 {(pUC19{gamma}3 wild-type (wt); [27 ]}. The 12 nucleotides corresponding to the FG loop (loop 6, L6) in CH1 (88–91 aa KPSN) were replaced with the 27 nucleotides encoding the C{kappa} epitope (40–48 aa WKIDGSERQ) to produce pUC19{gamma}3C{kappa}. Mutagenesis was performed using a method based on the QuikChangeTM kit (Stratagene, Medprobe, Oslo; #200518). The sample reactions were prepared essentially as indicated in the kit manual. In the reaction, two complementary primers, 5'-Q6CH1C{kappa} (cta cac ctg caa cgt gaa tca ctg gaa gat tga tgg cag tga acg aca aac caa ggt gga caa gag agt tg) and 3'-Q6CH1C{kappa} (caa ctc tct tgt cca cct tgg ttt gtc gtt cac tgc cat caa tct tcc agt gat tca cgt tgc agg tgt ag) were used (DNA Technology A/S, Aarhus, Denmark). A correct insertion of C{kappa}-encoding nucleotides was confirmed by sequencing (GATC Biotech AG, Konstanz, Germany). An ~0.9-kb HindIII-BglII fragment of pUC19{gamma}3C{kappa}, which contains the sequenced CH1 exon with the C{kappa} epitope, was subcloned into another pUC19{gamma}3wt to exclude possible amplification errors outside the sequenced fragments. The mutant C{gamma}3 gene of this new pUC19{gamma}3C{kappa} was subcloned as a 2.8-kb HindIII-BamHI fragment into the expression vector pLNOH2 [28 ] replacing the C{gamma}3 gene in the vector. The mutated vector is denoted pLNOH2C{kappa}.

Cloning of V region genes of 3C10 B cell hybridoma and construction of Troybodies
The rearranged V[diversity (D)]joining (J) of H- and light (L)-chain genes was polymerase chain reaction (PCR)-amplified from cDNA from the hybridoma 3C10 essentially as described [22 , 28 ]. Briefly, mRNA was isolated by use of the Dynabeads® mRNA DIRECTTM kit (Dynal, Oslo), and cDNA was synthesized using the First-Strand cDNA synthesis kit (Amersham Biosciences, Oslo). The specifically designed PCR primers (Sigma Genosys Ltd., UK) were 5'V{kappa}_3C10, tgt gca ttc cga cat tgt gct gac cca atc t; 3'V{kappa}_3C10, acg tac gtt cta ctc acg ttt gat ttc cag ctt ggt; 5'VH3C10, tgt gca ttc cga agt gaa gct ggt gga g; and 3'VH3C10, gac gta cga ctc acc tga gga gac tgt gag agt ggt. (Restriction enzyme sites for BsiWI and BsmI are shown underlined. The splice donor sites are shown in bold.) The amplified VDJ of the 3C10 H-gene was sequenced and subcloned upstream of the genomic wt human {gamma}3 C region gene in pLNOH2, creating a vector that encodes a chimeric mouse V, human {gamma}3 CD14-specific H-chain (pLNOH2{alpha}CD14). To obtain an identical H-chain but with the C{kappa} epitope, the wt {gamma}3 gene in pLNOH2{alpha}CD14 was replaced with the C{kappa}-mutated {gamma}3 gene from pUC19{gamma}3C{kappa} (4.5), resulting in vector pLNOH2{alpha}CD14C{kappa}. The amplified VJ of the 3C10 L-chain gene was sequenced and subcloned upstream from the human {kappa} C region gene in the pLNO{kappa} vector [28 ]. The vector is denoted pLNOK{alpha}CD14. The V sequences of 3C10 have been submitted to the EMBL GenBank, with Accession Numbers AY669065 and AY669066.

The V region genes conferring the specificity for the hapten 4-hydroxy-3-iodo-5-nitrophenylacetic acid (NIP) have previously been described [29 ]. When transfected into {lambda}1-expressing cells (J558L), the H-chain V region encoded by the vector pLNOH2 produces Ab with NIP specificity. Thus, by substituting the {gamma}3 gene in the pLNOH2 vector with the mutated C{kappa} {gamma}3 gene, a NIP-specific vector with the C{kappa} epitope was obtained (see above). To obtain NIP-specific Ig in cells not expressing {lambda}1, the C{kappa}-mutated pLNOH2 vector (pLNOH2{alpha}C{kappa}) was cotransfected with the plasmid pro145 encoding a mouse {lambda}1 chain (Celltech, Berks, UK).

Production and isolation of Troybodies and mAb
To obtain CD14-specific Troybodies, the pLNO{kappa}{alpha}CD14 and the pLNOH2{alpha}CD14C{kappa} vectors were cotransfected into NS0 cells by electroporation using an ECM® 830 apparatus (BTX, VWR International AS, Oslo) and the following conditions: 2 x 107 cells; 10–20 µg DNA; 200V; pulse length; 70 ms. H- and L-chains were expressed from the human cytomegalovirus promoter of the L- and H-chain vectors. To obtain NIP-specific Ab, pLNOH2{alpha}C{kappa} was electroporated into J558L, which constitutively expresses the mouse {lambda}1 L-chain but no H-chain.

Stably transfected clones were obtained by limiting dilution in culture medium supplemented with 0.8 mg/ml G418 (Gibco, Invitrogen, Carlsbad, CA), and SN were screened for Ig production by a human IgG3-specific ELISA. Mouse anti-human IgG3 (HP6047, Sigma-Aldrich) was used as coat Ab, and alkaline phosphatase-conjugated goat anti-human IgG (Fc-specific, Sigma-Aldrich) was the detection Ab. Ab-producing clones were expanded, and Ig were affinity-purified from cell SN by use of protein L-sepharose (Affitech, Oslo) or protein G-sepharose (Amersham Bioscience, Oslo). For transient expression of Ig, the vectors described above encoding H- and L-chains were cotransfected into HEK293E cells (4x105/well, cultured in 24-well plates), using lipofectamine 2000 (Invitrogen). The L-chain plasmid encoding {lambda}1 (pro145) was used to obtain NIP specificity in these cells. The SN were harvested on day 3, and the Ig concentrations were measured by IgG3-specific ELISA.

The 3C10 mAb (IgG2b{kappa}) was purified on an anti-mouse-{kappa} mAb (187.1)-coupled sepharose column. Purified anti-TLR4 mAb (HTA125, IgG2a{kappa}) and the purified anti-CD14 mAb 5C5 (IgG2a{kappa}) and 18D11 (IgG1{kappa}) were kind gifts from Terje Espevik (University of Science and Technology, Trondheim). Their isotype-matched controls were purchased from PharMingen (San Diego, CA).

Flow cytometry
To investigate CD14 expression on human cells, monocytes and monocyte-derived DCs were stained with anti-human-CD14-fluorescein isothiocyanate (FITC; clone 18D11, Diatec, Oslo). Mouse IgG1-FITC (Diatec) was used as isotype control. To investigate TLR4 expression, the same cell types were stained with HTA125 (or isotype-control IgG2a{kappa}) and anti-mouse-IgG-FITC (Zymed, Medprobe, Oslo).

To detect binding of the CD14-specific Troybodies, freshly isolated PBMC were stained with the CD14-specific Troybody and detected using goat-F(ab')2-anti-human-IgG-phycoerythrin (PE; SouthernBiotech, Birmingham, AL). L6-{lambda}2315 [27 ], a human IgG3 with NIP specificity and with the epitope {lambda}2315 in L6, was used as isotype-matched control. To assure correct gating of CD14-positive cells, anti-human-CD14-FITC was used. Staining PBMC with 3C10 and its isotype-matched, control mouse IgG2b was detected by goat-anti-mouse-IgG2b-FITC (SouthernBiotech).

mAb used for staining in the assay for APC maturation were anti-human-CD80-FITC, anti-human-CD86-PE (both PharMingen), anti-HLA-DR-APC (Diatec), and isotype-matched controls.

In all staining experiments, 10,000 cells were run on a FACScalibur (Becton Dickinson, San Jose, CA) and analyzed by CellQuest (Becton Dickinson).

T cell proliferation assay
APC were freshly isolated PBMC (7x104/w), purified monocytes (3x104/well), or monocyte-derived DCs (2x104/w) from HLA-DR4 (DRA1, B1*0401) donors. Irradiated APC (20 Gy) and mouse C{kappa}-specific, DR4-restricted human CD4+ T cells (4x104/well) were cultured in 96-well plates with titrated amounts of different Ab. After 48 h, the cultures were pulsed for 16–24 h with 1 µCi tritiated thymidine (3[H]dThd). The cultures were harvested, and [3H]dThd incorporated into DNA of proliferating cells was measured using a TopCount NXT scintillation counter (Packard, Meriden, CT).


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RESULTS
 
A C{kappa} epitope in CD14-specific mouse mAb 3C10 is efficiently presented on MHC class II molecules to human CD4+ T cells
To test whether CD14 is an efficient target for delivery of antigen to APC for MHC class II presentation to T cells, we exploited the fact that antigen processing of mouse Ig{kappa} yields a 40–48 aa C{kappa} epitope, which is presented on HLA-DR4 to cloned, C{kappa}-specific, human CD4+ T cells (T18 clone) [6 ]. As shown in Figure 1A , mouse mAb 3C10, specific for human CD14 [30 ], was much more efficiently presented by APC to C{kappa}-specific CD4+ T cells compared with isotype-matched, control mAb [the presentation of the latter presumably representing uptake via Fc receptors (FcRs) and constitutive pinocytosis]. The enhancement (left-shift of dose-response curves) of CD4+ T cell responses was dependent on the type of APC and was much more pronounced for monocytes and for PBMC (x103) than for DCs (x10). Likewise, the level of proliferation of T cells (3[H]dThd incorporation) was more pronounced with monocytes and PBMC than with DCs. These results most likely reflect the higher CD14 expression on monocytes than on monocyte-derived DCs [31 ] (Fig. 1B) . In addition to the 3C10 mAb, we tested two other CD14-specific mAb, 5C5 and 18D11, and obtained similar results (data not shown).



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  		Figure 1. mAb targeted to CD14 on APC is efficiently presented on MHC class II molecules to CD4+ T cells. (A) DR4+ PBMC, monocytes, or matured DC were cultured with titrated amounts of 3C10, a mouse IgG2b{kappa} mAb specific for CD14 ({diamondsuit}) or isotype-matched, control mAb ({cjs3752}), and cloned CD4+ T cells, specific for mouse C{kappa}40–48/DR4 (DRA1, B*0401). Proliferation of T cells was measured as incorporation of 3[H]dThd between 48 and 72 h. Similar experiments were performed for HTA125 mouse IgG2a{kappa} mAb targeting TLR4 ({blacktriangleup}) and isotype-matched control ({triangleup}). (B) Expression of CD14 and TLR4 on monocytes (d0) and immature (d5) and mature (d7) DC (filled histograms). Unshaded histograms represent staining with isotype-matched, control mAb.

As CD14 and TLR4 are part of the LPS receptor complex [9 ], we tested if a mouse mAb specific for TLR4, HTA125 [32 ], was as potently presented to the C{kappa}-specific CD4+ T cells as was the CD14-specific mAb. It is surprising that the TLR4 mAb was only slightly better presented than was the isotype-matched, control Ab and only when monocytes were used as APC (Fig. 1A) . Consistent with this, TLR4 was expressed on monocytes but not on DC (Fig. 1B) . The reason for the poor presentation when targeting TLR4 could be related to a low affinity of the HTA125 mAb for TLR4 or a relatively low expression of TLR4 on the cell surface (Fig. 1B) . As a result of the more impressive responses obtained with 3C10 anti-CD14 mAb than with the HTA125 anti-TLR4 mAb, we focused our further work on this specificity.

Presentation of CD14-specific 3C10 mouse mAb to C{kappa}-specific T cells requires conventional antigen processing
We next studied the requirements for processing the 3C10 mAb prior to presentation. Monocytes were incubated with CD14-specific 3C10 mAb, followed by addition of mouse C{kappa}-specific human T cells loaded with Ca2+-binding Fura-2. As can be seen from Figure 2 , anti-CD14-pulsed monocytes induced within minutes an increase in cytosolic Ca2+ measured in single T cells. The response was specific, as an IgG2a{lambda} mAb did not induce any responses.



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  		Figure 2. The anti-CD14 mAb 3C10 requires antigen processing for presentation on MHC class II molecules to C{kappa}-specific CD4+ T cells. 3C10 (100 µg/ml) was added to a semiconfluent layer of monocytes in the presence or absence of brefeldin A, chloroquine, or leupeptin. After incubation and washings, Fura-2/AM-loaded, C{kappa}-specific T cells were allowed to sediment onto the layer of mAb-pulsed monocytes. Images of cytosolic-free calcium in single T cells were recorded and analyzed. (A) Time-course represents mean of free cytosolic Ca2+ in all single T cells (n) in response to APC treated with mAb and inhibitors as indicated. (B) Fraction of C{kappa}-specific T cells giving a response 100 nM above their individual, basal Ca2+ level.

We proceeded to test if agents known to inhibit antigen processing could inhibit the ability of anti-CD14-pulsed monocytes to stimulate Ca2+ responses in T cells. Brefeldin A is a fungal metabolite known to fuse endoplasmic reticulum and Golgi and thus inhibit egress of nascent MHC class II molecules to the cell surface. Leupeptin is a peptide that inhibits proteases involved in antigen processing, whereas chloroquine raises the pH of endosomes. These inhibitors have been shown to abolish antigen processing without abolishing APC function [33 , 34 ]. As can be seen from Figure 2 , all three agents inhibited the ability of anti-CD14-pulsed monocytes to stimulate Ca2+ responses in single T cells. Thus, conventional antigen processing of anti-CD14 mAb was required for stimulation of C{kappa}-specific T cells. This result is consistent with previous studies demonstrating a processing requirement for Ig prior to recognition by CD4+ T cells [7 , 35 , 36 ].

Construction and production of human CD14-specific Troybodies
Having established that CD14 is an efficient target, we proceeded to construct recombinant Ab with specificity for CD14 and with a T cell epitope in the C region (Troybodies). The mouse V regions were cloned from the hybridoma 3C10. As a model T cell epitope, we used the mouse C{kappa}40–48 T cell epitope and inserted it into the FG loop, L6, in the CH1 domain of human {gamma}3, replacing the 4 aa encoding this loop. As a nontargeting control for the CD14-specific Troybody, we made a recombinant Ab with V regions having specificity for the hapten NIP. This control Ab has a mouse {lambda} L-chain, whereas the Troybody has a human {kappa} L-chain; neither of these L-chains stimulates T18 [6 ]. The recombinant CD14-specific Troybody is denoted {alpha}CD14.L6-C{kappa}, and the NIP-specific control Ab is denoted L6-C{kappa} (or peptide Ab; Table 1 ). As additional controls, we produced CD14-specific Ab with human {gamma}3 without any T cell epitope ({alpha}CD14.wt; Table 1 ) and CD14-specific Troybodies with other T cell epitopes such as telomerase (tel; unpublished results).


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  		Table 1. Troybody and Control Ab Used in This Studya

The constructs encoding the CD14-specific Troybody, the control peptide Ab, {alpha}CD14.wt, and {alpha}CD14.L6-tel (unpublished) were transiently transfected into HEK293E cells and stably transfected into NS0 or J558L cells. The recombinant Ab were secreted into the SN and could be detected by a human IgG3-specific sandwich ELISA. Recombinant Ig were purified from SN on protein G- or protein L-sepharose and had a correct size and L-H assembly as tested by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (data not shown).

The specificity of the CD14-specific Troybody was tested by flow cytometry. The CD14-specific Troybody was indeed able to bind to CD14 on monocytes (Fig. 3 ), as was {alpha}CD14.wt (data not shown), and the control peptide Ab (L6-C{kappa}) did not. Thus, the genetically added V regions endowed the Troybody with the correct specificity; moreover, insertion of the C{kappa} epitope in the CH1 domain did not abrogate secretion or correct folding or binding of the Troybody to CD14.



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  		Figure 3. Specificity of {alpha}CD14.L6-C{kappa} Troybodies. Human PBMC were stained with {alpha}CD14.L6-C{kappa} (solid line), L6-C{kappa} (dotted line), or L6-{lambda}2315 (shaded histogram; Table 1 ) and detected with PE-conjugated, anti-human IgG. Monocytes, gated on the basis of their appearance in a forward-/side-scatter dot plot, were analyzed.

Enhanced ability to induce specific T cell activation by CD14-specific Troybodies in vitro
To study if the human Troybodies could stimulate specific human T cells, titrated amounts of Troybodies or controls were added to irradiated human DR4+ APC (PBMC or monocytes) and tested for their ability to induce proliferation of the mouse C{kappa}-specific CD4+ human T cells (T18; Fig. 4 ). The dose-response curves show that the CD14-specific Troybody {alpha}CD14.L6-C{kappa} was at least 100 times more efficient on a molar basis at stimulating the mouse C{kappa}-specific T cells compared with the control peptide Ab L6-C{kappa} with specificity for the hapten NIP. Although purified recombinant Ab have the advantage of being pure, they could aggregate as a result of the purification procedure. By contrast, recombinant Ab in SN should not be aggregated but has the drawback that it also contains other proteins. However, none of these factors influenced the results, as similar data were obtained when using purified recombinant Ab and SN from cells transiently transfected with the Ig expression vectors (Fig. 4 and data not shown).



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  		Figure 4. CD14-specific Troybody efficiently delivers T cell epitopes to APC in vitro for T cell stimulation. Titrated amounts of {alpha}CD14.L6-C{kappa} ({blacksquare}) or the nontargeting L6-C{kappa} peptide Ab ({blacktriangledown}) were added to irradiated DR4+ APC [PBMC (left panel) and monocytes (right panel)] and C{kappa}-specific CD4+ T18 cells. Proliferation was measured as 3[H]dThd incorporation.

CD14-specific 3C10 mAb does not induce activation or maturation of APC
One obvious reason for the greatly enhanced responses to 3C10 mAb and CD14-specific Troybodies could be that mAb ligation of CD14 could result in up-regulation of APC function of monocytes. However, the 3C10 mAb did not induce secretion of TNF-{alpha} by monocytes, whereas the natural CD14 ligand LPS and the TLR2 ligand Pam3Cys did (data not shown). Moreover, 3C10 did not up-regulate CD80, CD86, or MHC class II molecules on monocytes after 42 h (data not shown). These data are consistent with previous reports, as the 3C10 mAb has been shown to be a blocking mAb that neutralizes the effect of LPS [11 , 37 ]. These results indicate that ligation of CD14 by 3C10 mAb does not up-regulate APC function.

CD14-specific V regions and the C{kappa} T cell epitope need to be physically linked
As stated above, the 3C10 anti-CD14 mAb did not induce TNF-{alpha}-secretion and up-regulation of CD80, CD86, or MHC class II, making it unlikely that ligation of CD14 enhanced APC function. To further exclude this possibility, we tested if coadministration of a CD14-specific Ab without the mouse C{kappa} epitope and a nonspecific Ab with the epitope could enhance MHC class II presentation of the T cell epitope to the same extent as the CD14-specific Troybody alone (Fig. 5 ). A CD14-specific, recombinant Ab, without the C{kappa} epitope and with a human constant {kappa} L-chain ({alpha}CD14.wt), failed to induce T cell proliferation. These results show that coadministration of {alpha}CD14.wt and the control peptide Ab L6-C{kappa} did not result in any proliferative responses, whereas the {alpha}CD14.L6-C{kappa} Troybody did (Fig. 5) . This result demonstrates that V regions targeting CD14 and the C{kappa} epitope need to be physically linked in the Troybody for T cell responses to be enhanced, which strongly suggests that the CD14-specific Troybody is channeled into the MHC class II loading compartment upon ligation of CD14.



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  		Figure 5. The targeting V regions and the T cell epitope need to be physically linked to enhance specific T cell stimulation. A proliferation assay was performed with irradiated PBMC as APC and C{kappa}-specific CD4+ T18 cells as responder cells. Titrated amounts of {alpha}CD14.L6-C{kappa} ({blacksquare}), {alpha}CD14.wt ({diamond}), nontargeted mAb L6-C{kappa} ({blacktriangledown}), or a combination of {alpha}CD14.wt and L6-C{kappa} ({circ}) were added to the cell culture. Proliferation was measured as 3[H]dThd incorporation.


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DISCUSSION
 
We have targeted a PRR of the innate immune system, CD14, and shown that mAb bound to CD14 is channeled into the MHC class II presentation pathway for stimulation of mouse C{kappa}-specific/DR4-restricted human CD4+ T cells. It has previously been shown that targeting other PRRs, such as TLR2 [7 ], mannose receptor [3 , 4 ], and DC-SIGN [5 , 6 ], leads to antigen uptake and presentation to T cells. Collectively, these results suggest that PRRs may be excellent targets for Ab-mediated delivery of T cell epitopes to APC for elicitation of T cell responses.

Natural ligands for PRRs may up-regulate expression of costimulatory molecules and secretion of IL-12 by APC, as shown for TLR2 [38 , 39 ]. Thus, for natural ligands, it is difficult to distinguish two factors that may contribute to more potent T cell activation: increased APC function and increased peptide loading of MHC class II molecules. The use of nonsignaling Ab makes it possible to distinguish between the two: If a peptide from a nonsignaling mAb is presented on MHC class II, it means that engagement of the PRRs on APC results in increased MHC class II presentation even in the absence of signaling through the receptor. We have previously shown this to be the case for TLR2 and demonstrate here that this also applies to CD14.

The present results show that ligand bound to CD14, in this case, a mAb, is channeled into the MHC class II presentation pathway. Consistent with this, monocytes have been shown to phagocytose Gram-negative bacteria by a CD14-dependent pathway [17 ], and CD14 is internalized rapidly to the Golgi area upon binding of 3C10 mAb [40 ]. It has also been shown that LPS is internalized via CD14-dependent mechanisms [41 ].

CD14 is part of a LPS receptor complex that includes MD-2 and TLR4. The LPS receptor complex was found to recycle rapidly between the Golgi and the plasma membrane [40 ]. It was therefore surprising that although CD14 was an efficient target for entry into the MHC class II pathway, TLR4 was apparently not. The poor capacity of the TLR4 mAb to induce proliferation of C{kappa}-specific T cells could have technical reasons or could indicate that TLR4 is a poor target for Ab-based vaccines. As for technical reasons, the particular anti-TLR4 mAb (HTA125) used could have insufficient specificity or affinity for TLR4 for processing and presentation to be revealed. Supporting such an argument, it has previously been demonstrated that various mAb specific for DC-SIGN differed in their abilities to elicit C{kappa}-specific T cell responses [6 ]. Thus, it is possible that other mAb specific for TLR4 could be better processed and presented than the HTA125 mAb. More likely, TLR4 may be a poor target as a result of its low expression on surfaces of APC. We have confirmed here that TLR4 is expressed only at low levels on the surface of monocytes [42 , 43 ] but not at all on DC, where TLR4 has been reported to have an intracellular location [44 ]. Moreover, Sabroe and coworkers [43 ] have shown that there is ~10 times more CD14 expression than TLR4 expression on the surface of monocytes. Consistent with the poor cell-surface expression of TLR4, it has been observed that only CD14 and not TLR4 functions as an endocytic receptor for LPS [45 ]. These previous and present results could suggest that TLR4 is not a suitable target for Ab-based vaccines, although it is clearly indispensable as a receptor for LPS [10 ]. This conclusion contrasts our previous findings that TLR2 might be a good target [7 ]; however, TLR2 is expressed at a much higher level on cell surfaces of APC than is TLR4 [43 ].

Bivalent, anti-CD14 mAb could cross-link CD14 on the cell surface, which could increase the responses. This might also be the case for natural ligands for CD14-like LPS, which could be part of larger bacterial fragments having many LPS molecules. Moreover, if the latter were the case, peptides from proteins in the LPS-containing bacterial fragments could be the donors of piggybacking bacterial peptide sequences. Thus, binding of bacterial fragments, via CD14, could result in bacterial peptides being presented on MHC class II molecules. Thus, when pathogen-associated molecular patterns bind to corresponding PRRs and elicit innate inflammatory-immune responses, adaptive CD4+ T cell responses may follow in the wake [7 ].

It has been suggested that CD14 serves as a receptor on monocytes for apoptotic cells [16 ], although the phosphatidylserine receptor may be of overriding importance [46 ]. If CD14 contributes to phagocytosis of apoptotic cells, the present results suggest that peptide sequences from apoptotic cells could be presented by MHC class II molecules and perhaps be cross-presented on MHC class I molecules, although the latter remains to be demonstrated. The present human Troybody is targeted to CD14, which is highly expressed on monocytes and macrophages, but less so on DC. Moreover, when monocytes develop into moncocyte-derived DC, the CD14 expression decreases [31 ] (Fig. 1A) . However, monocytes are thought of as predecessors of DC [31 , 47 ]. Thus, targeting CD14 on monocytes may be advantageous for efficient endocytic loading of cells, which later, as DCs, efficiently display processed peptides on MHC class II molecules on the cell surface for T cell recognition.

When tested in the same experiments, the 3C10 mouse Ig was more potent than the CD14-specific Troybody on a molar basis. There may be several reasons for the difference in potency between the natural 3C10 mAb and the recombinant CD14-specific Troybody. First, it may be caused by the new context of the transplanted C{kappa} T cell epitope. In the 3C10 Ab, the T cell epitope is situated in the constant part of mouse {kappa} L-chain, whereas in the Troybody, it is situated in the constant part of a human H-chain. The difference between the two Ab may reflect a distinction in the processing of different domains of the Ig and thus, the generation of the antigenic C{kappa} peptide for binding to DR4. Second, as the Troybody only contains the minimal length 9 aa (P1–P9) transplanted into CH1, the changes in the residues flanking the epitope could influence the accessibility to proteases involved in antigen processing [48 , 49 ]. Peptides eluted from MHC class II molecules often have a length of 13 aa or more, which is longer than the transplanted 9 aa C{kappa} minimal epitope. Thus, the flanking regions of C{kappa} in its position in CH1 might negatively influence its binding to DR4 or its stimulatory capacity for T cells. Finally, the difference in potency may be caused by differences in FcR-mediated uptake of 3C10 (mouse IgG2b) and {alpha}CD14.L6-C{kappa} Troybody (human IgG3).

It was thought previously that peptides, which correspond to T cell epitopes, could represent ideal subunits for safe vaccines. However, synthetic peptides have proved to be poor immunogens because of their small molecular size and short serum half-life. A more fruitful strategy might be recombinant proteins, including Ab, which carry integrated T cell epitopes [50 51 52 53 ]. In addition, targeting antigens to APC has been shown to increase immune responses [18 , 19 , 54 ]. To unify these concepts, we previously constructed recombinant mAb (Troybodies) with V regions specific for APC and T cell epitopes inserted into the C region. Troybodies in the mouse target APC and efficiently stimulate T cells in vitro and in vivo [22 23 24 25 ]. We have constructed here the first Troybody for human application. The enhanced stimulatory effect of CD14-specific Troybody was indeed dependent on CD14-specific V regions and integration of the T cell epitope in the C region, linked within the same molecule. Our results suggest that the Troybody strategy can also be applicable in humans. However, as the present CD14-specific Troybody was a chimeric molecule with mouse V regions and human C regions, a fully human Troybody should be constructed. In addition, the T cell epitope should be relevant to infectious or cancerous diseases in humans and not a model T cell epitope like the one used here.

The Troybody technology, here extended for human applications, offers flexibility that could be useful in the field of vaccine development. First, it allows design of reagents that target T cell epitopes of choice to any selected cell-surface molecule, such as CD14, on APC. Second, the strategy should allow the introduction of many different T cell epitopes into different loops of the same Ab so that a multivaccine can be generated.


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ACKNOWLEDGEMENTS
 
This work was funded by The Research Council of Norway and The Norwegian Cancer Society. We are grateful to Morten Flobakk, Hilde Omholt, Dr. Keith Thompson, and Dr. Elin Lunde for helpful discussions and advice.

Received August 30, 2004; revised November 5, 2004; accepted November 7, 2004.


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