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The Journal of Immunology, 2002, 169: 2337-2345.
Copyright © 2002 by The American Association of Immunologists

Enhancement of MHC Class II-Restricted Responses by Receptor-Mediated Uptake of Peptide Antigens1

Lolita Zaliauskiene{ddagger}, Sunghyun Kang{ddagger}, Kerri Sparks{ddagger}, Kurt R. Zinn{dagger}, Lisa M. Schwiebert{ddagger}, Casey T. Weaver* and James F. Collawn2,{ddagger}

Departments of * Pathology, {dagger} Radiology, and {ddagger} Cell Biology and the Comprehensive Cancer Center, University of Alabama, Birmingham, AL 35294


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Peptides, either as altered peptide ligands, competitors, or vaccines, offer an outstanding potential for regulating immune responses because of their exquisite specificity. However, a major problem associated with peptide therapies is that they are poorly taken up by APCs. Because of poor bioavailability, high concentrations and repeated treatments are required for peptide therapies in vivo. To circumvent this problem, we tested whether covalently coupling a peptide T cell determinant, OVA323–339, to transferrin (Tf) enhances APC uptake and presentation as monitored by Th cell activation. Functional analysis of the Tf-peptide conjugates revealed that the conjugates were presented 10,000- and 100-fold more effectively by B cells than intact Ag and free peptide, respectively. Furthermore, we demonstrate that the Tf-peptide conjugates are taken up by B cells through a receptor-mediated process and subsequently delivered to the lysosomal compartment. Using an adoptive transfer assay, we show that that the Tf-peptide complexes are 100-fold more effective in vivo than the free peptide in activating CD4+ T cells by following an early activation marker, CD69. Our results demonstrate that coupling peptides to Tf enhances peptide presentation, thereby making it possible to take full advantage of peptide-specific therapies in modulating T cell responses.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Major histocompatibility complex class II molecules bind peptide Ags and present them to Th cells, thereby promoting immune responses. The peptides are derived from proteolytically processed Ags that enter the cell through the endocytic pathway (reviewed in Ref. 1) by pinocytosis or via cell surface receptors (2, 3). The peptides bind to newly synthesized MHC class II {alpha}-{beta} chains before delivery to the cell surface for presentation to Th cells (reviewed in Ref. 4).

MHC class II molecules bind a number of peptides, including peptides derived from self proteins. Discrimination between self and foreign-derived peptides forms the basis for Th cell recognition of foreign Ags. A breakdown in this process results in autoimmune disease. In experimentally induced organ-specific models of autoimmune diseases, it is clear that pathogenic responses can be prevented by the administration of self-Ag (5, 6, 7). However, the ability of self Ags to compete for MHC class II binding and presentation is limited if uptake or internalization of the Ag only occurs through pinocytosis. Allen and coworkers (8) first demonstrated that Ag processing and presentation is improved by modifying the Ag so that it can be taken up efficiently via receptor-mediated endocytosis. They demonstrated that mannosylation of an Ag promoted Ag uptake through the mannose receptor, and was not inhibited by normal serum proteins like the native Ag was (8). This study suggested that efficient Ag uptake and presentation by receptor-mediated endocytosis enhances Th cell responses.

Once it became clear that susceptibility to the various autoimmune diseases was strongly associated with certain MHC class II molecules and the autoantigenic self peptides were identified, it became readily apparent that peptides could be used to block (9, 10, 11, 12) or modify (13, 14, 15, 16, 17, 18, 19, 20) T cell responses. For modulation of Th cell responses, peptides offer several advantages over intact Ags as immunogens or tolerogens. First, peptides require less stringent degradative conditions than native Ags (21). Second, with a smaller determinant, there is less likelihood of cross-reactivity between the peptide and other self-proteins. And third, peptides offer exquisite specificity over native Ags. Despite these advantages, the use of peptides has remained fairly limited, because they are rapidly cleared from the circulation (11) and poorly taken-up by APCs (22).

To increase the effectiveness of peptide presentation, investigators have coupled peptides to ligands specific for cell surface receptors found on the APCs (8, 23, 24). In most cases, Ag presentation was improved 10- to 100-fold in vitro. Abs have been the most common reagent for transporting peptides, and have been tested in vivo as well (25, 26); however, Abs have the potential for toxic side-effects (27), which may limit their use in the treatment of human autoimmune diseases.

Because internalized transferrin (Tf)3 receptors (TR) have been shown to intersect with newly synthesized MHC class II molecules in the biosynthetic pathway, they could potentially be used to deliver peptide Ags into the APC. In the present report, we tested how coupling peptide Ags to Tf affected Ag presentation to T cells both in vitro and in vivo. We used the OVA323–339 peptide as our model Ag and coupled it to Tf using a chemical cross-linker. Testing the Tf-OVA peptide conjugates in T cell activation assays revealed that the conjugates were over 10,000-fold more effective than intact Ag in vitro and ~100-fold more effective than soluble peptide both in vitro and in vivo. These findings suggest that coupling peptides to Tf dramatically enhances peptide presentation, thereby making peptide-directed strategies for enhancing or suppressing immune responses now feasible.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice and cell lines

DO11.10 TCR transgenic mice on a BALB/c background (28, 29) were bred in a specific pathogen-free facility and were screened at 3–4 wk of age for transgene expression by two-color flow cytometric analysis after staining of peripheral blood with anti-CD4 and the anti-clonotypic mAb, KJ1-26 (30). BALB/c mice were purchased from Frederick Cancer Research and Development Center (Frederick, MD) or bred in our facility (University of Alabama, Birmingham, AL). The OVA-specific T cell hybridoma DO-BW was kindly provided by Dr. O. Kanagawa (Washington University, St. Louis, MO) and grown in DMEM (Mediatech, Herndon, VA) supplemented with 2 mM glutamine, antibiotics, and 10% FBS. A20 B cells were provided by Dr. J. F. Kearny (University of Alabama) and grown in RPMI 1640 (Mediatech) with 2 mM glutamine, antibiotics, and 10% FBS. The IL-2-dependent T cell lines CTLL-2 and HT-2, as well as human cervix carcinoma-derived HeLa cells, were obtained from American Type Culture Collection (Manassas, VA).

Reagents

Human Tf and OVA were purchased from ICN Pharmaceuticals (Costa Mesa, CA) and Sigma-Aldrich (St. Louis, MO), respectively. Peptides were synthesized by New England Peptide (Fitchburg, MA). The amino acid sequences of the peptides were ISQAVHAAHAEINEAGR (OVA); CSSAESLKISQAVHAAHAEINEAGR (cathepsin S cleavable peptide (CS)-OVA); CGAGAGAGISQAVHAAHAEINEAGR (noncleavable peptide (NC)-OVA); and CGAGAGAGEQKLISEEDL (myc-tagged NC linker (NC-myc)).

Preparation of Tf conjugates

Tf-oval conjugates were prepared using the heterobifunctional reagent succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) (Pierce, Rockford, IL). Tf (5 mg) was reacted with 20-fold molar excess of SMCC in 50 mM HEPES buffer (pH 7.4) for 1 h at room temperature. SMCC-modified Tf was separated from unreacted cross-linker by gel filtration on a Sephadex G-50 (Sigma-Aldrich) column equilibrated in 50 mM HEPES buffer (pH 7.4). Ovalbumin (20 mg) was thiolated with 2-iminothiolane (Pierce) at a 1:20 molar ratio in 50 mM HEPES buffer (pH 7.4) for 1 h at room temperature under nitrogen. SMCC-modified Tf was then reacted with thiolated ovalbumin for 3 h at room temperature and stored at 4°C overnight. Cross-linked products were separated by gel filtration chromatography on a Sephacryl S-200 column (Amersham Pharmacia Biotech, Piscataway, NJ) equilibrated in PBS (pH 7.4). The Tf-oval conjugates were further divided into two pools: I (molecular mass ~1000–440 kDa) and II (molecular mass ~440–120 kDa) based on the elution of molecular mass standards under native conditions. Total protein concentrations were determined using the Bradford assay (31). Tf concentrations were determined by absorbance at 595 nm. The molar ratios of Tf to ovalbumin for pool I and II were 1:1.1 and 1:1.6, respectively.

Tf-peptide conjugates were prepared using the SMCC cross-linker. Tf (10 mg) was reacted with 5- to 30-fold molar excess of SMCC in 50 mM HEPES buffer (pH 7.4) for 1 h at room temperature. SMCC-modified Tf was purified by gel filtration. CS-OVA, NC-OVA, or myc-tagged linker peptides were added to SMCC-modified Tf at the same molar ratio as was used with the cross-linker. The reactions were incubated overnight at room temperature. Reaction products were separated by gel filtration and the number of cross-linkers and/or peptides coupled to Tf was determined by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry.

Dimeric Tf (dTf) and CS-OVA were cross-linked using SMCC as described above. dTf was prepared by reacting Tf (30 mg/ml) with a 10-fold molar excess of glutaraldehyde (Sigma-Aldrich) in 50 mM PO4, pH 7.4, for 1 h at room temperature with gentle mixing. The reaction was quenched with the addition of lysine (0.1 M). The product was dialyzed against PBS and purified by gel filtration chromatography. The purity was determined by mass spectroscopy.

Preparation of deglycosylated apotransferrin (apoTf)

apoTf (5 mg) was treated with N-glycanase (50 mU; Glyco, Novato, CA) at 37°C overnight. N-glycanase was removed by concentration of the sample (and repeated dilutions in PBS) using a Centricon 50 filter (Millipore, Bedford, MA). To confirm that the N-linked carbohydrates had been removed from the protein, the conjugated material was tested using mass spectroscopy before and after the enzymatic digestion. The difference in size was ~5 kDa, consistent with the absence of two N-linked oligosaccharides (32).

MALDI-TOF mass spectroscopy

dTf or Tf-OVA peptide conjugates were analyzed in the positive mode on a Voyager Elite mass spectrometer with delayed extraction technology (PerSeptive Biosystems, Framingham, MA). The acceleration voltage was set at 25 kV and 5–100 laser shots were summed to obtain average mass spectra. Sinapinic acid (Aldrich, Milwaukee, WI) dissolved in acetonitrile/0.1% trifluoroacetic acid (1/1) was used for the matrix. Samples were either diluted 1/10 or 1/1 with matrix and 1 µl was plated onto a smooth plate. The mass spectrometer was calibrated with BSA (Sigma-Aldrich).

Ag presentation assay

DO-BW (105) and A20 (105) cells were cultured in 0.2 ml of medium at 37°C in the presence of the indicated concentrations of Ag. After 24 h, the supernatant was removed and tested for IL-2 concentration using IL-2-dependent cell lines, CTLL-2 or HT-2. Briefly, HT-2 cells (5 x 103) were cultured with 50 µl of the supernatants for 48 h at 37°C; 1 µCi of [3H]thymidine was added during the last 24 h of incubation. Cells were harvested and counted in scintillation counter (Beckman Coulter, Fullerton, CA) for [3H]thymidine incorporation.

Tf competition assay

DO-BW cells (105) and A20 cells (105) were cultured with either 5 µM of OVA peptide or 0.5 µM of Tf-CS-OVA conjugates in the presence of various concentrations of Tf. After 24 h, supernatants were collected and added to CTLL-2 cells as described above. Background counts were determined by measuring [3H]thymidine incorporation without supernatant addition.

Scatchard analysis

HeLa cells were plated at a density of 1.0 x 105 cells/well in 24-well tissue culture plates 24 h before the assay. Cells were washed once with serum-free DMEM, then incubated in serum-free DMEM for 1 h at 37°C. 125I-labeled Tf or Tf-OVA in 0.1% BSA in PBS was added to triplicate wells at concentrations ranging from 4 to 0.3 µg/ml and incubated at 0°C for 60 min. Cells were washed four times with ice-cold 0.1% BSA in PBS, lysed with 1 M NaOH, and counted.

Proteolysis assay

Proteolysis of radiolabeled Tf or Tf conjugates was determined as described previously (33).

Indirect immunofluorescence

A20 cells were preincubated in serum-free medium (RPMI 1640) for 1 h and then incubated for 2 h in the presence of 100 µM of free Tf or Tf-NC-myc conjugate. Cells were washed and transferred onto microscope slides using a cytospin centrifuge. Double-label indirect immunofluorescence was performed as described previously (33). mAb 1D4B specific for mouse lysosome-associated membrane protein (LAMP)-1 was purchased from BD PharMingen (San Diego, CA). MHC class II-specific mAb (clone NIMR-4) was obtained from Southern Biotechnology Associates (Birmingham, AL). Myc tag-specific rabbit polyclonal IgG Ab c-Myc (A-14) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). mAb specific for human Tf was purchased from Serotec (Oxford, U.K.).

Imaging was performed on a Leica DMIRBE inverted epifluorescence/Nomarski microscope outfitted with Leica TCS NT laser confocal optics (Exton, PA). Argon and krypton lasers (488 and 568 nm laser lines, respectively), with double-dichroic DD 488/568 nm and emission band passes of 500–550 nm (for green fluorochrome) and 596–722 nm (for red fluorochrome), were used. Samples were imaged by simultaneous scanning. Optical sections (0.5 µ each) through the z-axis were generated using a stage galvanometer. Flattened maximum projections of image stacks were prepared using TCS NT confocal imaging software (Leica).

Adoptive transfer

CD4+ Th cells expressing OVA peptide323–339-specific TCR were purified from DO11.10 SCID transgenic mice spleens and lymph nodes by magnetic sorting using Dynabead mouse CD4+ (Dynal Biotech, Oslo, Norway), and labeled with 1 µM of the intracellular fluorescent dye, CFSE (Molecular Probes, Eugene, OR). Labeled CD4+ T cells (2–3 x 106 cells/animal) were resuspended in 100 µl of DMEM and injected i.v. into BALB/c recipients. Ag (free OVA or Tf-OVA) was administered 24 h later through the tail vein. Spleens were recovered 24 h following Ag injection and analyzed by flow cytometry (FACS) after staining for an early T cell activation marker CD69 (Caltag Laboratories, Burlingame, CA), and examining its expression in a CFSE-positive pool of T cells.

Biodistribution of 99mTc-labeled peptides and Tf-peptide conjugates

99mTc radiolabeling of peptides and Tf-peptide conjugates was accomplished with succinimidyl 6-hyrazino nicotinate hydrochloride (HYNIC). HYNIC was first conjugated to CS-OVA or NC-OVA peptides according to Abrams et al. (34) using a 2:1 molar ratio of HYNIC to peptide (2 h at room temperature), followed by purification on a C18 reverse-phase HPLC column (Vydac, Hesperia, CA). The halves of HYNIC-modified peptides were coupled to Tf as described above. 99mTc was then chelated to the HYNIC-modified peptides or the HYNIC-modified peptide-Tf conjugates with tricine as the transchelator using a previously published method (35). Radiochemical purity of the samples was tested by thin layer chromatography (36). 99mTc-labeled peptides and conjugates were introduced to BALB/c mice via tail vein injection. After 22 h, the mice were sacrificed and the radioactivity in different organs was measured by a gamma counter.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Preparation of Tf-oval and Tf-OVA peptide conjugates

To determine whether coupling an Ag to a carrier molecule would enhance Ag presentation by B cells to Th cells, we prepared a number of Ag complexes. We used Tf as our carrier protein and tested different forms of the model Ag OVA (Table IGo). We compared monomeric and dTf along with native OVA and peptides containing the OVA fragment 323–339. We wanted to test dTf because it has been previously established that cross-linking Tf alters the trafficking of the TR (37). The Ag complexes were prepared using the chemical cross-linker SMCC (see Materials and Methods).


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  		Table I. OVA ligands tested in this studya

 
The Tf-OVA conjugates were purified on a Sephacryl S-200 gel filtration column. The conjugates eluted as a single peak (fractions 12–20) with a molecular mass range of ~1000 to ~120 kDa based on the elution profile of molecular mass standards (blue dextran 2000 (2000 kDa), thyroglobulin (669 kDa), {alpha}-2 macroglobulin (725 kDa), ferritin (440 kDa), monomeric Tf (80 kDa), and OVA (45 kDa) under native conditions; data not shown). The Tf-OVA conjugates were divided into two pools, pool I (the molecular mass range of 1000–440 kDa) and pool II (molecular mass range, 440–120 kDa). The molecular mass estimations based on elution profiles were also confirmed by SDS-PAGE (data not shown).

For the Tf-OVA peptide conjugates, the OVA323–339 determinant was synthesized with an amino-terminal leader sequence that included a cysteine residue for coupling to the SMCC cross-linker and a seven-amino acid linker sequence that contains a cathepsin S site found in the native ovalbumin sequence, SSAESLK (Tf-CS-OVA, Table IGo). The Tf-OVA peptide conjugates eluted as single peaks at a molecular mass of ~82 kDa. Each of the conjugates was analyzed using mass spectroscopy to estimate the extent of modification. The peptide-Tf coupling ratio was approximately one peptide per Tf monomer (data not shown). To confirm that the complexes were being taken up via a receptor-mediated pathway, deglycosylated apoTf (iron-free form) was used as a negative control, because removal of the two N-linked oligosaccharides in apoTf reduces the ability of the apoTf to take-up iron and bind to the TR (32).

Tf-peptide conjugates are efficiently presented by A20 B cells

The efficiency of Ag uptake and processing in A20 B cells was tested using the different forms of OVA. A20 B cells and OVA-specific DO-BW T cell hybridoma cells were cocultured with graded concentrations of intact OVA, OVA coupled to Tf (Tf-oval pools I and II; Fig. 1GoA). We also compared free peptide (OVA), peptides coupled to monomeric Tf (Tf-CS-OVA) and deglycosylated apoTf (apoTfde-OVA; Fig. 1Go, B and C). DO-BW activation was measured by production of IL-2 (see Materials and Methods). Surprisingly, the results demonstrated that coupling oval to Tf (pools I and II) lowered the ED50 by <10-fold. Tf-CS-OVA conjugates were >10,000-fold (ED50 of ~0.0004 µM) and free OVA peptides were >100-fold (ED50 of ~0.04 µM) more effective than intact Ag. More importantly, the Tf-peptide conjugates were 100-fold more effective than free peptide. The results indicated that the form of Ag that was presented most efficiently was the Tf-peptide conjugates.



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  		FIGURE 1. T cell activation in response to Tf-OVA conjugates and Tf-OVA peptide conjugates. DO-BW T cells and A20 B cells were cultured together in the presence of native ovalbumin, Tf-OVA conjugates (A), or various Tf-OVA peptide conjugates (B and C). Tf-oval I and Tf-oval II are Tf-oval conjugates at two different molecular mass ranges (see Materials and Methods). Tf-NC-OVA and Tf-CS-OVA are Tf coupled to the OVA323–339 peptide containing a linker with a NC sequence or with a CS cleavage site, respectively. apoTfde-NC-OVA is deglycosylated apoTf coupled to OVA containing a NC linker sequence (see Table IGo for sequences). After 24 h, the supernatants were removed and the IL-2 production was measured by [3H]thymidine incorporation using the IL-2-dependent cell line, HT-2. A representative experiment of three is shown in each panel.

 
A second surprise was that the dTf (dTf-CS-OVA, Table IGo) was ~10-fold less effective than monomeric Tf for Ag delivery and presentation (data not shown), suggesting that cross-linking the receptor was not required for efficient presentation of the OVA T cell determinant. Although this result could be due to modifications of the dTf binding after glutaraldehyde fixation, the results did demonstrate that making Tf divalent certainly did not enhance delivery of the peptide cargo. Deglycosylated apoTf-NC-OVA conjugates were less effective than the free peptide (ED50, ~0.8 µM), indicating that the enhancement of presentation was not due to a nonspecific protein carrier effect. We performed two additional control experiments. In the first, we compared the native OVA determinant (residues 323–339) to the OVA peptides that included a leader sequence (both the NC-OVA and CS-OVA (Table IGo)) to insure that the leader sequence was not influencing peptide uptake and presentation. The results indicated that all three peptides were identical in their capacity to activate Th cells using A20 APCs (data not shown). In the second experiment, we compared OVA323–339 alone and OVA323–339 along with free Tf to confirm that the peptide needed to be coupled to Tf to see the enhanced presentation. The results indicate that the addition of the free Tf had no effect on presentation of OVA323–339 peptide (Fig. 1GoB), suggesting that the enhanced presentation of the OVA determinant required that it was coupled to the Tf ligand.

Tf-peptide conjugates with cathepsin S sites are more effectively presented to Th cells in vitro

To determine whether the cathepsin S proteolytic site in CS-OVA was important for peptide release, OVA peptides containing CS-OVA or NC-OVA were coupled to Tf and tested in T cell activation assays (Fig. 1GoC). Both Tf-CS-OVA and Tf-NC-OVA conjugates were modified to similar degrees, approximately two peptides per Tf molecule. The results show that conjugates containing the cathepsin S site were ~10-fold more effective than the Tf-NC-OVA conjugate. Because cathepsin S is important for processing of the MHC class II invariant chain and peptide loading (38), this finding suggests that the inclusion of this site in the linker sequence facilitated peptide release in a processing compartment. However, when the Tf-NC-OVA and Tf-CS-OVA conjugates were radiolabeled with 99mTc to determine the distribution of both conjugates in the body (see Materials and Methods), the NC Tf conjugate was more effectively delivered to mouse spleen and lymph nodes. Initial analysis of the Tf-peptide conjugates in vivo indicated that the CS-conjugate was released in the blood stream (data not shown). Therefore, because of this result, the NC Tf-OVA conjugate was used in all the in vivo experiments.

Uptake of the Tf-peptide conjugates occurs through the TR

To determine whether the Tf-peptide conjugates were taken up via the TR, we tested the ability of excess free Tf to block uptake and presentation of Tf-CS-OVA conjugates by A20 B cells. In this experiment, we cocultured A20 B cells and DO-BW T cells in the presence of either 5 µM of uncoupled OVA peptide (OVA323–339) or 0.5 µM Tf-CS-OVA conjugate along with increasing concentrations of free Tf. The results shown in Fig. 2Go indicate that in the presence of 100-fold excess of free Tf (50 µM), tritiated-thymidine uptake by the IL-2-dependent cell line CTLL-2 was lowered by 67%, suggesting that IL-2 production was dramatically decreased. In contrast, free Tf did not interfere with the activation of DO-BW using the uncoupled OVA. In fact, Tf stimulated IL-2 production in the presence of the OVA peptide, supporting the idea that Tf is required for T cell activation (39). Because the serum concentration of saturated Tf is ~6.5 µM (40), much lower than the concentrations we are testing in this study, this result suggests that the Tf-peptide conjugates would be effectively taken-up and presented in vivo even in the presence of the normal levels of endogenous Tf. The results provide strong evidence that the Tf-peptide conjugates are taken up by receptor-mediated endocytosis because Tf inhibited CTLL-2 growth in a dose-dependent manner.



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  		FIGURE 2. Uptake of Tf-OVA peptide conjugates occurs through the TR. DO-BW cells and A20 cells were cultured with 5 µM of OVA323–339 peptide or 0.5 µM of Tf-NC-OVA conjugate in the presence of various concentrations of native Tf (0.1–50 µM). The supernatants were assayed 24 h later for IL-2 content using HT-2 T cells as described in Fig. 1Go. The presence of increasing amounts of free Tf in the culture inhibited stimulation of DO-BW cells by Tf-OVA conjugates.

 
Tf-coupled peptides are delivered to the Ag processing compartment

To verify that peptides were targeted to the lysosome, we coupled a myc-tagged linker peptide to Tf and determined whether it was delivered to the lysosomal compartment where processing occurs. Because an Ab to the OVA323–339 determinant is not available, we followed the fate of a chemically cross-linked myc peptide in the A20 B cells. We used the same NC linker as was used in the Tf-NC-OVA conjugate because we wanted the peptide to remain associated with the Tf for as long as possible and avoid potential processing effects (Table IGo). The Tf-NC-myc conjugate was added in serum-free medium to A20 B cells for 2 h at 37°C, and the cells were prepared for confocal immunofluorescence analysis (see Materials and Methods). Free Tf was compared as a negative control. To establish that the Tf-peptide conjugates were being delivered to the prelysosomal/lysosomal processing compartment, we first tested whether the intracellular MHC class II molecules colocalized with LAMP-1, a marker for the processing compartment. The results shown in Fig. 3Go (top panels) demonstrate significant overlap (shown in yellow) and are consistent with the morphology of the LAMP-1 compartment in A20 cells seen by others (41). However, native Tf does not overlap with either MHC class II (Fig. 3Go, second row) or with LAMP-1 (third row), supporting the idea that the TR is only found in the early endosomal compartments. However, the Tf-NC-myc peptide conjugates show partial colocalization with markers of the later stages of the endocytic pathway, MHC class II (Fig. 3Go, fourth row) and LAMP-1 (fifth row). These results are similar to those of Maxfield and coworkers (37) who demonstrated that oligomerization of Tf altered the normal trafficking of the TR within the cell. To establish that the myc epitope was in the same compartment as the carrier Tf molecule, we also compared the distribution of the myc and Tf in the same cells, and found that they completely colocalized, suggesting that the peptide modification resulted in altered trafficking of the Tf ligand (data not shown). The data confirms that native Tf is delivered to the early stages of the endocytic pathway, whereas modified Tf is delivered to the lysosomal compartment.



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  		FIGURE 3. Tf-peptide conjugates are delivered to the MHC class II/LAMP-1+ compartment, whereas native Tf is not. A20 B cells were incubated with 50 µM of native Tf or Tf-NC-myc peptide for 2 h. Cells were fixed, permeabilized, and labeled with FITC-conjugated rat anti-mouse LAMP Ab 1D4B and rat anti-mouse MHC class II Ab (PE- or FITC-conjugated). Tf and Tf-myc localization was detected using either sheep anti-Tf Ab followed by Texas Red-labeled rabbit anti-sheep Ab or rabbit anti-myc antisera followed by Texas Red-labeled goat anti-rabbit. Colocalization of two proteins is indicated by yellow fluorescence.

 
Increasing the number of peptides bound per Tf molecule does not enhance T cell responses

Our results suggest that peptide Ags coupled to monomeric Tf were the most effective form of Ag presented by B cells to Th cells that we tested. In our first series of Tf-peptide conjugates, we specifically limited the coupling ratio to one peptide per Tf molecule. In the next series, we wanted to determine whether increasing the peptide coupling ratios affected T cell responses. To do this, we prepared a second set of Tf-CS-OVA conjugates with varying amounts of SMCC cross-linker and CS-OVA peptides. After each modification, the products were analyzed using MALDI-TOF mass spectrometry to assess the extent of coupling. Representative mass spectroscopic analyses are shown in Fig. 4GoA. In this analysis, the molecular mass of unmodified Tf was 78.8 kDa (Fig. 4GoA, upper panel). The molecular mass estimations appeared to vary <± 0.1 kDa. Next, Tf was modified with 5-fold molar excess of SMCC cross-linker and purified by gel filtration. Analysis of the modified Tf showed an increase in molecular mass of ~0.8 kDa (Fig. 4GoA, middle panel), which corresponds to an average of 2.3 cross-linkers bound per Tf molecule. This material was then reacted with 5-fold molar excess of CS-OVA peptide, and the gel-purified product was analyzed by mass spectroscopy (Fig. 4GoA, bottom panel). The major peak corresponded to a molecular mass of 84.7 kDa, which represents a Tf monomer with two peptides coupled to it. The presence of conjugates having no peptides (79.3 kDa), one (82.0 kDa), three (87.4 kDa), and four (90.1 kDa) was also detected in this sample. The mixture was not further purified, and in this example, the average number of peptides bound was taken to be approximately two peptides per Tf molecule.



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  		FIGURE 4. A, Mass spectroscopic analysis of Tf and Tf-peptide conjugates. Mass spectrometry analysis on Tf (top panel), Tf modified with SMCC (middle panel), and Tf-CS-OVA conjugates (bottom panel). Tf was modified with 5-fold molar excess of CS-OVA peptide (as described in Materials and Methods). Samples were taken from each step and analyzed in a MALDI-TOF mass spectrometer. The increase in mass showed that Tf was coupled with an average of 2.3 SMCC cross-linkers (middle panel) and 0, 1, 2, 3, or 4 CS-OVA peptides (bottom panel). In the bottom panel, the average coupling ratio was two peptides per Tf molecule. B, The coupling efficiency of cross-linkers and peptides to Tf. Tf was modified with 5-, 10-, 20-, or 30-fold molar excess of SMCC cross-linker and then incubated with corresponding amounts of CS-OVA peptide. The number of SMCC cross-linkers or peptides conjugated on Tf was assessed by MALDI-TOF mass spectroscopy as described in Materials and Methods.

 
Using this type of approach, we examined the coupling efficiency of the cross-linkers and peptides at different molar ratios using mass spectroscopy. Our goal was to determine the maximal number of peptides that could be effectively coupled to Tf. The results shown in Fig. 4GoB indicate that the number of cross-linkers added increased in a linear manner. Tf modified with 5-, 10-, 20-, or 30-fold molar excess of SMCC cross-linker coupled with an average of 3.6, 7.5, 15, or 24 cross-linker per Tf molecule, respectively, indicating that the cross-linker coupling efficiency was ~75%. Each SMCC-modified Tf was then reacted with an ~40% molar excess of CS-OVA peptide and the maximum number of peptides coupled to Tf leveled off at 9–10 peptides per monomer, suggesting that this may represent a practical limit for the coupling procedure. Though Tf has total 55 lysine residues (42), our results suggest that only a portion of those residues was accessible to the CS-OVA peptides. The results also implied that the cross-linkers (molecular mass ~334 Da) had better accessibility to the lysine residues than the larger peptides (molecular mass = 2580 Da).

After establishing the effective coupling ratios for the peptides, we prepared samples of Tf-peptide conjugates that averaged 1, 2.5, 5, and 8 peptides per Tf monomer, and tested them using an assay to determine the relative amount of Tf delivered to the lysosomal compartment (33). This assay necessitated the use of adherent cells, that were TR-positive, rather than A20 B cells. HeLa cells were incubated with 125I-labeled Tf-CS-OVA conjugates or native Tf at 37°C for 1 h to load the endocytic pathway with receptor-ligand complexes. The cells were then rapidly washed, and the reappearance of intact and degraded Tf-CS-OVA conjugates or Tf in the medium was monitored by measuring the TCA insoluble and soluble radioactivity. As shown in Fig. 5Go, the apoTf released into the medium was intact native protein, with only 1.8% in the TCA soluble pool. This suggested that only a small percentage of the Tf was delivered to the late endosomal/lysosomal compartment and was consistent with previous results using this type of analysis (33). In contrast, Tf-CS-OVA conjugates with 2.5, 5, and 8 peptides were less efficiently recycled because, after 2 h, 3.2, 5.3, and 11.4% of the radioactivity was detected in the TCA soluble pool. This indicated that at higher substitution levels, Tf delivery to the lysosomal compartment is more efficient. Interestingly, this low level of delivery to the lysosomal compartment as monitored by radioactivity in the TCA soluble pool is consistent with the partial level of colocalization of Tf-peptide conjugates with the LAMP-1-positive compartment seen in Fig. 3Go. Because B cells use conventional endocytic compartments for loading MHC class II molecules (43), our results suggest that the Tf conjugates with higher levels of substitution are more efficiently delivered to the lysosomal compartment than those with lower coupling ratios.



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  		FIGURE 5. Tf conjugates with higher level of substitution are more effectively delivered to the lysosomal compartment. HeLa cells were incubated with 125I-labeled Tf or Tf-CS-OVA peptide conjugates (2.5, 5, or 8 peptides/Tf; 4 µg/ml) at 37°C to load the endocytic pathway with radiolabeled ligand-receptor complexes. After 1 h, the label was removed, the cells were rinsed with ice-cold 0.1% BSA in PBS, and reincubated at 37°C in medium containing 50 µg/ml unlabeled Tf for various times. After each time point, the cell surface radioactivity (acid wash), internalized radioactivity (base lysis), and radioactivity released into the medium as TCA soluble or insoluble counts were determined. The TCA soluble counts represented the percentage of protein that reached the lysosomal compartment that was degraded and released into the medium (57 ).

 
Next, we tested whether more efficient Ag delivery to the lysosome correlated with more efficient B cell presentation and, more importantly, more efficient Th cell activation. To test this, we performed an in vitro T cell proliferation assay using samples of Tf-peptide conjugates that averaged 1, 2.5, 5, and 8 peptides per Tf monomer (Fig. 6Go). After a 24-h incubation with varying concentrations of the Tf-peptide conjugates, IL-2 production was measured by monitoring the proliferation of HT-2 cells. Surprisingly, we found that there was an inverse correlation between the number of peptides added and IL-2 produced. In the figure shown, the molar concentration was calculated based on the peptide concentration rather than on the Tf conjugate. In the experiment shown, the Tf-OVA conjugates with one peptide added were most effective at DO-BW T cell activation, while eight OVA peptide-Tf conjugates induced IL-2 production at a level similar to that seen with free OVA peptide.



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  		FIGURE 6. A, Peptide-Tf conjugates with high peptide coupling ratios are less effective in activating Th cells. DO-BW cells and A20 cells were incubated with Tf-CS-OVA conjugates of different peptide/Tf ratios (1, 2.5, 5, or 8 peptides/Tf molecule) at varying concentrations. After 24 h, the supernatants were assayed for the production of IL-2. B, Higher coupling ratios of peptide to Tf decrease Tf’s binding affinity for its receptor. 125I-labeled Tf or Tf-CS-OVA conjugates (2.5, 5, or 8 peptides/Tf molecule) were incubated at 0°C in triplicate wells containing 1.5 x 105 HeLa cells. After 1 h, the radiolabel was removed, the cells were rinsed three times with 0.1% BSA in PBS and the cell-associated radioactivity was determined. The binding affinities (Ka) were determined from the slopes of the least-squares fitted lines.

 
Because a higher level of peptide substitution on Tf did not translate into a more effective Ag delivery vehicle (as monitored by production of IL-2), we performed a Scatchard analysis using radiolabeled Tf-CS-OVA conjugates and native Tf as a control to test how the Tf binding affinity was affected by peptide cross-linking. In this experiment, increasing amounts (6–600 ng) of 125I-labeled Tf-CS-OVA or Tf were incubated with HeLa cells for 1 h at 0°C. A representative binding experiment is shown in Fig. 6GoB. The results indicate that unmodified Tf has an equilibrium dissociation constant of 3.0 x 10-9 M, which is comparable to that found previously (44, 45). For the Tf-CS-OVA conjugates with 2.5, 5, and 8 peptides bound, the affinities decreased to 39, 8.7 or 2.9% of that of unmodified Tf, indicating that as the level of peptide coupling increased, the binding affinity for the receptor decreased (Fig. 6GoB). Because highly modified Tf-peptide complexes had lowered affinities for the TR, it appeared that preparing conjugates with higher levels of substitutions offered no real advantage for Ag delivery.

Activation of adoptively transferred Ag-specific Th cells following i.v. administration of Tf-OVA peptide conjugates

Adoptive transfer experiments were performed to extend the in vitro studies on the use of Tf as a carrier molecule. In these experiments, CD4+ T cells from DO11.10 mice were tracked in naive BALB/c recipients after immunization with the various forms of the OVA peptide (29) (see Materials and Methods). After adoptive transfer, the OVA-specific DO11.10 T cells represented only ~0.1–0.2% of the total CD4+ T cells purified from the spleens of BALB/c mice. In our initial studies, adoptively transferred T cells were detected using mAb against OVA-specific TCR, KJ1-26.1 (30). Because the TCR is down-regulated during the first 24–48 h after activation (46) (C. T. Weaver, unpublished observations), as an alternative approach, we developed an assay for measuring OVA-specific T cell responses independently of TCR down-regulation. The CFSE has been widely used to follow the proliferation of cells in vivo (47). In this assay, we tail-vein injected CFSE-labeled DO11.10 CD4+ T cells into naive BALB/c recipients, and 24 h later i.v. administered OVA peptide, Tf-NC-OVA, or apoTfde-OVA. T cell activation was monitored by the expression of CD69, an early activation marker (48), 24 h after Ag administration. The results shown in Fig. 7GoA indicate that 91% of Ag-specific T cells were activated following injection of 0.02 nmol of the Tf-NC-OVA conjugate, while the same dose of free peptide (OVA) or the apoTf control was at a background level of T cell activation (~9%). The fact that apoTfde-OVA conjugate had similar effects on T cells as free OVA peptide suggested that T cell activation in vivo was due to Ag delivery via TR and was not a random carrier effect. The results shown in Fig. 7GoB represent the mean percent of CD69 expression ± SD from nine independent experiments. The administration of unmodified Tf indicated that the background activation in these adoptive transfer experiments was ~9–10% (Fig. 7GoB). T cell activation using Tf-NC-OVA peptide conjugates was ~100-fold (ED50, ~0.002 nmol) more effective than free peptide (ED50, ~0.2 nmol), and 50-fold more effective than apoTf (ED50, ~0.1 nmol). The results indicate that coupling peptides to Tf dramatically enhances Th cell activation not only in vitro, but also more importantly in vivo.



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  		FIGURE 7. Tf-peptide conjugates enhance T cell activation in vivo. The CFSE-labeled CD4+ T cells (purified from DO11.10/SCID transgenic animals) were adoptively transferred into BALB/c mice, and 24 h later, the recipients were immunized with different amounts of OVA323–339, Tf-NC-OVA, or deglycosylated apoTf-OVA (apoTfde-OVA) conjugate. Twenty-four hours after the Ag administration, spleens were harvested, and processed for flow cytometric analysis. A, CD69 expression on activated CD4+ T cells is enhanced by coupling the OVA determinant to Tf. Contour plots for CD69 expression is shown for three different concentrations of Ags. The numbers in the upper right corner of each panel indicate the percentage of CD69-positive-CFSE-positive T cells. The representative experiment of three is shown. B, The mean activation of adoptively transferred T cells after immunization with Tf-NC-OVA or OVA peptide alone. The results represent the mean ± SD from nine in vivo experiments. The level of T cell activation with native Tf is shown.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we demonstrate that coupling peptides to Tf enhances Ag delivery as monitored by Th cell activation. We show that T cell activation is enhanced in in vitro Th cell assays, but importantly, also occurs in vivo. Further, we show that uptake of the Tf-peptide conjugates occurs through the TR and that modified Tf-peptide conjugates are efficiently delivered to the lysosomal compartment where Ag processing occurs.

Tf has been used by a number of investigators to enhance Ag delivery, but the results have been conflicting (49, 50). Using Tf-cytochrome c conjugates, McCoy et al. (49) demonstrated that T cell activation was augmented when cytochrome c was coupled to Tf. In their studies, the protein-protein conjugates only appeared to reach the early endosome because cytochrome c was degraded, but the Tf portion of the conjugate was not. However, Pierce and coworkers (50), using the same Ag, found that coupling it to Tf did not enhance presentation unless cytochrome c or Tf was cross-linked with Abs or chemical cross-linkers. These authors suggested that monomeric Tf was inefficient because it only trafficked to the early stages of the endocytic pathway, and not to the lysosomal compartment where Ag processing occurs. These results suggested that there may be variability in Ag processing by different APC that could account for inconsistency in T cell presentation. However, both studies suggested that Ag delivery was limited to the early endosome (49, 50), and therefore, many Ags would not be effectively degraded and subsequently presented.

One mechanism for eliminating processing differences is to use peptide rather than intact Ags. Mauri et al. (51) used Tf-bound peptides to improve the sensitization of APCs in vitro. In these studies, despite the fact the peptide-Tf coupling ratio was 5:1, the conjugates were still more effective than free peptide. Given the success of peptide therapies in vitro and the failures in vivo (52) it is surprising that Tf-peptide conjugates were not tested in vivo. In our experiments, we determined that a Tf-peptide ratio of 1:1 was the most effective, and like Pierce and coworkers (50), concluded that coupling native Ag to Tf had little effect on Ag presentation. Based on our Scatchard analysis of the Tf-peptide conjugates, it is tempting to speculate that coupling a native Ag to Tf dramatically alters its receptor binding characteristics, and therefore prevents it from being taken up by an efficient receptor-mediated process.

Based on the Tf proteolysis assay, it is clear that more Tf reaches the lysosomal compartment when it has attached peptides to it, and that the level of conjugation directly affects lysosomal targeting efficiency. We have no direct way of correlating efficiency of lysosomal delivery to Ag presentation and subsequent Th cell activation because these modifications only affect lysosomal delivery incrementally. However, by following the myc peptide coupled to Tf, we were able to demonstrate that peptides do reach the lysosomal compartment in B cells, although we still do not know how much processing actually occurs there. The fact that Tf was also found in the lysosomes, whereas native Tf was not, suggests that at least some processing occurs in this compartment.

Using a modification of the adoptive transfer system developed by Jenkins and coworkers (29), we demonstrated that the Tf-peptide conjugates were >100-fold more effective than free peptide in activating adoptively transferred DO11.10 CD4+ T cells in BALB/c mice. Other groups have used different cell surface receptors (mannose receptor, Fc receptor) to modulate Ag delivery and T cell activation (25, 26). However, because the methods used to monitor the activation of Th cells were different, it is difficult to compare their results with ours.

It is worth noting that the assay used in this study for T cell activation in vivo, CD69 induction by Ag-specific T cells, does not discriminate between immunogenic and tolerogenic antigenic stimuli. We opted for this marker because it is reliably up-regulated early in T cell responses that precede either clonal proliferation or anergy (53, 54), and it is therefore a useful indicator for studies of Ag delivery, irrespective of the functional outcome of the response. Indeed, although not directly examined, the i.v. administration route used for our studies typically induces tolerance rather than immunity, suggesting that Tf-peptide conjugates might be excellent Ags for tolerance induction or perhaps activation of regulatory T cell responses. Additional studies will be needed to determine whether other routes of administration would also result in Tf-mediated enhancement of antigenic stimulation or if Tf conjugation modulates the functional outcome of the antigenic response.

The mechanism responsible for enhanced delivery is clearly dependent upon a receptor-mediated event, both in vitro and in vivo, because the deglycosylated apoTf was ineffective in both circumstances. Similar results were obtained by Stutzman and coworkers (49) using apoTf coupled to native cytochrome c, suggesting that specific uptake mechanisms are required for this effect, and more importantly, that this is not simply a protein carrier effect. Our initial studies using the apoTf-peptide conjugates revealed that in vitro the conjugates were no more effective than free peptide in activating Th cells. In contrast, when these same conjugates were tested in the adoptive transfer assay, the apo-complexes were just as effective as the holoTf-peptide conjugates (data not shown). Our interpretation of this result, was that the apoTf was able to recapture iron in vivo, and not in vitro. To test this idea, we relied on the fact that deglycosylated apoTf is not able to reload iron (32), and, therefore, would not bind to the TR. The results were as expected, and the deglycosylated apoTf-peptide conjugate was no more effective than the free peptide. However, whether this means that the complexes are exclusively taken up by TR in vivo cannot be determined in an animal model.

In summary, our results demonstrate that coupling peptide Ags to Tf improves peptide presentation and offers an exciting potential for the use of Tf as an efficient carrier molecule for delivery of competitor peptides (to ensure better competition with self Ags (8, 22)), altered peptide ligands (to modify the immune responses from pathologic to protective ones (55)), or agonist peptides (to inhibit Th1 and/or induce Th2 responses (56)).


   Acknowledgments
 
We thank Dr. John Kearny (University of Alabama) for providing the A20 B cells and Dr. Osami Kanagawa (Washington University, St. Louis, MO) for providing the DO-BW T cell hybridoma. We thank Albert Tousson (University of Alabama) for the help with laser confocal microscope imaging and Lori Coward (University of Alabama) for mass-spectroscopic analysis of the proteins.


   Footnotes
 
1 This work was supported by a Biomedical Science Grant from the Arthritis Foundation (to J.F.C.) and the Cancer Research Experiences for Students Summer Internship Program (to K.S.). The mass spectrometer was purchased by funds from National Institutes of Health Shared Instrumentation Grant No. S10RR11329 and from a Howard Hughes Medical Institute Infrastructure Support Grant to the University of Alabama (Birmingham, AL). Operation of the Shared Facility has been supported in part by National Cancer Institute Core Research Support Grant No. P30 CA13148-27 to the University of Alabama Comprehensive Cancer Center. Back

2 Address correspondence and reprint requests to Dr. James F. Collawn, Department of Cell Biology, University of Alabama, 1918 University Avenue, McCallum Basic Science Building 350, Birmingham, AL 35294-0005. E-mail address: jcollawn{at}uab.edu Back

3 Abbreviations used in this paper: Tf, transferrin; TR, Tf receptor; CS, cathepsin S cleavable peptide; NC, noncleavable peptide; NC-myc, myc-tagged NC linker; SMCC, succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate; MALDI-TOF, matrix-assisted laser desorption ionization-time of flight; dTf, dimeric Tf; apoTf, apotransferrin; LAMP, lysosome-associated membrane protein; HYNIC, succinimidyl 6-hyrazino nicotinate hydrochloride. Back

Received for publication February 8, 2002. Accepted for publication June 26, 2002.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
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