| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Departments of
*
Immunology,
Neurology, and
Internal Medicine, Mayo Medical and Graduate Schools, Mayo Clinic, Rochester, MN 55905; and
Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, MD 21205
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The central role that DC have in initiating an immune response has led to interest in DC-associated molecules that can regulate the biology of these cells or their interaction with T cells. Among the myriad of costimulatory molecules elaborated on DC, the role of B7 superfamily members in T cell activation and differentiation is well established (4, 5). Although both B7-1- and B7-2-transfected APCs can activate naive T cells, studies using Ab blockade and knockout mice have indicated a predominant role for the latter in naive T cell activation (6, 7, 8, 9, 10). The constitutive expression of B7-2 on DC suggests an important role for this costimulatory molecule in the unique ability of DC to activate naive T cells (11, 12). The rapid up-regulation of B7-2 on other professional APCs, however, has led to examination of other molecules on the surface of DC involved in T cell activation and differentiation.
DC-specific ICAM-grabbing nonintegrin (DC-SIGN) has been characterized as a DC-specific C-type lectin that promotes T cell priming through its interaction with ICAM-3 on the latter cells (13). DC-SIGN has also been shown to play a role in DC migration and DC-facilitated HIV trans infection of T cells.
Receptor activator of NF-
B (RANK) is a member of the TNFR superfamily that is expressed on DC. The interaction between RANK on DC with its cognate ligand, TNF-related activation-induced cytokine (TRANCE) expressed on activated T cells, enhances the immune response (14, 15). TRANCE-treated DC had increased survival as well as enhanced T cell-priming capacity (16, 17, 18). Interestingly, despite the immunostimulatory properties, ligation of RANK did not induce DC maturation in terms of surface costimulatory markers. The third DC-specific molecule is the recently identified B7 superfamily member B7-DC. Although B7-DC has less than 20% amino acid homology to classical B7-1 and B7-2, it maintains the Ig fold common to the B7 family members. B7-DC is most similar in amino acid sequence to B7-H1/PD-L1 (34% identity and 48% similarity), with which it shares a known receptor, PD-1. B7-DC, also termed PD-L2, as it is the second B7 superfamily member known to bind PD-1 on activated T cells, reportedly modulates both T cell proliferation and cytokine expression in vitro (19, 20). The role of DC-elaborated B7-DC in activating T cells, however, has yet to be established.
In the current study, we analyzed a small set of randomly selected sera from patients with monoclonal gammopathies for Abs that bind to mouse DC generated in vitro from bone marrow precursors. Based on previous studies identifying Abs that bind oligodendrocytes with subsequent physiologic sequela, we hypothesized that naturally occurring IgM Abs with potent biological properties are relatively common. The random screen of IgM Abs identified an Ab, serum-derived human IgM 12 (sHIgM12), that specifically cross-links B7-DC on mouse DC generated in vitro, as well as to their endogenous counterparts. The Ab has strong immunopotentiating activity on CD4 and CD8 T cells. Furthermore, the effects of sHIgM12 are dependent on the pentameric structure of the IgM Ab, as monomeric IgM subunits did not stimulate the immune response, and in fact inhibit the activity of the intact pentamer. These findings highlight the general ability of low affinity IgM Abs to cross-link cell surface receptors and mediate important biological signals. The immune potentiating effect of binding B7-DC on DC in particular, and naturally occurring IgM Abs in general, may be a valuable resource that can be harnessed for therapeutic use. We also find that the sHIgM12 Ab binds human DC derived from CD14+ cells from peripheral blood. This finding suggests the possibility that the sHIgM12 Ab could be used to modulate immunity in humans.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Human serum samples were obtained from the dysproteinemia clinic under the direction of R. Kyle and chosen solely by the presence of an Ig clonal peak of greater than 20 mg/ml. Sera were from 20 patients with a wide variety of conditions characterized by a monoclonal IgM spike, including Waldenstrom’s macroglobulinemia, lymphoma, and monoclonal gammopathy of undetermined significance. Sera were dialyzed against water, and the precipitates were collected by centrifugation (14,000 rpm/30 min) and dissolved in PBS. Solutions were centrifuged and chromatographed on a Superose-6 column (Amersham Biosciences, Piscataway, NJ). IgM fractions were pooled and analyzed by SDS-PAGE. Concentrations were determined by reading absorbance at 280 nm. IgM solutions were sterile filtered and cryopreserved.
The IgM/ Ab sHIgM12 was purified from a patient with Waldenstrom’s macroglobulinemia. The patient presented with a monoclonal spike of IgM in the serum, indicating the presence of an expanded B cell clone. Consistent with this view, unambiguous amino-terminal amino acid sequences of an H chain sequence and an L chain variable sequence were determined from Fv fragments of serum IgM by standard amino acid sequence analysis. The amino acid sequence was used to isolate a prevalent cDNA sequence from peripheral blood RNA. The sequence of the cDNA led to a prediction of the structure of the complementarity-determining region 3 of the putative mAb. This structure was verified subsequently by enzyme digestion of the protein, followed by mass-spectrometric analysis of the resulting fragments. Biotinylated sHIgM12 was prepared by adding biotin to carbohydrate moieties on serum-derived Ab. The polyclonal human IgM (pHIgM) Ab used as a control in these studies has been described (21).
Monomeric sHIgM12 was made from the pentameric form in 200 mM Tris, 150 mM NaCl, and 1 mM EDTA, pH 8.0, by reduction with 5 mM DTT (Sigma-Aldrich, St. Louis, MO) for 2 h at room temperature. Subsequent alkylation was performed for 1 h on ice with 12 mM iodacetamide. IgM monomers were isolated by chromatography on Superdex-200 column equilibrated with PBS, and characterized by reducing and nonreducing SDS-PAGE.
Mice and reagents
C57BL6/J, C3H/HeJ, and BALB/c strains of mice were obtained from Jackson ImmunoResearch Laboratories (West Grove, PA). B7-DC knockout and litter mate control bone marrow was acquired from D. Pardoll, Johns Hopkins University (Baltimore, MD). The knockout mice were generated by disruption of the second exon of the B7-DC gene on a 129/SvJ genetic background. The bone marrow was derived from animals of mixed genotype, as the knockout line in the process of being backcrossed to C67BL/6. The B7-DC status of DC derived from the bone marrow cells was confirmed by flow cytometry. B7-DC-deficient DC did not express epitopes recognized by rat anti-murine B7-DC-Ab (TY-25) nor by DC-reactive human Ab sHIgM12. OT-I and D0.11 transgenic strains of mice (22, 23) were bred and maintained at the Mayo Clinic animal facility according to the protocol approved by the Institutional Animal Care and Use Committee, Mayo Clinic. Chicken albumin was obtained from Sigma-Aldrich. The peptides used in the current study were synthesized at the Mayo Protein Core Facility, Mayo Clinic. Appropriate fluorophore-coupled anti-CD11c (HL-3), anti-B220 (RA3-6B2), anti-CD80 (16-10A1), anti-CD86 (GL-1), anti-CD44 (IM7), anti-CD69 (H1.2F3), anti-CD3e (145-2C11), anti-Mac1 (M1/70), pan-NK Ab (DX-5), anti-Kb (AF6-88.5), and anti-I-Ab (KH74) were obtained from BD PharMingen (San Diego, CA). FITC-coupled goat anti-human IgM Ab was obtained from Jackson ImmunoResearch Laboratories. Anti-DEC-205 Ab was obtained from Serotec (Oxford, U.K.). Forty-eight hours before staining, 293T cells were transiently transfected with 2 µg of plasmid and FUGENE-6 (Roche Applied Sciences, Indianapolis, IN), according to manufacturer’s instructions. B7-DC plasmid and the ligand for developing a hamster anti-murine B7-DC mAb (8C10H9H3) have been described (19). The Kb-SIINFEKL tetramer coupled to APC was prepared as described (24).
Generation of immature and mature murine DC in vitro
DC from the mouse bone marrow were isolated using an established protocol (25). Briefly, bone marrow was isolated from the long bones of the hind legs. Erythrocytes were lysed by treatment with ammonium chloride/potassium bicarbonate/EDTA at 37°C. The remaining cells were plated at the density of 1 x 106/ml in six-well plates (BD Biosciences, San Jose, CA) in RPMI 10 containing 10 µg/ml of murine GM-CSF and 1 ng/ml of murine IL-4 (PeproTech, Rocky Hill, NJ). The cells were incubated at 37°C with 5% CO2. After 2 days of culture, the cells were gently washed and replaced with RPMI 10 containing the same concentration of GM-CSF and IL-4 for another 5 days. At this stage, greater than 90% of the cells in culture were CD11c positive in most cultures. When indicated, DC from 7-day cultures were matured for an additional 48 h following addition of 10 µg/ml of LPS (Difco, Detroit, MI) or 50 µM CpG. DC maturation status was verified by flow cytometry by staining with class-II, CD80- and CD86-specific Abs. Unless otherwise indicated, all experiments were performed using bone marrow-derived DC.
Human DC were derived from CD14+ mononuclear cells isolated from peripheral blood using magnetic bead sorting (Miltenyi Biotec, Auburn, CA). The isolated cells were incubated with IL-4 and GM-CSF (R&D Systems, Minneapolis, MN) for 9 days (immature cells) or where incubated in the presence of LPS (10 µg/ml) or IL-1B/TNF-
(1870 and 2200 U/ml, respectively), or IFN-
(2000 U/ml), starting on day 7 of culture and harvested on day 9. Immature DC were CD83 negative, while a distinct population of CD83+ cells appeared in matured cultures by day 9.
Flow cytometry
Cells were washed with FACS buffer (0.5% BSA and 0.1% sodium azide in PBS) and centrifuged into a 96-well plate (Nunc, Rochester, NY). The indicated Abs were added to the wells for a 30-min incubation on ice. After three washes, cells were fixed with 1% paraformaldehyde and analyzed on a FACSCalibur (BD Biosciences-PharMingen, San Diego, CA). Data were analyzed using CellQuest software (BD Biosciences-PharMingen). In PD-1 competition experiments, bone marrow-derived DC were incubated with PD-1.Ig (10 µg) for 20 min on ice before addition of sHIgM12 (10 µg). Cells were subsequently washed twice and stained with FITC-conjugated anti-human IgM secondary Ab. In the reciprocal experiment, DC were first incubated with sHIgM12 before addition of PD-1.Ig. Following washes, cells were stained with FITC-conjugated anti-mouse IgG secondary Ab.
Isolation of endogenous DC
DC were isolated from the spleen and thymus of mice, as described (26). Briefly, the tissue was cut into small pieces and incubated with RPMI containing 2 mg/ml of collagenase, 100 µg/ml of DNase (Sigma-Aldrich), and 2% FCS for 20 min at 37°C. Subsequently, 0.031 M EDTA (pH 7.5) was added for 5 min. The cells were subjected to erythrocyte lysis using ammonium chloride/potassium bicarbonate/EDTA at 37°C, counted, and used for flow cytometry.
In vitro activation of naive T cells
Naive mouse splenocytes were harvested from mice and plated in triplicate. Responder cells (3 x 105) were stimulated in vitro for 3 days with titrated doses of Ag or Ag-pulsed DC, as indicated. The plated cells were pulsed with [3H]thymidine 18 h before harvest.
Adoptive transfer of DC and T cells
DC derived from 7-day bone marrow cultures were pulsed overnight in 1 µM of the class I-restricted peptide SIINFEKL or the class II-restricted peptide ISQAVHAAHAEINE, or with 1 mg/ml of the protein chicken OVA. The sHIgM12 or the control Ab was coincubated with the peptide in the cultures at a 10 µg/ml concentration. The cells were harvested the next day, washed three times in PBS, and injected i.v. at 107 cells/mouse for in vivo priming of T cells. DC and T cells were administered in separate injections. Spleen cells were harvested 2 or 7 days after adoptive transfer and either analyzed directly by flow cytometry or incubated in culture with various concentrations of OVA for 3 additional days. Cultures were pulsed with [3H]thymidine overnight before harvesting and evaluation for 3H incorporation as a measure of activation.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
DC were generated in culture by incubation of mouse bone marrow cells in medium supplemented with GM-CSF and IL-4. Cells from 7-day cultures were incubated with purified Ab isolated from human sera and stained with fluoresceinated goat anti-human Ab and Abs specific for cell surface molecules typically expressed on DC. As shown in Fig. 1, the human Ab sHIgM12 binds cells that express high levels of CD11c, class II, and CD86 (as well as the DC marker DEC-205; data not shown). pHIgM, as well as the remaining tested mAbs from patients with gammopathies or from EBV-transformed cell lines, did not bind appreciably to the DC populations.
|
DC isolated from tissues were examined to establish whether the determinant bound by sHIgM12 Ab is expressed by naturally differentiated DC. DC freshly isolated from spleen, thymus, and bone marrow all expressed the marker recognized by the sHIgM12 Ab (Fig. 2). In contrast, most bone marrow cells, splenic B and T cells, as well as splenic macrophages do not express appreciable amounts of the determinant recognized by sHIgM12 Ab (data not shown). B cells, T cells, NK cells, and macrophages were activated with LPS or Con A to assess whether activated lymphoid or monocytic cells express the Ag. None of the activated cells from these lineages bound sHIgM12 (data not shown). Therefore, we concluded that the sHIgM12 Ab binds a cell surface molecule expressed selectively among cells of the immune system by DC, and that this determinant is expressed increasingly as the DC mature and become activated.
|
The sHIgM12 Ab potentiates dendritic Ag-presenting function
The Ag-presenting functions of the DC were assessed in vitro. Ab-treated DC were pulsed with peptide Ag and used to stimulate naive Ag-specific T cells freshly isolated from mice expressing transgenic TCRs. DC pulsed with the class I-binding peptide SIINFEKL and incubated overnight with polyclonal IgM Ab activated naive OT-I CD8 transgenic T cells. DC treated with the same peptide and incubated with the sHIgM12 mAb overnight were 
10-fold more effective activators of naive T cells as judged by the number of pulsed DC required to induce the incorporation of [3H]thymidine (Fig. 3A). BALB/c bone marrow-derived DC pulsed with ISQAVHAAHAEINE peptide activated naive T cells freshly isolated from the DO11.10 TCR transgenic mouse even more effectively. Greater than 100-fold more DC treated with polyclonal IgM Ab were needed to activate T cells to levels observed with sHIgM12 Ab-treated DC (Fig. 3B). These experiments demonstrate that treatment of DC with an IgM Ab that binds to certain surface determinants is capable of dramatically enhancing T cell activation functions of DC.
|
To visualize what was happening to T cells in vivo, C57BL/6 Ag-pulsed, Ab-treated DC and OT-I transgenic T cells were adoptively transferred into C57BL/6 hosts. In these experiments, OT-I T cells were identified using Kb:SIINFEKL tetramers as a probe. Spleen cells were recovered 7 days after transfer, and tetramer-positive T cells were analyzed by flow cytometry to determine their activation state. T cells stimulated in vivo by DC pretreated with sHIgM12 expressed substantially higher levels of the activation markers CD44 and CD69 as compared with T cells stimulated by DC pretreated with PBS (Fig. 4). DC not pulsed with Ag had no effect on activation of transgenic T cells upon adoptive transfer, whether pretreated with sHIgM12 Ab or not (data not shown). These experiments demonstrate that sHIgM12 Ab treatment potentiates the ability of DC to activate T cells in vivo.
|
The immunomodulatory properties of the Ab sHIgM12, as well as its relative binding specificity for DC, led us to examine candidate target molecules known to have both characteristics. RANK is expressed on DC and has been shown to have immunostimulatory properties when it binds the cognate ligand TRANCE (14, 15). Furthermore, RANK ligation leads to immunopotentiation without further maturation of the DC, akin to the aforementioned observations with sHIgM12. Despite binding DC, however, sHIgM12 did not bind TGF-
-activated RAW264.7 preosteoclast cell line that expresses high levels of RANK (30 ; and confirmed in our hands by the specific binding of a TRANCE fusion protein to their cell surface; data not shown).
The role DC-SIGN plays in the initial interaction between DC and T cells makes this molecule another attractive candidate. The surface expression of DC-SIGN, however, is down-regulated by 50% upon LPS maturation, while the epitope bound by sHIgM12 is up-regulated (13). Therefore, it was not likely that Ab sHIgM12 binds to DC-SIGN.
The third candidate DC-specific molecule is the recently identified B7 superfamily member B7-DC. To determine whether the cognate epitope for sHIgM12 on the surface of DC is B7-DC, soluble murine PD-1.Ig fusion protein was coincubated with bone marrow-derived DC before staining with sHIgM12. The binding of PD-1 fusion protein attenuated subsequent staining of sHIgM12 to 50% of the mean fluorescent intensity in comparison with noncompetitive levels (Fig. 5A, left panel). The reciprocal experiment shows that sHIgM12 can also block subsequent PD-1 binding to DC (Fig. 5A, middle panel). The higher avidity of the pentameric IgM Ab may contribute to the higher levels of competition with PD-1, as binding of the latter to DC is only 20% of baseline levels following preincubation with sHIgM12. Furthermore, preincubation of CD11c+ DC with a hamster IgG mAb specific for murine B7-DC (8C10H9H3) blocked sHIgM12 staining to background levels (Fig. 5A, right panel), whereas sHIgM12 binding decreased anti-B7-DC Ab staining by 52% (data not shown). To assess directly whether sHIgM12 is binding B7-DC, 293T cells transfected with plasmid-encoding murine B7-DC were stained with sHIgM12 or control Ab, demonstrating a high level of sHIgM12 staining in >95% of transfectants, as determined by flow cytometry (Fig. 5B). As the PD-1 receptor has been shown to have dual specificity for B7-DC and a second B7 family member, B7-H1, also expressed on activated DC, we analyzed P815 cells transfected with B7-H1 to determine whether the epitope for sHIgM12 is conserved between the two family members (31). P815 cells transfected with murine B7-H1 did not bind to sHIgM12 (left panel), whereas it did bind as expected to PD-1-Ig (right panel, Fig. 5C). Therefore, the ligand for sHIgM12 on DC is B7-DC. The lack of cross-reactivity of the Ab with a related superfamily member is not surprising as the B7-DC and B7-H1 only share 38% identity in their amino acid sequences.
|
To directly assess whether sHIgM12 Ab immunopotentiates DC function by interactions with B7-DC, the T cell stimulatory capacity of DC from B7-DC knockout mice was investigated following Ab treatment. Bone marrow-derived DC generated from B7-DC-deficient or littermate control mice were pulsed overnight with OVA protein and soluble Ab treatment. DC were subsequently washed several times before coincubation with TCR transgenic OT-1 T cells. Treatment of wild-type DC with sHIgM12 enhanced T cell proliferation 5-fold compared with DC treated with pHIgM control Ab. Potentiation of T cell proliferation following treatment with sHIgM12 Ab was equivalent to that following treatment of DC with anti-CD40 Ab (Fig. 6A). sHIgM12 treatment of B7-DC-deficient DC, however, did not enhance T cell proliferation relative to pHIgM control Ab treatment (Fig. 6B). DC from B7-DC-deficient mice, however, were still responsive to Ab treatment against CD40 (Fig. 6B).
|
As B7-DC has been reported by Latchman et al. (20) to inhibit T cell proliferation and cytokine secretion, we evaluated whether the observed enhancement in CD4 and CD8 T cell proliferation (Fig. 3) was due to Ab blockade of the inhibitory interaction between B7-DC/PD-1. DC were treated in the same manner as those used in T cell proliferation assays; in brief, DC were pulsed overnight with OVA and Ab treatment, followed by several washes. The treated DC were then stained with FITC-conjugated anti-human IgM Ab for residual sHIgM12 or pHIgM binding. Fig. 7A shows that sHIgM12 could not be detected on the surface of washed DC. A separate aliquot of the treated and washed DC, however, was stained with additional sHIgM12 Ab (Fig. 7B) or anti-B7-DC Ab (Fig. 7C) at similar levels, demonstrating the presence of B7-DC on the surface of sHIgM12-treated cells after the IgM Ab was washed off. Therefore, following overnight treatment with sHIgM12 and washing, the Ab did not remain bound to the DC and did not alter the overall level of cell surface B7-DC at the time the DC were used in the T cell proliferation assays or adoptively transferred into mice. A separate aliquot of the DC treated above stimulated OT-I splenocyte proliferation to a similar level as shown in Fig. 3A. Also, addition of sHIgM12 Ab to cultures during the incubation of DC and T cells did not influence the level of stimulation, providing further indication that sHIgM12 does not block positive signals between B7-DC and ligands expressed by naive T cells (data not shown).
|
Our hypothesis is that IgM Abs, even when they have low affinity binding sites, have the ability to activate cells because they can cross-link receptor complexes on the cell surface. To test this view, monomeric fragments of sHIgM12 were evaluated for their ability to bind DC, potentiate DC function, and block the ability of intact Ab to potentiate function. Although sHIgM12 monomers bound DC less intensively than intact pentamers (data not shown), the binding sites of the monomers were still active as coincubation with monomers effectively blocked staining of DC with intact pentamers (Fig. 8A). Blocking of the ability of pentameric sHIgM12 Ab to bind DC was essentially complete. Although overnight treatment of DC with intact sHIgM12 Ab enhanced the ability of these cells to stimulate naive OT-I T cells, incubation of DC with IgM monomers of sHIgM12 did not potentiate their ability to activate the T cells (as judged by incorporation of [3H]thymidine; Fig. 8B). Importantly, the monomeric Ab fragments were able to block the ability of intact IgM to potentiate DC Ag-presenting function (Fig. 8B). Our interpretation of these experiments is that monomers effectively compete with pentamers for B7-DC on the surface of DC and that saturation of the binding sites with monomers is not sufficient to potentiate the ability of DC to activate T cells. This finding supports our view that sHIgM12 Abs potentiate DC by cross-linking B7-DC on their cell surface.
|
Our previous experience using IgM Abs that bind cells in a tissue-specific pattern suggests that Abs reacting with one species can display similar binding characteristics to cells in other species. We, therefore, tested human DC to determine whether they too might be bound by the sHIgM12 Ab. As shown in Fig. 9, 9-day immature human DC derived in culture from CD14+ peripheral blood cells following incubation in IL-4 and GM-CSF bind sHIgM12 Ab weakly, but not control pHIgM Ab. Upon stimulation with IL-1/TNF- or LPS, but not IFN-, the DC in the cultures matured, as evidenced by expression of the DC marker CD83 (not shown). As was observed in the mouse DC cultures, matured DC tended to express more ligand recognized by the sHIgM12 Ab than did immature DC. Because appropriate reagents are not available for the analysis of B7-DC expression on human DC, we are not able to verify that the ligand recognized on the human DC is in fact B7-DC. Nonetheless, these findings are suggestive that the sHIgM12 Ab might be useful for clinical manipulation of the immune response in people.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
We have analyzed the ability of IgM Abs to bind with relative specificity to defined cell types and to induce important physiological changes in two different model systems. We previously showed that certain IgM Abs bind oligodendrocytes and enhance their ability to repair the myelin wraps on axons in demyelinated lesions (32, 33, 34). Recent in vitro studies indicate that these Abs induce intracellular signals, resulting in a calcium flux within the targeted cells. We interpret these findings as evidence that the IgM Abs are cross-linking surface structures on oligodendrocytes, generating a signal that pushes the cells toward regeneration of the myelin wraps within demyelinated lesions.
An important feature of this earlier study was that functional IgM Abs were identified from a small (fewer than 100) bank of Abs that were not selected by immunization. Although most Abs did not bind oligodendrocytes demonstrably, a few did. Furthermore, these Abs seemed to have relative specificity for glial cells. This means that the frequency of IgM Abs that bind oligodendrocytes in an unselected repertoire is remarkably high. These observations led us to the hypothesis that IgM Abs that bind to a variety of different cell types might be present in natural repertoires and that these Abs may have the ability to affect important biological changes in targeted cells. To further develop this hypothesis, we sought to identify Abs that bind specifically to a second cell type, DC, and to determine whether Abs of this kind could be used to induce important biological changes in these cells.
Surprisingly, the random screen of a small number of IgM Abs led to the identification of Ab sHIgM12, a reagent that has strong immunopotentiating properties upon cross-linking B7-DC. B7-DC is a new member of the B7 family of costimulatory molecules that has recently been identified to have relative specificity for expression on DC (19). B7-DC has been shown to have a costimulatory role in the activation and proliferation of naive T cells. B7-DC costimulated CD4+ T cell proliferation even more strongly than B7-1 (19). Furthermore, B7-DC fusion protein increased secretion of IFN-, a Th1-type lymphokine, but not IL-4 or IL-10. Our study extends these observations to indicate a functional role for DC-expressed B7-DC in costimulating T cell proliferation and activation. Treatment of DC in vitro with the sHIgM12 Ab and Ag potentiates Ag-presenting functions beyond what is normally seen with Ag-pulsed DC not treated with Ab or treated with irrelevant Ab. DC treated with sHIgM12 Ab displayed 10- to 100-fold increased ability to activate naive Ag-specific T cells in vitro and an enhanced ability to prime splenic T cells upon adoptive transfer into naive animals. sHIgM12 treatment of DC in the absence of Ag, however, did not activate transgenic T cells, indicating that its ligand, B7-DC, is indeed a costimulatory molecule and not a mitogenic signal.
The mechanism of activation appears to involve cross-linking B7-DC on the cell surface of the DC. This hypothesis is supported by the finding that B7-DC-deficient DC are immunopotentiated by treatment with anti-CD40 Ab, but not by treatment with sHIgM12 Ab. Furthermore, monomeric fragments of the sHIgM12 Ab did not potentiate the immune response in wild-type DC, and were able to block the ability of intact pentamer to do so. The ability of monomer to block pentamer-mediated immunopotentiation of Ag presentation by DC can be explained by interference of binding. We have confirmed that the monomers indeed block binding of pentamers by flow cytometry. IgM pentamers are well known for their low affinity reactivity with Ags that have repeating structural motifs. The availability of 10 identical binding sites on IgM Abs allows avidity to compensate for low affinity in generating measurable interactions between the Abs and their ligands. Molecules expressed on the surface of cells function as repeating epitopes because targeted molecules can become juxtaposed in the dynamic membrane. The pentameric structure, therefore, also provides the opportunity for cross-linking cell surface molecules, an event often associated with the initiation of signaling cascades.
Other details of the mechanism underlying Ab-mediated potentiation of DC functions remain to be determined. The Ab could either act directly on DC by cross-linking cell surface determinants, changing the array of intracellular signals that regulate cell physiology, or by blocking an inhibitory interaction between DC and T cells (e.g., B7-DC and PD-1) (20). We favor the former possibility for two reasons. First, the Ab potentiates T cell responses in circumstances in which it has been incubated with DC and washed away before T cells are present. The immunostimulatory effect of sHIgM12 on T cell proliferation was observed in washed DC 24 h after Ab treatment, despite the lack of subsequent staining with anti-human IgM-FITC-conjugated secondary Ab (Fig. 9A). Furthermore, sHIgM12 treatment did not modulate the expression of B7-DC on DC before their use in T cell proliferation assays (Fig. 9, B and C). Second, the monomeric IgM fragments block immunopotentiation and pentamer binding. Therefore, we strongly suspect that the Ab cross-links an important cell surface determinant by binding B7-DC, up-regulating the ability of the treated DC to activate naive T cells. The potentiating effect of Ab treatment could enhance DC function by up-regulation of cell surface molecules, by stimulating the secretion molecules that influence T cell activation, and/or by enhancing DC survival in culture.
The identification of an Ab that enhances DC Ag-presenting functions after treatment in vitro has potential clinical ramifications. DC are being widely used in immunotherapy protocols to activate endogenous responses against tumor and viral Ags. Ab treatment of cultured DC pulsed with Ag could lead to improved immunization strategies. We have shown a 10-fold enhancement of CD8+ responses and a greater than 100-fold increase in CD4+ T cell responses stimulated by DC treated with mAb sHIgM12. This degree of immune potentiation could boost the efficacy of existing DC-based strategies.
An interesting question is raised by our studies. Do naturally occurring Abs that bind endogenous cells and induce signals have physiological significance in situ? This has been an issue of historical interest (35). The general view is that natural Abs have functions in housekeeping, facilitating the cleanup of cellular debris. Abs are not generally described that naturally function as ligands for endogenous receptors as a consequence of binding mediated by their Ag binding sites. One possibility for the dearth of reports similar to ours is that the concentration of the relevant naturally occurring Abs may be below detectable levels. Only in cases involving substantial clonal expansion of Abs in the class we are describing would noticeable biological changes occur in vivo. The presence of sporadic paraneoplastic physiological manifestations involving a wide variety of tissues in patients with IgM lymphomas, Waldenstrom’s macroglobulinemia, or monoclonal gammopathies of undetermined significance supports this assertion. Reports of clinical manifestations would be expected, and there are a number of reports that fit this profile (36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46). Similarly, polyclonal expansion of B cell populations in disease states such as Sjogren’s syndrome could result in the appearance of clinical symptoms associated with cell-specific binding of autoantibodies (42, 43). Several of the biologically active Abs that we have described are xeno-Abs for which issues relating to tolerance are not a factor. However, differences in the avidity of secreted pentameric IgM Abs and monomeric IgM receptors arrayed on the surface of B cells could result in a subset of Abs with weak affinity for self molecules. When expressed on the surface of B cells, the binding of the receptor to self determinants could be too weak to induce tolerance, but when secreted, these isogenic pentameric Abs could be reactive to these same self-encoded molecules. Autoimmunity might not become an issue unless the particular Ab were expanded in the repertoire serendipitously. In line with this view, we have described mouse Abs that bind and activate mouse oligodendrocytes (33, 34) and human Abs that bind to human oligodendrocytes (47), and in the case reported in this study, human Abs that bind to human DC. We conclude that the existence of a wide variety of human IgM Abs with reactivity to important human receptors is a reasonable supposition. Furthermore, we propose that Abs of this kind will not be difficult to identify. Because some of the human Abs bind not only to rodent cells used to investigate mechanisms of action, but also to their human counterparts, these Abs might also be useful clinically.
|
Footnotes |
|---|
2 S.R. and L.T.N. contributed equally to this work.
3 Address correspondence and reprint requests to Dr. Larry R. Pease, Department of Immunology, Mayo Medical and Graduate Schools, 200 First Street SW, Rochester, MN 55905. E-mail address: pease.larry{at}mayo.edu
4 Abbreviations used in this paper: DC, dendritic cell; DC-SIGN, DC-specific ICAM-grabbing nonintegrin; pHIgM, polyclonal human IgM; RANK, receptor activator of NF-B; sHIgM12, serum-derived human IgM 12; TLR, Toll-like receptor; TRANCE, TNF-related activation-induced cytokine.
Received for publication September 18, 2002. Accepted for publication December 10, 2002.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
1. J. Cell. Biochem. 83:320.[Medline]
/CD8 interaction on the surface of T cells. J. Immunol. 167:821.This article has been cited by other articles:
|
|
 |
|
  J.-S. Chung, T. Yudate, M. Tomihari, H. Akiyoshi, P. D. Cruz Jr., and K. Ariizumi Binding of DC-HIL to Dermatophytic Fungi Induces Tyrosine Phosphorylation and Potentiates Antigen Presenting Cell Function J. Immunol., October 15, 2009; 183(8): 5190 - 5198. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. D. Pavelko, M. J. Hansen, and L. R. Pease CTL Activation Using the Natural Low-Affinity Epitope 222-229 from Tyrosinase-Related Protein 1 Leads to Tumor Rejection Cancer Res., April 1, 2009; 69(7): 3114 - 3120. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Kobayashi, K. Iijima, S. Radhakrishnan, V. Mehta, R. Vassallo, C. B. Lawrence, J.-C. Cyong, L. R. Pease, K. Oguchi, and H. Kita Asthma-Related Environmental Fungus, Alternaria, Activates Dendritic Cells and Produces Potent Th2 Adjuvant Activity J. Immunol., February 15, 2009; 182(4): 2502 - 2510. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Radhakrishnan, L. N. Arneson, J. L. Upshaw, C. L. Howe, S. J. Felts, M. Colonna, P. J. Leibson, M. Rodriguez, and L. R. Pease TREM-2 Mediated Signaling Induces Antigen Uptake and Retention in Mature Myeloid Dendritic Cells J. Immunol., December 1, 2008; 181(11): 7863 - 7872. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Radhakrishnan, R. Cabrera, E. L. Schenk, P. Nava-Parada, M. P. Bell, V. P. Van Keulen, R. J. Marler, S. J. Felts, and L. R. Pease Reprogrammed FoxP3+ T Regulatory Cells Become IL-17+ Antigen-Specific Autoimmune Effectors In Vitro and In Vivo J. Immunol., September 1, 2008; 181(5): 3137 - 3147. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. D. Pavelko, K. L. Heckman, M. J. Hansen, and L. R. Pease An Effective Vaccine Strategy Protective against Antigenically Distinct Tumor Variants Cancer Res., April 1, 2008; 68(7): 2471 - 2478. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Okazaki and T. Honjo PD-1 and PD-1 ligands: from discovery to clinical application Int. Immunol., July 2, 2007; (2007) dxm057v1. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Radhakrishnan, K. R. Wiehagen, V. Pulko, V. Van Keulen, W. A. Faubion, K. L. Knutson, and L. R. Pease Induction of a Th1 Response from Th2-Polarized T Cells by Activated Dendritic Cells: Dependence on TCR:Peptide-MHC Interaction, ICAM-1, IL-12, and IFN-{gamma} J. Immunol., March 15, 2007; 178(6): 3583 - 3592. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Radhakrishnan, L. T. Nguyen, B. Ciric, V. P. Van Keulen, and L. R. Pease B7-DC/PD-L2 Cross-Linking Induces NF-{kappa}B-Dependent Protection of Dendritic Cells from Cell Death J. Immunol., February 1, 2007; 178(3): 1426 - 1432. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. A. Blocki, S. Radhakrishnan, V. P. Van Keulen, K. L. Heckman, B. Ciric, C. L. Howe, M. Rodriguez, E. Kwon, and L. R. Pease Induction of a gene expression program in dendritic cells with a cross-linking IgM antibody to the co-stimulatory molecule B7-DC FASEB J, November 1, 2006; 20(13): 2408 - 2410. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Zhang, Y. Chung, C. Bishop, B. Daugherty, H. Chute, P. Holst, C. Kurahara, F. Lott, N. Sun, A. A. Welcher, et al. Regulation of T cell activation and tolerance by PDL2 PNAS, August 1, 2006; 103(31): 11695 - 11700. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Radhakrishnan, E. Celis, and L. R. Pease B7-DC cross-linking restores antigen uptake and augments antigen-presenting cell function by matured dendritic cells PNAS, August 9, 2005; 102(32): 11438 - 11443. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Bayry, S. Lacroix-Desmazes, M. D. Kazatchkine, O. Hermine, D. F. Tough, and S. V. Kaveri Modulation of Dendritic Cell Maturation and Function by B Lymphocytes J. Immunol., July 1, 2005; 175(1): 15 - 20. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. R. Sloma, M. J. Hansen, A. A. MacDougall, V. P. Van Keulen, R. B. Jenkins, and L. R. Pease A Class I Transgene Reveals Regulatory Events on Chromosome 1 Marking Peripheral T Cell Differentiation and Memory J. Immunol., June 15, 2005; 174(12): 7564 - 7572. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Shin, K. Yoshimura, T. Shin, E. B. Crafton, H. Tsuchiya, F. Housseau, H. Koseki, R. D. Schulick, L. Chen, and D. M. Pardoll In vivo costimulatory role of B7-DC in tuning T helper cell 1 and cytotoxic T lymphocyte responses J. Exp. Med., May 16, 2005; 201(10): 1531 - 1541. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. L. Riley and C. H. June The CD28 family: a T-cell rheostat for therapeutic control of T-cell activation Blood, January 1, 2005; 105(1): 13 - 21. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Radhakrishnan, L. T. Nguyen, B. Ciric, D. Flies, V. P. V. Keulen, K. Tamada, L. Chen, M. Rodriguez, and L. R. Pease Immunotherapeutic Potential of B7-DC (PD-L2) Cross-Linking Antibody In Conferring Antitumor Immunity Cancer Res., July 15, 2004; 64(14): 4965 - 4972. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Radhakrishnan, K. Iijima, T. Kobayashi, M. Rodriguez, H. Kita, and L. R. Pease Blockade of Allergic Airway Inflammation Following Systemic Treatment with a B7-Dendritic Cell (PD-L2) Cross-Linking Human Antibody J. Immunol., July 15, 2004; 173(2): 1360 - 1365. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Matsumoto, H. Inoue, T. Nakano, M. Tsuda, Y. Yoshiura, S. Fukuyama, F. Tsushima, T. Hoshino, H. Aizawa, H. Akiba, et al. B7-DC Regulates Asthmatic Response by an IFN-{gamma}-Dependent Mechanism J. Immunol., February 15, 2004; 172(4): 2530 - 2541. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
, Represent the response of OT-I splenocytes to peptide-pulsed, polyclonal IgM Ab-treated cells. B, MHC class II-restricted response was assessed by pulsing Ab-treated DC with an MHC class II-binding peptide (ISQAVHAAHAEINE). Splenocytes from the cognate DO.11 TCR transgenic mice were used as responders. •, Represent the response of DO.11 splenocytes to peptide-pulsed, sHIgM12-treated DC. 
), pHIgM control Ab (•), or anti-CD40 (
) Abs. Each data point was done in triplicate.
, Represents the monomeric sHIgM12-treated group. 
, Represent the monomeric-sHIgM12, followed by native sHIgM12-treated group. The presented data depict one of two independent comparable experiments.