MHC class II loading of high or low affinity peptides directed by Ii/peptide fusion constructs: implications for T cell activation

Tone F. Gregers1, Burkhard Fleckenstein2,3, Frode Vartdal3, Peter Roepstorff2, Oddmund Bakke1 and Inger Sandlie1

1 Division of Cell and Molecular Biology, Department of Biology, University of Oslo, N-0316 Oslo, Norway 2 Protein Research Group, Department of Biochemistry and Molecular Biology, University of Southern Denmark, DK-5230 Odense M, Denmark 3 Institute of Immunology, Rikshospitalet, The National Hospital, University of Oslo, N-0027 Oslo, Norway

Correspondence to: T. F. Gregers; E-mail: t.f.gregers{at}bio.uio.no
Transmitting editor: D. Tarlinton


    Abstract
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
CD4+ T cells recognize peptides presented on the cell surface of antigen presenting cells in the MHC class II context. The biosynthesis and transport of MHC class II molecules depend on the type II transmembrane invariant chain (Ii) and are tightly regulated processes. Ii is known to bind to the MHC class II peptide-binding groove via its class II-associated Ii peptide (CLIP) region early in the biosynthetic pathway to prevent premature peptide binding. In this study we have genetically exchanged CLIP with peptides of either high or low affinity for the class II peptide binding groove and utilized the properties of Ii to manipulate MHC class II loading. An inducible promoter controlled expression of the Ii/peptide fusion constructs, and presentation at different expression levels was studied. Both peptides were excised from Ii and presented on MHC class II molecules as shown by liquid chromatography–tandem mass spectrometry, but the high affinity peptide was presented more efficiently than the low affinity peptide. Both peptides were efficient in eliciting T cell responses at high Ii/peptide concentration independent of the duration of T cell stimulation. The peptides were also able to elicit an IL-2 response at low expression levels; however, the kinetic differed as the T cells required longer duration of T cell contact to reach a significant T cell response. This probably reflects the number of class II/peptide complexes at the cell surface and is discussed.

Keywords: antigen concentration, antigen presentation, invariant chain, peptides, processing


    Introduction
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Peptides bound to the heterodimeric MHC class II molecules are presented at the cell surface where the class II/peptide complex may stimulate CD4+ T cells and elicit an immune response. The type II transmembrane glycoprotein invariant chain (Ii) is known to have several important roles in regulating antigen presentation (for review see 1). Upon synthesis in the endoplasmic reticulum (ER) Ii forms trimers, each of which associates with three class II dimers to form nonamers (2). This association prevents binding of immunogenic peptides to class II in the ER due to the class II-associated Ii peptide (CLIP) fragment that binds in the peptide-binding groove (3,4). Ii also facilitates endosomal transport of MHC class II due to the two leucin-based sorting signals found in its cytoplasmic tail (57). In endosomal compartments, Ii is sequentially degraded by proteases until its final degradation product CLIP is left in the groove (for review see 8). CLIP is released from the groove by the catalytic action of human leucocyte antigen (HLA)-DM and exchanged for immunogenic peptides derived from proteins that have entered the endosomal–phagosomal system (9). Mainly peptides with a high affinity for the peptide binding groove are presented by class II due to the editing function of HLA-DM (10); however, presentation of low affinity peptides also occurs. Therefore, it is important to increase our knowledge of both high and low affinity peptides, how they interact with MHC class II and how they stimulate the T cells.

In this study we have utilized an HLA-DM negative cell line that expresses MHC class II and Ii constructs where the CLIP region is genetically exchanged for peptides with either high or low affinity for the peptide binding groove. It is well established that cells expressing Ii constructs with T cell epitopes in the CLIP region are able to efficiently present the peptides to specific T cells (1113). We show here that this approach leads to efficient loading of class II with both peptides; however, the presentation efficiency differs between the two peptides. We have utilized this fact to investigate under what circumstances these peptides are recognized by specific T cells.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Antibodies, T cell hybridomas and ligands
BU43 is a mouse monoclonal IgM antibody specific for the luminal C-terminal part of human Ii (Binding Site, Birmingham, UK), while H116-32 is a mouse monoclonal IgG1 antibody specific for I-Ak, and was a gift from Dr F. Momburg (Heidelberg, Germany). The mouse monoclonal IgG1 antibody Vic-Y1, a gift from Dr W. Knapp (Vienna, Austria), is specific for the N-terminal Ii tail (14) and was conjugated with Alexa-594 by using Alexa Fluor 594 Protein Labeling Kit from Molecular Probes (Eugene, OR). Goat anti-mouse IgG Alexa 488 was obtained from Molecular Probes (Leiden, The Netherlands), while Texas Red (TR)-conjugated goat anti-mouse IgM antibody was from Southern Biotechnology Association, Inc. (Birmingham, UK). The T cell hybridoma 3A9 which is specific for the mouse MHC class II molecules I-Ak in complex with amino acid 46–61 (NTDGSTDYGILQINSR) of HEL and the synthetic HEL 46–61 peptide were obtained from Dr R. Germain (NIH).

DNA constructs
cDNA that encodes the p33 form of human Ii in pSV51 and pMEP4 (Invitrogen, The Netherlands) has been described previously (15,16). Amino acids 88–104 of Ii, corresponding to the CLIP region, were genetically exchanged with either hen egg lysozyme (HEL) 46–61 or HEL 46–61, D52A (HDA) by polymerase chain reaction-spliced by overlap extension (PCR-SOEing). Ii 1–87 and Ii 105–216 were amplified separately in a first round of PCR using the following primers (see Table 1 for sequences): primers A and C or A and D were used to amplify Ii 1–87 HEL or Ii 1–87 HDA, respectively. Primers C' and B or D' and B were used to amplify Ii 105–216 HEL or Ii 105–216 HDA, respectively. Primers C, C', D and D' contained tags encoding HEL 46–61 or HEL 46–61, D52A. Then the products from the first PCRs were spliced after overlap extension in a second PCR that amplified the final Ii constructs using primers A and B, giving Ii HEL and Ii HDA. All primers were obtained from DNA Technology (Aarhus, Denmark). Ii HEL and Ii HDA were subcloned as KpnI–BamHI fragments into the heavy metal inducible expression vector pMep4, which contains the hygromycin resistance gene. The insertion of new sequences in the CLIP region was verified by cutting with the additional restriction sites BamHI and NdeI in the Ii HEL and Ii HDA construct, respectively, and the amplified PCR Ii constructs were sequenced (GATC Biotech, Konstanz, Germany).


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Table 1. PCR primers used
 
Cell culture
Madin–Darby canine kidney strain II (MDCK) cells were grown in complete medium: DMEM (Bio Whittaker, MD) supplemented with 10% FCS (Integro, Zaandam, Holland), 2 mM glutamine, 25 U/ml penicillin and 25 µg/ml streptomycin (all from Bio Whittaker). The 3A9 T cell hybridomas were grown in RPMI 1640 supplemented with 10% FCS (Integro), 25 U/ml penicillin and 25 µg/ml streptomycin, 0.11 µg/ml Na–pyruvate, 1% non-essential amino acids (Bio Whittaker) and ß-mercaptoethanol. All cells were incubated at 5% CO2 in a 37°C incubator.

Transfections
MDCK cells are MHC class II, HLA-DM and Ii-negative epithelial cells. MDCK cells stably transfected with mouse I-Ak {alpha} and ß (MA cells) and inducible human Ii (MAI cells) have been described previously (17). MA cells were triple transfected with Ii HEL (MAIH) or Ii HDA (MAIHDA) in pMep4 using the DNA–calcium phosphate procedure, giving rise to stable colonies as described previously (18). Positive clones were selected in the presence of 200 µg/ml G418 (for I-Ak selection) and 300 µg/ml hygromycin (for Ii selection) (Gibco/BRL). Resistant clones were induced with 25 µM CdCl2 for 16 h to induce Ii expression. Triple positive clones were identified by immunofluorescence microscopy.

Immunofluorescence microscopy
For total Ii labeling, transfected cells were grown on glass coverslips, induced to express Ii and fixed in 3% PFA for 20 min at room temperature. Fixed cells were impermeabilized with 0.2% saponin and incubated first for 30 min at room temperature with the anti Ii antibody Vic Y1-Alexa 594 diluted in PBS containing saponin. For double labeling of I-Ak and Ii, transfected cells were grown as above and incubated with the anti Ii antibody BU 43 for 1 h at 37°C to detect internalized Ii. The cells were then fixed, impermeabilized and incubated with the anti I-Ak antibody H116-32 diluted in PBS and saponin for 30 min at room temperature. BU 43 and H116-32 were detected by the secondary antibodies goat anti-mouse IgM-TR and goat anti-mouse IgG-Alexa 488, respectively. The coverslips were washed and mounted in DAKO® fluorescent mounting medium (Carpinteria, CA). Confocal images were acquired with a Leica TCS-NT digital scanning confocal microscope equipped with a Zeiss LSM 410 (Carl Zeiss Inc., Thornwood, NY) with a 100x Zeiss PlanApochromat oil immersion objective NA 1.4. Immunofluoresence images were acquired with Zeiss Axioplan 2 microscope equipped with Axiocam HRc camera and 20x Axioplan-NEOFLUAR air objective NA 0.5 (Carl Zeiss Inc.). Images were processed using Adobe Photoshop (Adobe Systems Inc.).

Isolation of I-Ak bound peptides
MAIH and MAIHDA cells were grown in complete medium to 1 x 108 cells. For the last 16 h of growth, cells were cultured in 25 µM CdCl2 to induce high Ii expression. The cells were washed three times in ice-cold PBS and lysed in ice-cold lysis buffer (50 mM Tris–HCl pH 7.5, 150 mM NaCl, 1% NP-40) containing a cocktail of protease inhibitors (4 µg/ml PMSF, 2 µg/ml antipain, 2 µg/ml leupeptin and 1 µg/ml pepstatin A) for 30 min. The lysates were centrifuged at 10,000 g for 20 min at 4°C to remove cell nuclei and cellular debris, and the supernatants were stored at –20°C until used. Peptide-loaded MHC class II molecules were purified from the supernatant essentially as described previously (19). Shortly afterwards, 3 mg/ml of the anti-I-Ak antibody H116-32 was covalently coupled to 10 ml Protein A-Sepharose CL-4B (PA) (BioRad Laboratories, Hercules, CA) using the dimethyl pimelimidate cross linking method described previously (20). The cell lysates were passed successively through Sepharose CL-4B (18 ml; Amersham Pharmacia Biotech, Uppsala, Sweden), Protein A-Sepharose CL-4B (4.5 ml, Amersham Pharmacia Biotech) and finally the Protein A-Sepharose CL-4B coupled to H116-32 (1 ml). The H116-32 column was washed successively with PBS containing 0.5% NP-40/0.02% NaN3, PBS containing 0.05% NP-40/0.02% NaN3 and finally with PBS. Peptides were released directly from the H116-32 column with 2 ml aqua dest/0.1% TFA (pH 2.34) at 20°C for 15 min. Eluate was centrifuged through Microcon YM-10 filters (Millipore, Bedford, MA) with a nominal molecular weight limit of 10 kDa. The filtrate was concentrated by vacuum centrifuge and stored at –70°C until further analysis.

LC-MS/MS analysis of peptide mixtures eluted from I-Ak
For both MAIH and MAIHDA, the dried peptide mixtures of one vial was dissolved in 10 µl 5% formic acid. Half of each sample was loaded on a home-packed capillary column (Zorbax C18, 5 µm, 10 cm, 75 µm ID) for capillary liquid chromatography (Dionex, LC Packings, Denmark), which was online-coupled to a quadrupole time-of-flight Ultima mass spectrometer (Micromass, Manchester, UK). Peptides were eluted at 200 nl/min by an increasing concentration of acetonitrile (2%/min gradient). A MS-TOF survey spectrum was recorded for 1 s. The three most intense ions present in the MS-TOF spectrum were selected and fragmented by collision-induced dissociation in the second quadrupole (4.4 s per MS/MS spectrum). Masses of the generated b- and y-type fragment ions (21) were interpreted manually to determine the sequence of the presented peptides.

Response factors for peptides predominantly presented by I-Ak
Two pairs of synthetic peptides, DGSTDYGILQINSRG/DGSTAYGILQINSRG and GSTDYGILQINSRG/GSTAYGILQ INSRG (purchased from Eurogentec, Herstal, Belgium) were compared regarding ionization efficiencies in nano-electrospray ionization mass spectrometry in order to determine differences in the response factors for each peptide pair (Quadrupole time-of-flight instrument; Micromass). The individual peptide concentrations were determined by amino acid analysis (Pharmacia, Biochrom 20), and equimolar mixtures of each peptide pair were applied in mass spectrometry. Spectra were recorded using different cone and capillary voltages and varying acetonitrile concentrations (from 0 to 30%). Signal intensities for the D-containing peptide and its A-substitution analog were compared.

Antigen presentation assay
MDCK cells stably transfected with I-Ak alone or in combination with Ii constructs were used as antigen presenting cells (APCs). The cells were seeded at 5 x 103 cells/well in 96-well plates to avoid polarization. The cells were allowed to adhere overnight before they were incubated with various concentrations of CdCl2 to induce expression of Ii. Mostly 5 x 104 3A9/well were added and incubated further at 37°C for 24 h before the supernatants were harvested. The production of IL-2 in the supernatant was determined using the IL-2-dependent CTLL-2 cell line. Proliferation of CTLL-2 was measured by pulsing with 1 µCi [3H]thymidine and, after overnight incubation, the cells were harvested and thymidine incorporation was counted in a Matrix96 ß-counter (Packard Instruments Company, Meriden, CT). The counting efficiency is only ~20–30% of ordinary scintillation fluid-based counters. Results were obtained from triplicates of at least three independent experiments.


    Results
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
CLIP may be replaced with HEL-derived epitopes without loss of Ii function
The CLIP region (amino acids 88–104) of human Ii was genetically replaced by a well known T cell epitope derived from HEL (HEL 46–61). A variant of this epitope was also introduced into CLIP to obtain a low affinity epitope in this region [HEL 46–61, D52A (HDA)] (22) (Fig. 1). Both constructs were generated by PCR-SOEing, and the Ii HEL and Ii HDA constructs were subcloned downstream of the heavy metal inducible promoter in the expression vector pMep4. MDCK cells stably transfected with mouse I-Ak under a constitutive promoter [MA cells (17)] were transfected with either Ii HEL or Ii HDA to produce the cell lines MAIH and MAIHDA, respectively. Adding heavy metals to the cell culture thus controlled the gene expression of the Ii variants (16). The cell lines were first shown to express equal amounts of Ii HEL and Ii HDA compared to Ii wild-type (MAI cells) by immunoprecipitation at different CdCl2 concentrations (data not shown). Figure 2(A) shows the total Ii expression in the transfectants following CdCl2 induction and shows clearly that the Ii expression level increases significantly from 1 to 25 µM of CdCl2.



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Fig. 1. Substitution of the CLIP region. The CLIP 81–108 sequence is shown. The core CLIP (italic) was genetically exchanged by the epitopes HEL 46–61 and HEL 46–61 (D52A) by PCR-SOEing. The arrows indicate the putative cathepsin cleavage sites.

 


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Fig. 2. Expression of Ii HEL and Ii HDA in response to CdCl2 induction, vesicle enlargement and colocalization with I-Ak. (A) MAIH and MAIHDA cells were grown on coverslips and incubated with 0, 1 and 25 µM CdCl2 overnight. The cells were then fixed in 3% PFA and labeled with the anti Ii antibody Vic Y1-Alexa 594 and analyzed by immunofluorescence microscopy. (B) MAI, MAIH and MAIHDA cells were grown on coverslips and incubated with 25 µM CdCl2 overnight. Then the cells were incubated with the anti Ii antibody BU43 for 1 h at 37°C, fixed in 3% PFA and labeled with the anti I-Ak antibody H-116–32. The cells were further labeled with goat anti-mouse IgG-Alexa 488 and goat anti-mouse IgM-TR, and analyzed by confocal immunofluoresence microscopy. Red channel: anti-Ii, green channel: anti-I-Ak. Bar: 20 µm.

 
Ii wild-type is known to induce the formation of enlarged endosomes in transfected cells upon high levels of expression (2325). We have recently shown that one single amino acid substitution in the cytoplasmic tail of Ii is sufficient to prevent this endosomal modification (26). The mutant phenotype gives a normal MHC class II distribution, but demonstrates a partly impaired Ii function with a decrease in antigen presentation (17). Thus, formation of large vesicles is an indication of a functional Ii. Therefore, we have tested whether or not Ii induces the formation of enlarged vesicles after substitution of the CLIP region. As seen in Fig. 2(B), both Ii HEL and Ii HDA induce the formation of enlarged vesicles and resemble Ii wild-type. In addition, both constructs localize together with I-Ak, indicating that the CLIP substitutions did not interfere with the intracellular distribution of MHC class II.

I-Ak is loaded with both high and low affinity peptides
Presentation of antigenic peptides within the Ii molecule has been reported to be very efficient (11,27). However, the exact nature of the peptide presented by MHC class II has never been examined. To investigate the HEL and HDA sequence displayed by I-Ak, 1 x 108 MAIH and MAIHDA cells were induced to express high levels of Ii and lysed in 1% NP-40. I-Ak/peptide complexes were purified by affinity chromatography and peptides were released as described in Methods. The sequence of the released peptides was determined by LC-MS/MS analysis. For MAIH, six peptides deriving from processing of the HEL peptide were identified (Table 2), and the most intense signals were found for peptides DGSTDYGILQINSRG and GSTDYGILQINSRG. The MS/MS spectrum of peptide DGSTDYGILQINSRG is presented in Fig. 3(A), showing the characteristic series of y-type ions used to deduce the amino acid sequence. In all peptides the C-terminus of the HEL peptide is prolonged by either one (G) or two (GA) amino acid residues derived from Ii. In contrast, for MAIHDA, only three HEL-derived peptides were identified, two of them corresponding to the two most intense ones found for MAIH.


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Table 2. Peptides presented by cell lines MAIH and MAIHDA as identified by LC-MS/MS analysis
 


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Fig. 3. LC-MS/MS analysis of peptides eluted from I-Ak from cell line MAIH. (A) MS/MS spectrum of peptide DGSTDYGILQINSRG (parent ion: MH2+ = 798.4). The y-type fragment ions are most prominent (due to the position of the R residue) and are assigned from y2 to y12. In addition the b-type fragment ions are present as minor signals (not shown). The amino acid sequence can be directly deduced from the mass difference between two neighboring y- (or b-) ions. (B) TIC of the LC-MS/MS run from 20 to 47 min. No distinct compounds could be directly assigned. (C) Selective monitoring of m/z = 798.39 for the doubly charged peptide DGSTDYGILQINSRG. A specific signal at 40.29 min is obtained whose intensity (1.19 x 104) was used for quantification. This monitoring was done for all peptides listed in Table 2.

 
To obtain more quantitative results on those peptides predominantly presented by cell lines MAIH and MAIHDA, response factors for the two peptide pairs DGSTDYGILQINSRG/DGSTAYGILQINSRG and peptides GSTDYGILQINSRG/GSTAYGILQINSRG were accurately determined. The pairs differ only by an aspartic acid compared to the alanine residue. Peptide GSTAYGILQINSRG showed a 1.6 (± 0.2) times higher signal intensity in nano-electrospray mass spectrometry than peptide GSTDYGILQINSRG. For peptide DGSTAYGILQINSRG only a slightly higher response was found when compared with its D-containing analog (factor 1.2 ± 0.1). The difference in signal intensities between D- and A-containing peptides was essentially independent of the experimental conditions.

After correcting the signal intensities of peptides DGSTDYGILQINSRG and GSTDYGILQINSRG by the factor 1.2 and 1.6, respectively (Table 2), it could be estimated that about eight times more HEL peptides were released from I-Ak of cell line MAIH compared with the corresponding HDA peptides presented by MAIHDA. From the total ion count of the LC-MS/MS run no distinct compounds could be assigned (Fig. 3B). However, selected ion monitoring of the m/z-values of the identified peptides showed clear peaks in the total ion chromatograms (TIC) (e.g. monitoring of m/z = 798.39; Fig. 3C) whose intensities were also used for quantification. Intensities for peptides DGSTDYGILQINSRG and GSTDYGILQINSRG were corrected as described above. This evaluation resulted in about 6-fold more HEL-derived peptides than HDA peptides (Table 2).

Both MAIH and MAIHDA stimulate the T cell hybridoma 3A9 equally efficiently
We next investigated whether the MAIH and MAIHDA cell lines were able to stimulate the T cell hybridoma 3A9. This CD4+ T cell hybridoma recognizes HEL 46–61 in the context of I-Ak (28). As controls, we used MAI cells as well as MA cells expressing I-Ak alone incubated with the synthetic HEL 46–61 peptide.

MDCK cells have an inherent ability to polarize when the cells are grown to confluence, giving an apical and a basolateral surface where MHC class II molecules mainly are transported to the basolateral surface (29). To avoid polarization, different concentrations of APC were investigated for their capacity to stimulate 3A9 as follows: different numbers of MA, MAI, MAIH and MAIHDA were seeded in a 96-well plate and allowed to grow overnight. They were then induced to express Ii for 16 h. 3A9 were subsequently added to the cells and, in addition, the MA cells were incubated with synthetic HEL 46–61 peptide. After ~24 h, IL-2 production from 3A9 was measured indirectly by proliferation of the CTLL-2 cell line. As shown in Fig. 4(A), MAIH did stimulate 3A9 in all cases. However, 5 x 103 cells/well gave the most efficient response. MA incubated with peptide also stimulated 3A9 but not to the same extent as MAIH. Thus this confirms that the approach taken, where peptides are placed in the CLIP region, results in optimal presentation of the HEL epitope on the cell surface. 3A9 did not recognize MAI or MA alone, nor did 3A9 produce any IL-2 alone (Fig. 4A and data not shown). However, MAIHDA were approximately equally efficient in stimulating 3A9 as MAIH. MA cells incubated with synthetic HDA peptides also activated the 3A9 cells (data not shown), indicating that the T cells recognize the HDA peptide even though the major anchor residue was exchanged with an alanine.



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Fig. 4. Presentation of HEL and HDA to 3A9. Different numbers (A) MA, MAI, MAIH and MAIHDA, (B) MAIH and MAIHDA or (C) 5 x 103 MAIH and MAIHDA cells were grown in 96-well plates and incubated with 25 µM CdCl2 overnight. 5 x 104 3A9/well (A and B) or different amounts of 3A9/well (C) were added to the cells; however, MA cells were in addition incubated with 10 µg/ml HEL 46–61 peptide (p) (A). The cells were incubated at 37°C for an additional 24 h. Supernatants were harvested and IL-2 production was measured by proliferation of the CTLL-2 cell line. [3H]Thymidine incorporation was quantified by a Matrix96 ß-counter. Results were obtained from triplicates.

 
Since peptides with low affinity for the MHC class II groove are known to be suboptimal T cell activators, we further wanted to compare the ability of MAIH and MAIHDA to induce IL-2 production from 3A9 at different densities of available APC or T cells. First, different numbers of MAIH or MAIHDA cells were induced to express high levels of Ii constructs, then incubated with 5 x 104 3A9 and examined for IL-2 production. Figure 4(B) shows no significant difference between the two cell lines even at low APC levels. In the next experiment, 5 x 103 MAIH or MAIHDA cells were induced to express high levels of Ii HEL and Ii HDA respectively, and then incubated with increasing numbers of 3A9, without any significant difference between the two cell lines (Fig. 4C). The results indicate that at high concentrations of antigen the two cell lines have the same ability to activate 3A9, regardless of the APC or T cell concentrations in the wells.

The expression level of Ii fusion constructs determines the capacity to stimulate 3A9
To investigate how the concentration of the Ii/peptide constructs affected the presentation efficiency of the HEL and HDA peptides, different concentrations of CdCl2 were added to the MAIH and MAIHDA cells prior to 3A9 addition. Between 5 and 25 µM of CdCl2, no significant difference in T cell stimulation was observed between MAIH and MAIHDA cells (data not shown). However, titration of the CdCl2 concentration between 0.05 and 2.5 µM, giving different, but small amounts of available peptides in the context of Ii, demonstrated a difference between MAIH and MAIHDA (Fig. 5A).



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Fig. 5. Ii expression level and T cell incubation time. MAIH and MAIHDA cells were grown in 96-well plates and incubated with (A) different concentrations, (B) 1 µM or (C) 25 µM CdCl2 overnight, and then incubated further with 5 x 104 3A9. IL-2 production in the supernatant was measured after overnight incubation (A) or after time points as indicated (B–C), by proliferation of CTLL-2. [3H]Thymidine incorporation was quantified by a Matrix96 ß-counter. Results were obtained from triplicates.

 
We next wanted to investigate how the incubation time with 3A9 might affect the ability of MAIH and MAIHDA cells to induce IL-2 production. MAIH and MAIHDA were incubated with 1 µM CdCl2 overnight before the 3A9 was added. Medium was then harvested at different time points and IL-2 production was measured. Already after 4–6 h some HEL-induced activation was observed. However HDA-induced activation was not significant until 8–10 h after addition of 3A9 (Fig. 5B).

In conclusion, the results indicate that lower antigen concentration and shorter duration of T cell contact is sufficient for HEL peptide-induced activation, whereas HDA peptide seems to require higher concentration and longer incubation with the T cells. At high expression level of both Ii HEL and Ii HDA (25 µM CdCl2 overnight) no significant difference was observed between the two cell lines, as both gave significant T cell stimulation at 4–6 h (Fig. 5C).


    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
In this study we have utilized an inducible expression system to investigate in detail the differences in high and low affinity MHC class II binding peptides, their loading efficiency and ability to activate T cells. We have studied how peptide concentrations and duration of T cell contact with APCs affect T cell activation. Both the low and high affinity peptides were delivered to MHC class II as genetic fusion with human Ii, such that the CLIP region was substituted with one of the peptides. The Ii/peptide fusion genes were transfected into I-Ak expressing MDCK cells under the control of an inducible heavy metal promoter and fusion protein production was induced with CdCl2. It has previously been shown that mouse I-Ak both interacts and is transported together with human Ii intracellularly (30), and that human Ii facilitates I-Ak mediated antigen presentation (17).

As model antigen we utilized the well known HEL protein, which has been used extensively in studies of antigen presentation. The dominant T cell epitope is amino acids 46–61 (28), where Asp in position 52 confers high affinity state to the interaction between I-Ak and the peptide. An Asp52 to Ala substitution results in lower binding strength and reduced SDS stability and half-life of the I-Ak/peptide complex (22,31).

We show that both HEL and HDA (D52A substitution) peptides may be incorporated into the Ii molecule without affecting the function of Ii, as both Ii HEL and Ii HDA like Ii wild-type induce the formation of enlarged endosomes upon high level of expression. In addition, both constructs are transported to the cell surface where they internalize and then colocalize with I-Ak in intracellular vesicles, demonstrating that Ii and I-Ak molecules reach the same compartment, a prerequisite for efficient peptide loading. Nested sets of both HEL and HDA peptides were released from I-Ak and characterized by HPLC (data not shown) and LC-MS/MS, demonstrating that the peptides were correctly excised from the Ii molecule. The most prominent peptides that derived from the HEL/HDA sequence had residue D48 or G49 in the N-terminal end and were extended with an Ii-derived G105 in the C-terminus [(D)GSTDGYGILQINSRG/(D)GSTAGYGILQ INSRG]. It is important to note that these fragments, HEL/HDA 48–61 plus one or two C-terminal residues are identical to the two most prominent fragments previously recovered from B lymphoma cells cultured with exogenous lysozyme (32).

The cathepsins F, L and S are involved in processing of Ii and are known to be important for the generation of the CLIP fragment (reviewed in 33). These cathepsins are endopeptidases which seem to favor certain sequence motifs such as X-{Phi}-X*X (where X is any amino acid, {Phi} is a hydrophobic residue and * indicates the cleavage site) (34). These proteases might be involved in processing of the Ii/peptide fusion molecule, as putative cleavage sites exist between the HEL-derived residues T47 and D48 and also between the Ii-derived residues L107 and P108 (indicated by arrows in Fig. 1). However, the MS studies show that peptides never had more than one Ii-derived GA extension in the C-terminus. Furthermore, only a minor fraction of the degradation products started at residue T47, indicating that non-specific exopeptidases probably further trim the length of the peptide. Despite the exopeptidase activity, the peptides are never shorter than G105 from Ii or G49 from HEL, indicating that MHC class II peptide binding groove protects the peptide from further degradation within 3–4 amino acids prior to and 10–11 residues after the major P1 D52 anchor as previously described for peptides derived from intact HEL proteins (32).

Peterson et al. (35) have previously reported that the amount of the HDA peptide presented from recombinant HEL with the D52A substitution is one tenth of the wild-type peptide presented from HEL protein. This is in agreement with our results as quantification of the I-Ak released peptides revealed that the HEL peptide was presented six to eight times as efficiently as the HDA peptide onto I-Ak. This could be due to the following possibilities: first, a low affinity peptide might generate an unfavorable conformation of the class II/peptide complex. This might result in less efficient transport to the cell surface (36,37) and increased lysosomal degradation (37). Second, once at the cell surface, the class II/peptide complex might recycle and be subjected to new rounds of peptide loading in the recycling compartments (38,39), thereby exchanging the low affinity peptide with peptides of higher affinity for the peptide binding groove. We cannot at the moment distinguish between these possibilities; however, they will eventually result in fewer I-Ak/HDA complexes at the cell surface compared with the number of the more stable I-Ak/HEL complexes.

Despite this, at high levels of expression, MAIHDA cells are equally efficient in stimulating the IL-2 release as the MAIH cells, suggesting that both cell lines present sufficient numbers of class II/peptide complexes at the cell surface to induce a T cell response. In addition, we show that although we need a certain number of both T cells and APCs to detect significant T cell activation, MAIHDA is at least as effective as MAIH in stimulating 3A9 when the expression level of the Ii fusion constructs is high. The same result was found at medium levels of Ii HEL and Ii HDA. On the other hand, small amounts of MAIHDA cells were still able to activate the T cells at low Ii HDA expression level; however, not to the same extent as the MAIH cell line expressing Ii HEL. Class II/peptide complexes are known to be concentrated in cholesterol- and sphingolipid-rich raft microdomains, which allow efficient antigen presentation especially at low ligand densities (40). In addition, certain class II/peptide complexes are enriched in tetraspan microdomains in an antigen dependent manner (41), and it has been proposed that these microdomains are involved in immunological synapse formation. (42). It is therefore tempting to speculate that I-Ak/HEL at low levels are enriched in such tetraspan microdomins and that the MAIH cells are able to engage several T cells at a certain time point, whereas I-Ak/HDA complexes less efficiently localize to such domains due to unfavorable conformation induced by the low affinity peptide. This will eventually lead to less efficient immunological synapse formation and thus engagement of fewer T cells at a certain time point. Therefore, longer duration of T cell contact is required for the low affinity peptide/MHC class II complex to achieve sufficient amount of IL-2 production. However, it remains to be established whether these class II/peptide complexes localize differentially to plasma membrane microdomains.

In this study we have utilized a model system based on the biological function of Ii where the CLIP region is exchanged by the dominant T cell epitope from HEL (HEL 46–61) and a low affinity variant of this peptide (HEL 46–61, D52A). We believe that the results obtained may be utilized to characterize T cell epitopes in general. High affinity peptides in the context of Ii may activate the T cells even at low level, while low affinity peptides require higher levels of expression to induce an IL-2 response. Thus by regulating the expression level of Ii/peptide fusion constructs different types of epitopes may be studied.


    Acknowledgements
 
We thank Dr Bjarne Bogen for providing his lab facilities for the CTLL-2 assays. We also appreciate the technical assistance from Eva Boretti, Dr Anne B. Vogt for helpful discussions and Dr Nicolas Barois for critical reading of the manuscript. T. F. G. obtains a PhD fellowship from The Norwegian Cancer Society, and B. F. has a postdoctoral fellowship from EMBIO, University of Oslo. F. V., I. S. and O. B. labs are generally funded by grants from The Norwegian Cancer Society and The Norwegian Research Council.


    Abbreviations
 
APC—antigen presenting cell

CLIP—class II-associated Ii peptide

ER—endoplasmic reticulum

HEL—hen egg lysozyme

HLA—human leucocyte antigen

Ii—invariant chain

LC—liquid chromatography

MS/MS—tandem mass spectrometry

MDCK—Madin–Darby canine kidney

PCR-SOEing—PCR-spliced by overlap extension

TIC—total ion chromatogram


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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