The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Liu, K.-J.
Right arrow Articles by Kim, B. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Liu, K.-J.
Right arrow Articles by Kim, B. S.
The Journal of Immunology, 1998, 160: 4161-4168.
Copyright © 1998 by The American Association of Immunologists

Hybrid Membrane IgM with the Transmembrane Region of I-A{alpha} Facilitates Enhanced Presentation of Distinct Epitopes to T Cells1

Ko-Jiunn Liu2,*, Michael Schwen*, Philip W. Tucker{dagger} and Byung S. Kim3,*

* Department of Microbiology-Immunology, Northwestern University Medical School, Chicago, IL 60611; and {dagger} Institute for Cellular and Molecular Biology, University of Texas, Austin, TX 78712


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The role of B cell Ag receptors (membrane Ig or mIg) in the efficient Ag presentation to T cells, including the requirement of mIgM-associated Ig{alpha}/Igß, remains unclear. We report here that mIgM, substituted with greater than two-thirds of the NH2-terminal A{alpha} transmembrane (TM) regions of the MHC class II molecule, are capable of mediating the efficient presentation of specific Ag to some (Group 1) but not all (Group 2) T cell hybridomas. In contrast, the generation of epitopes recognized by the Group 2 hybridomas can be mediated only by the wild-type mIgM. Tyrosine phosphorylation appears to be necessary for the enhanced Ag presentation to Group 2 hybridomas, while it does not for Group 1 hybridomas. In addition, differential sensitivity of Ag processing to leupeptin, different duration required for epitope generation/presentation, as well as the involvement of distinct epitopes for stimulation of these groups of T cell hybridomas were observed. These results suggest that transport of the mIgM/Ag complexes to an endocytic compartment(s) for generation of certain T cell epitopes may be mediated by the N-terminal TM sequence of mIgM, independent of Ig{alpha}/Igß association. This function can be replaced by two-thirds of the NH2-terminal TM region of A{alpha} chain of class II molecules.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The B cell Ag receptor complex consists of membrane-bound Ig (mIg)4 recognizing specific ligands and Ig{alpha}/Igß dimers transducing signals generated after mIg ligation (reviewed in 1 . Binding of Ag to the Ag-specific mIg is followed by the internalization of the mIg/Ag complex, leading to the intracellular transport of this complex to the endocytic compartments, where the specific Ag is processed for subsequent presentation to T cells. CD4+ Th cells recognize Ag in the form of short peptides (10–30 residues) loaded on MHC class-II molecules (2). Generally, such peptides are generated after internalization and enzymatic processing of externally produced Ag in the endocytic compartments of APCs. It has been shown that Ag is much more efficiently presented to T cells by the B cells specific for the Ag (3, 4). Thus, the efficient Ag presentation by B cells expressing mIgM specific for the Ag may be due to the ability of mIg to deliver Ag into appropriate processing and loading compartments. In addition, ligation of mIg on B cells also initiates a complex cascade of signaling events, including activation of protein tyrosine kinases and Ca2+ mobilization (1, 5, 6). We (7, 8, 9) and others (10, 11) have shown that the TM domain and cytoplasmic (CY) tail of mIgM are important in the efficient presentation of specific Ag by B cells. However, the role(s) of signal transduction events as well as mIg in the enhanced presentation of specific Ag are still unclear.

Based on subcellular fractionation and identification of various markers associated with endocytic compartments, it has recently been proposed that the compartments involved in Ag processing and peptide loading are separate (12, 13, 14, 15, 16). This putative loading compartment appears to contain mIg as well as class II molecules lacking Ii association, although, depending on the cell line, the markers are somewhat different (13, 14). The completion of transport of mIg/Ag complex to this compartment appears to take 60 to 120 min (13, 14, 15). In addition, Ig molecules complexed with Ag are also found in the peptide-loading compartment, strongly suggesting that the Ig-mediated enhanced Ag presentation may involve targeting the Ag complexes to this compartment (13, 15). However, formation of newly synthesized class II-Ii complexes and peptide loading to class II molecules are found in multiple endocytic compartments, including early endosomes, late endosomes, as well as immature lysosomes (17). Therefore, the role of mIg in the Ag processing and presentation via class II molecules remains unclear.

Previously, we have demonstrated that a BCL1 line, transfected with a DNA construct encoding mIgM that specifically recognizes phosphorylcholine (PC), was able to present PC-conjugated hen egg white lysozyme (PC-HEL) more efficiently over unconjugated HEL to an HEL-specific T cell hybridoma (7, 8, 9). Variant Ig transfectants, expressing the µ-chain with substitutions in the TM regions with equivalent sequences from I-Ab{alpha} chain of the MHC class II molecule, failed to display this preferential presentation of PC-HEL at low concentrations. However, these variant mIgM transfectants appeared to function normally for receptor-mediated endocytosis of ligands (Refs. 8 and 18, and K.-J. Liu and B. S. Kim, unpublished observations). Subsequent subcellular fractionation analysis revealed that the ligands internalized through the variant mIgM receptors with A{alpha}TM substitutions were not efficiently transported to a dense compartment comigrated with lysosomal markers, in contrast to the wild-type mIgM (9). Ligation of the chimeric receptors also failed to induce a significant level of tyrosine phosphorylation of cellular proteins (9, 19). Therefore, the mIgM-mediated enhancement of specific Ag presentation may involve the efficient transport of the mIg/Ag complexes to the lysosome-like dense compartments, and possibly, the activation of protein tyrosine kinases.

Using transfectants expressing limited amino acid substitutions in the µ-TM region, other investigators (10, 11, 20, 21) have also investigated the role of the TM region in mIg-mediated enhanced Ag presentation to T cells. While many conclusions derived from these studies are similar, there are some discrepancies among the studies. For example, Patel and Neuberger (20) concluded that the association of Ig{alpha}/Igß is necessary and sufficient for the enhanced specific Ag presentation. In contrast, we (9) and others (11) recently showed that the association of Ig{alpha}/Igß alone may not be sufficient. Furthermore, a recent observation, that mIgG1 without the CY tail can also mediate the efficient Ag presentation in the absence of Ig{alpha}/Igß association (22), opens a question for the role of this association in mIgM-mediated, enhanced Ag presentation.

To further understand the differences in the findings, we have examined the ability of our BCL1-transfectants to present OVA and PC-OVA to several different OVA-specific T cell hybridomas, including DO-11.10, previously used by other investigators (23). We report here that the requirement for mIgM-mediated specific Ag presentation depends on the type of epitopes recognized by the T cell populations. Two types are identified: T cell epitopes generated in the endosomes accessible to variant mIgM containing A{alpha}TM regions and that generated in endocytic compartments accessible only to the wild-type mIgM. The fact that generation of separate sets of T cell epitopes is differentially affected by inhibitors of Ag processing and tyrosine kinase supports the above contention. These results suggest that processing of specific Ag internalized by the mIgM may involve multiple endocytic compartments and different proteases. Therefore, one major role of mIgM in specific Ag presentation may be efficient targeting of the Ag to separate endocytic compartments to ensure maximal processing and the consequent presentation of various epitopes to multiple T cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents

All chemicals and reagents, unless otherwise noted, were obtained from Sigma Chemical (St. Louis, MO).

Antigens

Conjugation of PC to OVA was performed as described previously (24). Briefly, OVA was dissolved in saline borate buffer (pH 9.5) and then incubated with p-diazonium phenylphosphorylcholine at room temperature for 2 h and then further incubated at 4°C overnight. The PC-conjugated OVA were dialyzed against PBS and stored at -20°C until use. The synthetic peptide representing a tryptic fragment of OVA (amino acids 323–339) was a generous gift from Dr. Stephen D. Miller (Northwestern University Medical School, Chicago, IL).

Antibodies

Cell lines secreting a murine anti-T15-Id mAb, AB1.2 (IgG1) (25), and an isotype-matched control mAb, P3, were obtained from the American Type Culture Collection (Rockville, MD). The FITC-labeled goat anti-mouse IgG1 was obtained from Caltag Laboratories (South San Francisco, CA).

T15-Id mIg transfectants

The transfectants carrying wild-type and variant mIgM gene constructs were generated as described previously (7). Briefly, the BALB/c-derived B cell lymphoma, BCL1 (26), was transfected by electroporation (27) with various expression constructs. The wild-type construct contained murine genomic sequences of productively rearranged VHS107-Cµ and V{kappa}22-C{kappa} Ig chains (28) encoding an IgM molecule with PC-binding specificity and the T15-Id. The B186, SC, and TM2 mIgM transfectants were generated by domain shuffle mutagenesis (7), where all or a portion of the 1.7-kb µTM exons containing fragments were replaced with sequences encoding equivalent regions of the MHC class II I-Ab {alpha}-chain (29). The mIgM transfectants were maintained in RPMI 1640 medium supplemented with 10% FCS (Bioproducts for Science, Indianapolis, IN), hypoxanthine (15 µg/ml), thymidine (10 µg/ml), adenine (25 µg/ml), xanthine (250 µg/ml), gentamicin (5 µg/ml), and mycophenolic acid (0.5 µg/ml). The amino acid sequences of the spacer, TM domain, and CY tail of the wild-type and substituted variant mIgM are shown in Figure 1Go.



View larger version (18K):
[in this window]
[in a new window]
  		FIGURE 1. Characterization of the mIgM constructs used in this paper. The amino acid sequences of the spacer, TM domain, and cytoplasmic tail from the wild-type and the variant mIgM are shown. Boxed areas represent the substituted spacer, TM, and cytoplasmic regions from I-Ab {alpha}-chain (B186, SC, TM1, TM2 variants). The previously reported properties of these chimeric mIgM-transfectants, namely Ig{alpha}/Igß association with the chimeric receptors (7, 19), Ca2+ mobilization upon receptor ligation (7, 19), and enhanced Ag presentation using an HEL-specific T cell hybridoma (7, 9), were summarized in this table.

 
T cell hybridomas

The BALB/c-derived, OVA-specific T cell hybridomas (22) DO-11.10, 3DO-54.8, 3DO-54.6, 3DO-26.1, 3DO-36.6.1, and 4DO-63.10 were obtained from Dr. Philippa Marrack (National Jewish Hospital and Research Center, Denver, CO).

Denaturation and enzyme digestion of OVA

The procedure of denaturation and enzyme digestion of OVA was adapted from previous reports (23, 30). Briefly, OVA (300 mg) was dissolved in 20 ml of 0.1 M Tris-buffer (pH 8.2). Solid urea was added to a final concentration of 8 M, and 2-ME was added to a final concentration of 0.2 M. The mixture was incubated for 12 h at room temperature. Sulfhydryl groups were then alkylated by addition of iodoacetic acid to a final concentration of 0.3 M. The pH was maintained at 7 by titration with NaOH. After incubation for 2 h at room temperature, the mixture was dialyzed against 5 mM Na2HPO4 and then dialyzed against 0.1 M NH4HCO3 (pH 8.2). For trypsin digestion, the denatured OVA was treated 2x with N-tosyl-L-phenylalanine chloromethyl ketone (TPCK)-treated trypsin (1% w/w) at 0 and 4 h. After incubation at 37°C overnight, the reaction was terminated by lyophilization. For {alpha}-chymotrypsin digestion, denatured OVA in NH4HCO3 was treated with enzyme (2% w/w) for 4 h at 37°C, and the reaction was terminated by addition of 1 mM TPCK to the mixture, followed by lyophilization. The enzyme-digested OVA was resuspended in PBS and stored at -20°C until use.

Ag presentation to T cell hybridomas

The presentation of Ag by the untransfected parental cells and the mIgM transfectants to the T cell hybridomas was determined by the production of IL-2 in the culture supernatant. Briefly, T hybridoma cells (5 x 104 cells/well) were cocultured with 105 cells/well of the untransfected parental cells or mIgM transfectants in the presence of various concentrations of Ag or PBS at 37°C for 20 to 24 h in 96-well culture plates. The production of IL-2 in the culture supernatant was determined by its ability to support the proliferation of an IL-2-dependent cell line, CTLL-2 (31), based on the [3H]TdR uptake of the indicator cells. [3H]TdR uptake was measured by liquid scintillation counting (Beckman Instruments, Fullerton, CA).

Inhibition of Ag processing with protease inhibitors and fixation of APC

For inhibition of Ag presentation with protease inhibitors, 1 x 105 cells (in 100 µl) of the wild-type or B186 variant mIgM transfectants were first incubated with 2 mM of leupeptin or 40 mM of NH4Cl in 96-well plates for 30 min at 37°C. Various concentrations of Ag or PBS, as well as 5 x 104 T hybridoma cells, were then added to the culture in a final concentration of 1 mM leupeptin or 20 mM NH4Cl. After 22 to 24 h at 37°C, the culture supernatant was assayed for the production of IL-2 as described earlier. For fixation of APC, mIgM transfectants were first washed twice with HBSS and twice with PBS. Cells were then incubated with 0.5% paraformaldehyde in PBS for 5 min at 37°C. The reaction was stopped by addition of an excess amount of ice-cold 0.5% glycyl glycine. The fixed cells were washed twice with cold 10% FCS in PBS, resuspended in RPMI 1640 medium, and incubated at 37°C for 1 h. After washing, the fixed cells (2 x 105 cells) were cocultured with Ag or PBS, and T cell hybridomas (5 x 104 cells) for 22 to 24 h. The production of IL-2 by the T cell hybridomas was determined as described earlier.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
mIgM with the A{alpha}TM region mediates Ag-specific presentation to some but not all T cells

We have shown previously that the N-terminal 1/3 region of µTM is involved in the Ag-specific, enhanced presentation of PC-HEL to T cells by B cells, using various transfectants expressing anti-PC mIgM and an HEL-specific T cell hybridoma (7, 8, 9). However, other investigators have shown that the C-terminal region is important for such specific Ag presentation, using an OVA-specific T cell hybridoma (10, 11). To explore the possibility that the difference between these systems reflects the type of T cells and/or T cell epitopes involved, the ability of mIgM transfectants (Figs. 1Go and 2Go) to present OVA and PC-OVA was tested using several OVA-specific T cell hybridomas (23), including DO-11.10 used previously by other investigators (10, 11). Since distinct Ag presentation pathways may be potentially utilized by different MHC class-II molecules, three hybridomas (DO-11.10, 3DO-54.8, and 3DO-54.6) restricted with I-Ad and three hybridomas (3DO-26.1, 4DO-63.10 and 3DO-36.6.1) restricted with I-Ed (23) were selected. The wild-type mIgM transfectant (BCg3R), but not the parental cells (BCL1), was able to specifically present PC-OVA to these T cell hybridomas, at a concentration 100-fold lower than that required for OVA (Fig. 2Go). These results are consistent with the previous reports (9, 10, 11).



View larger version (31K):
[in this window]
[in a new window]
  		FIGURE 2. Ag presentation to various OVA-specific T cell hybridomas. The untransfected BCL1 parental cells, as well as the wild-type (BCg3R) and variant (B186) mIgM transfectants (1 x 105 cells), were cultured with 5 x 104 cells of the I-Ad-restricted (DO-11.10, 3DO-54.8, or 3DO-54.6), or the I-Ed-restricted (3DO-26.1, 4DO-63.10, or 3DO-36.6.1), OVA-specific T cell hybridomas in the presence of various concentrations of PC-OVA (open circles) or OVA (closed circles) for 22 to 24 h. The culture supernatant was assayed for the production of IL-2 based on [3H]TdR uptake of an IL-2-dependent cell line, CTLL-2. The inserts represent the wild-type or variant T15-Id mIgM. Separation of Group 1 and Group 2 hybridomas was based on the enhanced Ag-specific stimulation of hybridomas presented by B186 variant carrying the spacer, TM, and CY regions of I-A{alpha} chain.

 
However, to our surprise, the B186 transfectant, carrying a variant mIgM substituted with the spacer, TM and CY of A{alpha}, was also able to preferentially present PC-OVA to some (including DO-11.10) but not all T cell hybridomas. The OVA-specific T cell hybridomas could be divided into two groups, based on their responses to specific Ag presentation by the transfectants. Both the wild-type and the B186 variant mIgM transfectants were able to present PC-OVA at a low Ag concentration to a group (Group 1) of hybridomas (DO-11.10, 3DO-54.8, 3DO-26.1, and 4DO-63.10). In contrast, only the wild-type mIgM transfectant was able to preferentially present PC-OVA to the second group (3DO-54.6 and 3DO-36.6.1) of hybridomas. Therefore, it appears that while the wild-type transfectant is able to preferentially present specific Ag to both groups of the T cell hybridomas, the B186 variant can preferentially present only to Group 1 hybridomas. These results indicate that the functions of µTM in Ag processing and/or trafficking the complexes into endocytic compartments are partly shared with A{alpha}TM. In addition, the preferential presentation of specific Ag by the B186 transfectant to T cells does not appear to be restricted by the type (I-A or I-E) of class II molecules.

N-terminal 2/3 A{alpha}TM is sufficient to mediate the enhanced Ag presentation

To further define the region of A{alpha}TM responsible for the enhanced Ag processing in the B186 variant, additional transfectants carrying various levels of substitution of µTM with A{alpha}TM regions were tested. These include SC mIgM substituted with A{alpha}TM only in the TM domain, TM1 in the N-terminal 2/3 TM, and TM2 in the N-terminal 1/3 TM (Fig. 1Go). As shown in Figure 3GoA, the SC and TM1 transfectants also display efficient specific Ag presentation to the Group 1 T cell hybridomas. However, this enhanced function of A{alpha}TM for Ag-processing is lost in the TM2 transfectant that expresses mIgM substituted with only the N-terminal 1/3 A{alpha}TM (Fig. 3GoB). Thus, it is likely that the 2/3 A{alpha}TM sequence in the TM1 transfectant is sufficient for such µ-A{alpha}TM-mediated preferential presentation of PC-OVA. Since the wild-type mIgM transfectant also yields a similar enhancement of T cell stimulation, A{alpha}TM and µTM regions appear to share a function for enhanced Ag presentation to Group 1 hybridomas. In contrast, substitution of mIgM with the A{alpha}TM regions abolishes the enhanced Ag processing/presentation to Group 2 hybridomas (Fig. 3Go).



View larger version (36K):
[in this window]
[in a new window]
  		FIGURE 3. Ag presentation to Group 1 and Group 2 T cell hybridomas by the SC, TM1, and TM2 transfectants. A, The wild-type (BCg3R) and variant (B186, SC, and TM1) mIgM transfectants were cultured with either a Group 1 (3DO-54.8) or Group 2 (3DO-54.6) I-A-restricted hybridoma, in the presence of 50 or 150 µg of PC-OVA for 22 to 24 h. B, The untransfected BCL1 parental cells, as well as the wild-type (BCg3R) and variant (B186, SC, and TM2) mIgM transfectants, were cultured with Group 1 hybridomas, in the presence of various concentrations of PC-OVA (open circles) or OVA (closed circles) for 22 to 24 h. None of the hybridomas reactive to OVA was preferentially stimulated by TM2. A representative result with 3DO-26.1 hybridoma is shown here. The production of IL-2 in the culture supernatant was measured as described above.

 
Requirement of tyrosine phosphorylation is different for the generation of distinct epitopes

Previously, we and others demonstrated that the ability of mIgM to transduce signals may not be sufficient for enhanced Ag presentation (7, 9, 10, 11). To verify the necessity of tyrosine kinase activity for enhanced Ag presentation to Group 1 hybridomas, the effect of herbimycin-A, a tyrosine-kinase inhibitor, on Ag presentation by the transfectants was examined (Fig. 4Go). Interestingly, the level of T cell activation was rather increased for Group 1 hybridomas following treatment with herbimycin. In contrast, such a treatment completely abolished the ability to stimulate Group 2 hybridomas. These results indicate that tyrosine phosphorylation is not necessary for enhanced Ag presentation by B cells to Group 1 hybridomas, while it appears to be necessary for Group 2 hybridomas.



View larger version (18K):
[in this window]
[in a new window]
  		FIGURE 4. Effect of herbimycin-A on Ag presentation to the representative Group 1 and Group 2 hybridomas. Wild-type mIgM transfectants were divided in two groups (8 x 106 cells/3 ml) and incubated 14 h in either 2 µM of herbimycin A or without herbimycin A. Both groups of transfectants were washed and placed (5 x 104 cells/culture) in an assay. Both groups were cocultured for 24 h with the group 1 hybridoma, 3DO 54.8 (5 x 104 cells/culture) or the group 2 hybridoma, 3DO 54.6, in the presence of various concentrations of PC-OVA.

 
Generation of epitopes recognized by the two groups of T cell hybridomas is differentially inhibited

The above results suggest that certain T cell epitopes from OVA may be generated in the endosomes accessible to the variant mIgM, while others may be generated in other compartments accessible only to the wild-type mIgM. To test this possibility, the effect of two protease inhibitors (NH4Cl and leupeptin) on the processing/presentation of OVA and PC-OVA was initially examined, using the wild-type mIgM transfectant as APC. The generation of epitopes recognized by Group 1 hybridomas (DO-11.10, 3DO-26.6.1, and 3DO-54.8) was sensitive to both leupeptin and NH4Cl at low concentrations of PC-OVA (specific Ag), as well as high concentrations of OVA (nonspecific Ag). In contrast, stimulation of Group 2 hybridomas (3DO-36.1 and 3DO-54.6) with specific Ag was not inhibited by leupeptin treatment, but was sensitive to NH4Cl. Figure 5GoA shows an example of the differential sensitivity, using I-Ad-restricted 3DO-54.8 and 3DO-54.6 hybridomas. Presentation of PC-OVA to Group 1 hybridomas (e.g., 3DO-54.8) by the B186 variant was also inhibited by leupeptin, similar to the presentation by the wild-type mIgM transfectant (Fig. 5GoB). These results strongly suggest that epitopes recognized by the two separate groups of OVA-specific T cell hybridomas are produced in distinct endocytic compartments after processing with different proteases.



View larger version (35K):
[in this window]
[in a new window]
  		FIGURE 5. Ag presentation to various T cell hybridomas in the presence of leupeptin or ammonium chloride. The wild-type mIgM transfectant (1 x 105 cells in 100 µl) was incubated with PBS (control), ammonium chloride (40 mM), or leupeptin (2 mM) for 30 min at 37°C. Various T hybridoma cells (5 x 104 cells in 100 µl) were then added to the culture, with either low Ag concentrations of PC-OVA (3 to 15 µg/ml) or high Ag concentrations of OVA (150 to 200 µg/ml). Results with DO-11.10 and 3DO-36.6.1 hybridomas are shown here. B, The wild-type and the B186 variant mIgM transfectants (1 x 105 cells in 100 µl) were incubated with PBS or 2 mM of leupeptin for 30 min at 37°C. The OVA-specific T cell hybridoma, 3DO-54.8 (5 x 104 cells in 100 µl), was then added to the culture with either a low concentration of PC-OVA (5 µg/ml) or a high concentration of OVA (150 µg/ml).

 
Generation of epitopes for the two groups of hybridomas requires different levels of Ag processing

The possibility that additional Ag processing is required to generate the epitopes recognized by Group 2 T cell hybridomas was further examined (Fig. 6GoA). Both groups of the hybridomas were stimulated with native OVA, denatured OVA, chymotrypsin-digested OVA, and trypsin-digested OVA, in the presence of fixed or unfixed APC. Group 1 hybridomas (e.g., 3DO-54.8) displayed a high level of response to trypsin-digested OVA, and a reduced, but significant, level of response to denatured OVA in the presence of fixed APC. In contrast, Group 2 hybridomas (e.g., 3DO-54.6) showed undetectable levels of response to either denatured OVA or trypsin digests of OVA.



View larger version (33K):
[in this window]
[in a new window]
  		FIGURE 6. Ag presentation to Group 1 and Group 2 hybridomas by fixed or nonfixed wild-type mIgM transfectants. A, Paraformaldehyde-fixed (2 x 105 cells) or nonfixed (1 x 105) wild-type mIgM transfectants were incubated with 5 x 104 T cell hybridomas (3DO-54.8 or 3DO-54.6) in the presence of 1 mg/ml of native OVA (OVA), denatured OVA (d-OVA), trypsin-digested OVA (trypsin), or PBS. B, The OVA-specific T cell hybridomas (5 x 104 cells) were cocultured with 1 x 105 of nonfixed or 2 x 105 of paraformaldehyde-fixed wild-type mIgM transfectants and various concentrations of the OVA323–339 peptide.

 
To verify the potential differences in the epitopes recognized by the different groups of T cell hybridomas, a synthetic peptide (OVA323–339) representing a tryptic fragment of OVA was used to stimulate the T cell hybridomas (Fig. 6GoB). Both groups of T cell hybridomas (3DO-54.8 and 3DO-54.6) responded to OVA323–339 in the presence of intact APC. However, only Group 1 hybridomas (DO-11.10 and 3DO-54.8), but not the Group 2 hybridoma (3DO-54.6), were able to respond to the peptide presented by fixed APC (Fig. 6GoB). Therefore, it is most likely that further processing of OVA323–339 is required for optimal stimulation of the Group 2 hybridoma, as shown with OVA digests (Fig. 6GoA). These results strongly suggest that the fine specificity of epitopes recognized by these two groups of T cell hybridomas is different. For the generation of epitopes recognized by Group 1 T cells, degradation by a trypsin-like protease appears to be sufficient. However, further processing of the products appears to be required for stimulation of Group 2 hybridomas.

Longer Ag processing time is required for Group 2 than Group 1 hybridomas

If the generation of these T cell epitopes recognized by the different T cell hybridoma groups is a sequential event, the epitopes recognized by Group 1 hybridomas could be produced before the epitopes for Group 2 hybridomas. To verify the time sequence of the epitope generation, the wild-type mIgM transfectant was pulsed with PC-OVA for varying time intervals and then chemically fixed with paraformaldehyde. The fixed APC were subsequently cultured with the representative T cell hybridomas (3DO-54.8 and 3DO-54.6) of the different groups. Figure 7Go clearly indicates that the generation of the epitopes for Group 1 hybridomas is much faster (30–60 min) than that of the epitopes for Group 2 hybridomas, which takes longer than 120 min. These results suggest that a sequential deliberation of mIg/Ag complexes into separate endocytic compartments may be necessary for processing of certain T cell epitopes. Thus, µTM is likely to facilitate the transport of Ag/mIg complexes to these compartments.



View larger version (21K):
[in this window]
[in a new window]
  		FIGURE 7. Duration of Ag processing time for epitopes recognized by Group 1 and Group 2 hybridomas. Wild-type mIgM transfectants were incubated with either 50 µg/ml OVA or 50 µg/ml PC-OVA and incubated at 37°C. At 10 min, 30 min, 1 h, and 2 h, the mIgM transfectants were fixed with paraformaldehyde. The fixed transfectants (5 x 104 cells/culture) were then cocultured with 5 x 104 cells/culture of Group 1 (3DO 54.8) or Group 2 (3DO 54.6) hybridomas for 24 h to stimulate the T cells.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Stimulation of T cells can be achieved following Ag presentation by a variety of APC types. However, an Ag-specific B cell can function as a much more efficient APC. The mIg/Ag complexes appear to be ultimately transported from the plasma membrane to lysosome-like compartments of B cells (9, 32), and different degradation products of the ligands may be produced as the mIg/ligand complexes travel through various steps of the endocytic pathway (33, 34). Both specific and nonspecific Ags may be transported to the endocytic compartments, and proper epitopes for various T cell populations can be generated during these processes. However, the machinery for the intracellular transport of a nonspecific Ag may be inefficient as compared with that of specific Ag recognized by mIg (35). However, this difference may represent quantitative differences in the Ag pathways, since similar sensitivities to the inhibitors are observed for processing of specific and nonspecific Ag (Fig. 5Go).

Previous studies (7, 8, 9, 10, 11) have suggested that mIg-mediated increased uptake of specific Ag is not sufficient for efficient Ag presentation to T cells. Proper delivery and processing of the internalized Ag may also be essential (9, 10, 36). While the wild-type transfectant is able to preferentially present PC-OVA to all the OVA-specific T cell hybridomas, the variant transfectants (B186 and SC) carrying A{alpha}TM can preferentially present PC-OVA only to Group 1 hybridomas (Figs. 2Go and 3Go). However, such a variant mIgM is not able to efficiently target its ligands to dense lysosomal compartments (9). Therefore, these results collectively suggest that certain T cell epitopes on OVA are generated in a separate endocytic compartment that is accessible to the variant mIgM transfectants. The generation of epitopes recognized by the other (Group 2) OVA-specific T cell hybridomas may require transport of Ag to a lysosome-like dense compartment, which can be mediated only by the wild-type mIgM (Ref. 9, and Figs. 2Go and 3Go). The generation of these two sets of T cell epitopes in different endocytic compartments appears to involve Ag processing by different proteases, since a differential sensitivity to leupeptin is detected for the generation of such epitopes (Figs. 5Go and 6Go).

The ability of endosomes to generate epitope(s) recognized by Group 1 OVA-specific T cell hybridomas is further supported by reports from McCoy et al. (37, 38). These investigators have shown that OVA internalized into early endosomes through TfR is efficiently presented to a Group 1 T cell hybridoma, 3DO-54.8. In addition, the mIg/Ag complexes are found primarily in the early endosomes, colocalized with TfR (37, 39). Moreover, the presentation of OVA targeted to early endosomes via TfR is inhibited by chloroquine (38), which functions similarly to NH4Cl (Fig. 4Go). These suggest that the early endosomes accessible to TfR are able to properly process OVA for this Group 1 hybridoma. This compartment also contains class II molecules as well as proteolytic enzymes that are involved in Ag processing, peptide-loading, and recycling of the molecules to the surface for Ag presentation (40). These early endosomes appear to be accessible to the mIgM/Ag complexes, including variant mIgM/Ag complexes where the epitope peptides for Group 1 hybridomas are generated and possibly loaded to the class II molecules. Thus, the initial Ag processing for Group 1 hybridomas may occur in an early endosomal compartment of B cells. However, the epitopes for Group 2 hybridomas appear to be generated in a dense endocytic compartment after further transport of specific Ag, based on subcellular fractionation (9) and the duration required for Ag processing (Fig. 7Go). Therefore, the processing of OVA may involve sequential degradation events mediated by separate proteases in different endocytic compartments. Together, mIgM is likely to facilitate transport of the Ag complexes to various endocytic compartments for extensive processing, and the early transport events may be shared with class II molecules to promote efficient Ag presentation.

The potential role of Ig{alpha}/Igß association to mIgM in B cell receptor-mediated specific Ag presentation is controversial. Since these chimeric µ-A{alpha}TM transfectants, except TM2, do not form mIgM/Ig{alpha}/Igß complexes (19), the association of Ig{alpha}/Igß is unlikely to be required for the specific Ag presentation to Group 1 hybridomas (Figs. 2Go and 3Go). However, this contradicts the conclusion (20) that signal transduction via Ig{alpha}/Igß is necessary and sufficient for enhanced Ag presentation to a Group 1 hybridoma, DO-11.10, using a chimeric µ-chain replaced by H-2KbTM and IgßCY. Furthermore, the µ-chains carrying TM substitutions and the cytoplasmic tails of Ig{alpha} (YS/VV-Ig{alpha}CY) or Igß (YS/VV-IgßCY) did not restore the enhanced Ag presentation (11) deficient in the TM substitutions. Therefore, this signal is not likely to be sufficient for the enhanced Ag presentation. FcR/Ig{alpha}CY and FcR/IgßCY recombinant molecules are known to facilitate enhanced Ag-specific presentation by targeting the Ag complexes toward a recycling compartment for MHC II and TfR (41). Therefore, the Igß function for enhanced Ag presentation (20) may actually represent targeting to such a recycling compartment. This would be consistent with our results, suggesting that the epitope(s) recognized by Group 1 hybridomas, including DO-11.10, are likely generated in early recycling endosomes. However, the FcR/Ig{alpha}CY chimeras were able to target Ag to a subsequent compartment utilizing newly synthesized class II molecules (41). This may be the same compartment where Group 2 epitopes are loaded.

The relationship between signal transduction and enhanced Ag presentation is not yet clear, including the involvement of tyrosine phosphorylation. B186, SC as well as TM1 containing various degrees of substitutions in the µTM with A{alpha}TM, display deficiencies in tyrosine phosphorylation upon ligation of mIgM (9, 19). However, these APCs provide enhanced Ag presentation to Group 1, but not to Group 2, hybridomas (Figs. 2Go and 3Go). Therefore, tyrosine phosphorylation is unlikely to be involved in targeting Ag complexes to the early recycling endosomes, where epitopes for Group 1 hybridomas might be generated. In contrast, such phosphorylation may be necessary for targeting to the dense late-endosomes/lysosomes to generate epitopes for Group 2 hybridomas (Fig. 4Go). If generation of the epitopes is sequential at the separate endocytic compartments, alterations in the TM regions leading to the blocking of the initial transport of the complexes will inhibit the enhanced Ag presentation to both groups of hybridomas. This possibility is strongly supported by our results. All the functional TM substitutions (B186, SC, and TM1) abrogate Ag-specific presentation only to Group 2, but not to Group 1, hybridomas alone. TM2 has apparently lost the function for both groups, suggesting that this variant may be deficient in the initial transport of the mIg/Ag complexes to the recycling compartment(s) involved in processing of epitopes for Group 1 hybridomas. This is consistent with a recent report indicating that a chemical blocking of the recycling compartment function inhibits further Ag processing (42).

The fact that both mIgM molecules with µTM or A{alpha}TM can enhance Ag processing/presentation to T cells is very intriguing. Class II molecules with homologous A{alpha}TM regions (A{alpha}/Aß-A{alpha}TM) are able to generate intracellular cAMP levels similar to the wild-type molecules (43). Thus, the above µ-A{alpha}TM transfectants (e.g., B186, SC) expressing A{alpha}TM dimers may also generate such a signal, which in turn may be able to enhance Ag processing (44). The potential involvement of the costimulatory molecules was excluded, since all the transfectants constitutively displayed similar high levels of B7 molecules on the surface when determined with CTLA4-Ig (data not shown). In addition, the TM region of class II molecules has been suggested to play an important role in the transport of the molecules to the plasma membranes (45, 46). Therefore, A{alpha}TM in our mIgM constructs is most likely involved in the transport of the mIg/Ag complexes to early endosome-like compartments. Such transport mediated by A{alpha}TM may be similar to that provided by µTM.

Taken together, the physiologic role of mIg in the enhanced presentation of specific Ag appears to efficiently direct Ag to a specific endocytic pathway. During the transport of specific Ag, it can be processed by various proteases in multiple endocytic compartments. Depending on the nature of Ag, some T cell epitopes may become available in the early endosomes, while others may need to be generated in the subsequent endocytic compartments. With the efficient targeting by mIg to travel through many endocytic compartments, Ag can be maximally processed, ensuring the presentation of many potential T cell epitopes on the Ag. This may greatly increase the capacity for the activated B cells (through binding of specific Ag to mIg) to interact, activate, and receive help from multiple T cells with diverse reactivity. Different TM segments of the µ-chain may be involved in the targeting into different endocytic compartments. One of the steps may be shared with A{alpha}TM of the MHC class II molecules, targeting the Ag complexes to the same compartment with class II molecules to provide efficient peptide-loading and subsequent presentation.


   Footnotes
 
1 This work was supported by U.S. Public Health Service Research Grant R01 AI15446. Back

2 Present address: Department of Medicine, University of Washington, Seattle, WA 98195. Back

3 Address correspondence and reprint requests to Dr. Byung S. Kim, Department of Microbiology-Immunology, Northwestern University Medical School, 303 E. Chicago Ave., Chicago, IL 60611. E-mail address: Back

4 Abbreviations used in this paper: mIg, membrane-bound Ig; TM, transmembrane; CY, cytoplasmic; PC, phosphorylcholine; HEL, hen egg white lysozyme; PC-HEL, PC-conjugated HEL. Back

Received for publication October 14, 1997. Accepted for publication December 22, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Reth, M.. 1992. Antigen receptors on B lymphocytes. Annu. Rev. Immunol. 10:97.[Medline]
  2. Germain, R. N., D. H. Margulies.. 1993. The biochemistry and cell biology of antigen processing and presentation. Annu. Rev. Immunol. 11:403.[Medline]
  3. Rock, K. L., B. Benacerraf, A. K. Abbas. 1984. Antigen presentation by hapten-specific B lymphocytes. I. Role of surface immunoglobulin receptors. J. Exp. Med. 160:1102.[Abstract/Free Full Text]
  4. Lanzavecchia, A.. 1985. Antigen-specific interaction between T and B cells. Nature 314:537.[Medline]
  5. Stevens, T. L., J. H. Blum, S. P. Foy, L. Matsuuchi, A. L. DeFranco. 1994. A mutation of the µ transmembrane that disrupts endoplasmic reticulum retention: effects on association with accessory proteins and signal transduction. J. Immunol. 152:4397.[Abstract]
  6. Cambier, J. C., C. M. Pleiman, M. R. Clark. 1994. Signal transduction by the B cell antigen receptor and its coreceptors. Annu. Rev. Immunol. 12:457.[Medline]
  7. Parikh, V. S., G. A. Bishop, K. J. Liu, B. T. Do, M. R. Ghosh, B. S. Kim, P. W. Tucker. 1992. Differential structure-function requirements of the transmembranal domain of the B cell antigen receptor. J. Exp. Med. 176:1025.[Abstract/Free Full Text]
  8. Liu, K.-J., V. S. Parikh, P. W. Tucker, B. S. Kim. 1993. Role of the B cell antigen receptor in antigen processing and presentation: involvement of the transmembrane region in intracellular trafficking of receptor/ligand complexes. J. Immunol. 151:6143.[Abstract]
  9. Liu, K.-J., V. S. Parikh, P. W. Tucker, B. S. Kim. 1994. Surface immunoglobulins mediate efficient transport of antigen to lysosomal compartments resulting in enhanced specific antigen presentation by B cells. Eur. J. Immunol. 24:2755.[Medline]
  10. Shaw, A. C., R. N. Mitchell, Y. K. Weaver, J. Campos-Torres, A. K. Abbas, P. Leder. 1990. Mutations of immunoglobulin transmembrane and cytoplasmic domains: effects on intracellular signaling and antigen presentation. Cell 63:381.[Medline]
  11. Mitchell, R. N., K. A. Barnes, S. A. Grupp, M. Sanchez, Z. Misulovin, M. C. Nussenweig, A. K. Abbas. 1995. Intracellular targeting of antigens internalized by membrane immunoglobulin in B lymphocytes. J. Exp. Med. 181:1705.[Abstract/Free Full Text]
  12. Qiu, Y., X. Xu, A. Wandinger-Ness, D. P. Dalke, S. K. Pierce. 1994. Separation of subcellular compartments containing distinct functional forms of MHC class II. J. Cell Biol. 125:595.[Abstract/Free Full Text]
  13. Amigorena, S., J. R. Drake, P. Webster, I. Mellman. 1994. Transient accumulation of new class II MHC molecules in a novel endocytic compartment in B lymphocytes. Nature 369:113.[Medline]
  14. Tulp, A., D. Verwoerd, B. Dobberstein, H. L. Ploegh, J. Pieters. 1994. Isolation and characterization of the intracellular MHC class II compartment. Nature 369:120.[Medline]
  15. West, M. A., J. M. Lucocq, C. Watts. 1994. Antigen processing and class II MHC peptide-loading compartments in human B-lymphoblastoid cells. Nature 369:147.[Medline]
  16. Peters, P. J., G. Raposo, J. J. Neefjes, V. Oorschot, R. L. Leijendekker, H. J. Gueze, H. L. Ploegh. 1995. Major histocompatibility complex class II compartments in human B lymphoblastoid cells are distinct from early endosomes. J. Exp. Med. 182:325.[Abstract/Free Full Text]
  17. Catello, F., R. N. Germain. 1995. Extensive trafficking of MHC class II-invariant chain complexes in the endocytic pathway and appearance of peptide-loaded class II in multiple compartments. Immunity 2:73.[Medline]
  18. Parikh, V. S., C. Nakai, S. J. Yokota, R. B. Bankert, P. W. Tucker. 1991. COOH terminus of membrane IgM is essential for an antigen-specific induction of some but not all early activation events in mature B cells. J. Exp. Med. 174:1103.[Abstract/Free Full Text]
  19. Michnoff, C. H., V. S. Parikh, D. L. Lelsz, P. W. Tucker. 1994. Mutations within the NH2-terminal transmembrane domain of membrane immunoglobulin (Ig) M alters Ig{alpha} and Igß association and signal transduction. J. Biol. Chem. 269:24237.[Abstract/Free Full Text]
  20. Patel, K. J., M. S. Neuberger. 1993. Antigen presentation by the B cell antigen receptor is driven by the alpha/beta sheath and occurs independently of its cytoplasmic tyrosines. Cell 74:939.[Medline]
  21. Pleiman, C. M., N. C. Chien, J. C. Cambier. 1994. Point mutations define a mIgM transmembrane region motif that determines intersubunit signal transduction in the antigen receptor. J. Immunol. 152:2837.[Abstract]
  22. Knight, A. M., J. M. Lucocq, A. R. Prescott, S. Ponnambalam, C. Watts. 1997. Antigen endocytosis and presentation mediated by human IgG1 in the absence of Ig{alpha}/Igß dimer. EMBO J. 16:3842.[Medline]
  23. Shimonkevitz, R., J. Kappler, P. Marrack, H. Grey. 1983. Antigen recognition by H-2-restricted T cells. I. Cell-free antigen processing. J. Exp. Med. 158:303.[Abstract/Free Full Text]
  24. Jang, Y. S., K. H. Lim, B. S. Kim. 1991. Analysis of T cell reactivities to phosphorylcholine-conjugated hen egg lysozyme in C57BL/6 mice: hapten-conjugate specificity reflects an altered expression of a major carrier epitope. Eur. J. Immunol. 21:1303.[Medline]
  25. Kearney, J. F., R. Barletta, Z. S. Quan, J. Quintans. 1981. Monoclonal vs. heterogeneous anti-H-8 antibodies in the analysis of the anti-phosphorylcholine response in BALB/c mice. Eur. J. Immunol. 11:877.[Medline]
  26. Gronowicz, E. S., C. A. Doss, F. D. Howard, D. C. Morrison, S. Strober. 1980. An in vitro line of the B cell tumor BCL1 can be activated by LPS to secrete IgM1. J. Immunol. 125:976.[Abstract]
  27. Chu, G., H. Hayakawa, P. Berg. 1987. Electroporation for the efficient transfection of mammalian cells with DNA. Nucleic Acids Res. 15:1311.[Abstract/Free Full Text]
  28. Guise, J. W., P. L. Lim, D. Yuan, P. W. Tucker. 1988. Alternative expression of secreted and membrane forms of immunoglobulin µ-chain is regulated by transcriptional termination in stable plasmacytoma transfectants. J. Immunol. 140:3988.[Abstract]
  29. Webb, C. F., C. Nakai, P. W. Tucker. 1989. Immunoglobulin receptor signaling depends on the carboxyl terminus but not the heavy-chain class. Proc. Natl. Acad. Sci. USA 86:1977.[Abstract/Free Full Text]
  30. Chesnut, R. W., R. O. Endres, H. M. Grey. 1980. Antigen recognition by T cells and B cells: recognition of cross-reactivity between native and denatured forms of globular antigens. Clin. Immunol. Immunopathol. 15:397.[Medline]
  31. Kappler, J. W., B. Skidmore, J. White, P. Marrack. 1981. Antigen-inducible, H-2-restricted, interleukin-2-producing T cell hybridomas: lack of independent antigen and H-2 recognition. J. Exp. Med. 153:1198.[Abstract/Free Full Text]
  32. Davidson, H. W., M. A. West, C. Watts. 1990. Endocytosis, intracellular trafficking, and processing of membrane IgG and monovalent antigen/membrane IgG complexes in B lymphocytes. J. Immunol. 144:4101.[Abstract]
  33. Davidson, H. W., C. Watts. 1989. Epitope-directed processing of specific antigen by B lymphocytes. J. Cell Biol. 109:85.[Abstract/Free Full Text]
  34. Watts, C.. 1997. Capture and processing of exogenous antigens for presentation on MHC molecules. Annu. Rev. Immunol. 15:821.[Medline]
  35. Kakiuchi, T., M. Watanabe, N. Hozumi, H. Nariuchi. 1990. Differential sensitivity of specific and nonspecific antigen-presentation by B cells to a protein synthesis inhibitor. J. Immunol. 145:1653.[Abstract]
  36. Mitchell, R. N., A. C. Shaw, Y. K. Weaver, P. Leder, A. K. Abbas. 1991. Cytoplasmic tail deletion converts membrane immunoglobulin to a phosphatidylinositol-linked form lacking signaling and efficient antigen internalization functions. J. Biol. Chem. 266:8856.[Abstract/Free Full Text]
  37. McCoy, K. L., M. Noone, J. K. Inman, R. Stutzman. 1993. Exogenous antigens internalized through transferrin receptors activate CD4+ T cells. J. Immunol. 150:1691.[Abstract]
  38. McCoy, K. L., D. Gainey, J. K. Inman, R. Stutzman. 1993. Antigen presentation by B lymphoma cells: requirements for processing of exogenous antigen internalized through transferrin receptors. J. Immunol. 151:4583.[Abstract]
  39. Barnes, K. A., R. N. Mitchell. 1995. Detection of functional class II-associated antigen: role of a low density endosomal compartment in antigen processing. J. Exp. Med. 181:1715.[Abstract/Free Full Text]
  40. Salamero, J., M. Humbert, P. Cosson, J. Davoust. 1990. Mouse B lymphocyte specific endocytosis and recycling of MHC class II molecules. EMBO J. 9:3489.[Medline]
  41. Bonnerot, C., D. Lankar, D. Hanau, D. Spehner, J. Davoust, J. Salamero, W. H. Fridman. 1995. Role of B cell receptor Ig{alpha} and Igß subunits in MHC class II-restricted antigen presentation. Immunity 3:335.[Medline]
  42. Pond, L., C. Watts. 1997. Characterization of transport of newly assembled, T cell-stimulatory MHC class II-peptide complexes from MHC class II compartments to the cell surface. J. Immunol. 159:543.[Abstract]
  43. Harton, J. A., A. E. Van Hagen, G. A. Bishop. 1995. The cytoplasmic and transmembrane domains of MHC class II ß chains deliver distinct signals required for MHC class II-mediated B cell activation. Immunity 3:349.[Medline]
  44. Faassen, A. E., S. K. Pierce. 1995. Cross-linking cell surface class II molecules stimulates Ig-mediated B cell antigen processing. J. Immunol. 155:1737.[Abstract]
  45. Cosson, P., J. S. Bonifacino. 1992. Role of transmembrane domain interactions in the assembly of class II MHC molecules. Science 258:659.[Abstract/Free Full Text]
  46. Wade, W. F., J. Davoust, J. Salamero, P. Andre, T. H. Watts, J. C. Cambier. 1993. Structural compartmentalization of MHC class II signaling function. Immunol. Today 14:539.[Medline]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Liu, K.-J.
Right arrow Articles by Kim, B. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Liu, K.-J.
Right arrow Articles by Kim, B. S.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS