In vivo induction of tolerance by an Ig peptide is not affected by the deletion of FcR or a mutated IgG Fc fragment
Moustapha El-Amine1,
Jennifer A. Hinshaw1,2 and
David W. Scott1,2
1 Department of Immunology, American Red Cross, J. Holland Laboratory, Rockville, MD 20855, USA 2 Department of Immunology, George Washington University Medical Center, Washington, DC 20037, USA
Correspondence to: D. W. Scott, Department of Immunology, American Red Cross Holland Laboratory, 15601 Crabbs Branch Way, Rockville, MD 20855, USA. E-mail: scottd{at}usa.redcross.org
Transmitting editor: D. R. Littman
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Abstract
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To induce tolerance to a variety of epitopes, we have designed a gene therapy approach in which peptides or antigens are expressed in frame on a soluble IgG fusion protein scaffold and delivered via retroviral gene therapy in B cells in vivo. Initially, tolerance to the
repressor cI sequence p1102 or its immunodominant epitopes (e.g. p1226 or p7388) was elicited in both T cells and B cells when lipopolysaccharide (LPS) blasts are transduced and injected into naive or even primed recipients. While a role of secreted Ig fusion protein in this process is not clear, we have previously demonstrated the importance of antigen presentation on MHC class II of B cell antigen-presenting cells (APC) for tolerance induction. To further examine the role of the Ig and especially of the Fc portion of the IgG in tolerogenesis, we transduced LPS blasts from FcR
II/, Fc
RI/, Fc
RIII/, FcR/ or naive mice with retroviral vectors expressing IgG1102,
IgG1102 (mutated construct on position 297 of the Fc portion) or IgG1226. When these transduced LPS blasts from FcR knockout mice were injected into normal (or knockout) syngeneic recipient mice, they induced tolerance both to the immunodominant epitopes and the full-length protein in that the antibody responses to the immunodominant epitopes were reduced. In this paper, we show that this tolerance resides at both the T and B cell level. Moreover, mutation of residue 297, which affects IgG functions including FcR binding, did not alter the tolerogenicity of the construct. These results suggest that the Fc portion of the IgG molecules is not required for humoral nor for cellular tolerance induction using the IgGantigen tolerogens.
Keywords: B cell, FcR, gene therapy, IgGantigen, retroviral vector, T cell, tolerance
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Introduction
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The use of IgG carriers to induce immune tolerance to specific peptides or antigens has been studied for several years (14). We have developed a gene-therapy-based approach in which murine IgG1 heavy chains are subcloned in-frame with antigens (peptides or full-length proteins) in retroviral vectors (5). B cells transduced with these retroviral vectors efficiently induce B cell and T cell tolerance in vivo (58). We originally expected that the long half-life of IgG carriers, their ability to disseminate between intra- and extravascular spaces, and their interaction with a variety of antigen-presenting cells (APC) by FcR, may contribute to their efficacy in tolerance induction. Interestingly, deletion of the IgG heavy chain reduced the efficacy and affected the persistence of the tolerant state (6). However, the amount of Ig fusion protein in the circulation of recipients of IgG fusion protein-transduced B cells never correlated with the degree of unresponsiveness (5). Thus, the role of the Fc and FcR in tolerance induction remained unclear.
To further understand the mechanisms of tolerance induction by Ig tolerogens, we recently tested whether MHC class II knockout or B cell knockout (µMT) donors could serve as a source of tolerogenic APC in our gene therapy model. Our data was consistent with the clear involvement of B cells and the necessity of presentation on MHC class II molecules (8). Moreover, no evidence for a role of regulatory cytokines such as IL-10 has been observed (8). This work demonstrated the importance of presentation in our model of tolerance induction, but did not shed light on the possibility that the IgGantigen (Ag) molecules still can be assembled, secreted and taken up by a neighboring APC and re-presented on MHC class II molecules of the transfected B cell. This process could involve FcR present on most APC. In this paper we investigate the role that FcR play in tolerance induction by IgGAg tolerogens. Our results show that neither T cell nor B cell tolerance is influenced by FcR family members on APC.
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Methods
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Mice
CB6 F1/J (BALB/c x C57Bl/6) and C57Bl/6 mice were purchased from the Jackson Laboratory (Bar Harbor, ME), while Fcer1g/Fcgr2b (FcR double-knockout on a C57Bl/6 x 129 F1 background), B6 x 129 F1 (wild-type for FcR double-knockout on a C56Bl/6 x 129/J background) and Fcgr2b (Fc
RII knockout on a BALB/c background) mice were purchased from Taconic Laboratory (Germantown, NY). BALB/c mice were purchased from NCI (Frederick, MD). Fcer1g (Fc
RI, Fc
RIII and Fc
RI knockout, on C57Bl/6 background) are a kind gift from Dr Dragana Jankovi
(NIAID, NIH, Bethesda, MD). All animals were initially used at 68 weeks of age and housed in pathogen-free micro-isolator cages in our animal facility.
Peptides and antigens
The major antigenic peptides of p1102 in H-2d and H-2b mice, residues 1226 (LEDARRLKAIYEKKK) and residues 7388 (VEEFSPSIAREIYEMY) conjugated or not to BSA respectively (6), were synthesized in the New England Peptide (Fitchburg, MA) and were purified to >95% homogeneity by HPLC. In addition, 1226BSA was used as an immunogen in B cell tolerance experiments since the BSA carrier can provide help for 1226 B cells [(2,9) and see below].
Retroviral constructs and virus-producer cell lines
The p1226IgG construct contains only the major I-Ad-restricted 1226 peptide in frame in the IgG1 heavy chain, as described previously (58). In addition, constructs containing ovalbumin (OVA), p1102 (full-length cI
repressor) in-frame with the IgG1 heavy chain were also engineered for experimental use. A further construct
p1102 (mutated at position 297 Asn
Ala of the murine IgG1 C region, using the QuickChange Site-Directed Mutagenesis kit; Stratagene, La Jolla, CA) was created for an IgG with no Fc-binding capability (10). However, the failure of the
p1102IgG to bind to FcR could not be validated for our fusion proteins because the transduced cells did not produce sufficient quantities for analysis (M. Litzinger and D. Scott, data not shown). As the original murine IgG1 heavy chain binds with high affinity to the NIP hapten when assembled with
light chain, the recombinant p1226IgG and p1102IgG fusion protein, can be detected with a NIP-gelatin binding ELISA, although this is an underestimate of secreted IgG fusion protein (5).
Virus-producer cell lines were prepared by lipofection of second-generation GP-E86 packaging cell lines with p1226IgGMBAE, p1102IgGMBAE,
p1102IgGMBAE or OVAIgGMBAE retroviral constructs respectively, and were found to be helper virus-free and to contain
105106 neomycin-resistant NIH 3T3 c.f.u./ml, using methods as described previously (5,6,8).
Retroviral-mediated gene transfer to lipopolysaccharide (LPS)-stimulated B cell blasts
Retroviral-mediated gene transfer into bacterial LPS (Escherichia coli 055:B5; Sigma)-stimulated splenic B cells has been described elsewhere (4,5,7). Briefly, primary culture of splenocytes was stimulated for 24 h with 30 µg/ml of LPS. Cells were then cultured (3 x 106 cell/ml) for an additional 24 h with supernatant enriched with retroviruses of the indicated peptideIgG in the presence of 3 µg/ml polybrene and LPS. Naive wild-type mice (or knockout mice, see below) then received 2 x 107 B cell blasts i.p.
Immunologic protocols
Five days after adoptive transfer of transduced B cells, mice were immunized s.c. in one footpad and at the base of the tail with 25 µg of recombinant p1102 protein or 25 µg of p1226 plus 25 µg OVA (as a specificity control) emulsified 1:1 in complete Freunds adjuvant (CFA). For testing B cell tolerance, mice were immunized with 25 µg of 1226 coupled to BSA, as a helper carrier, in CFA. Two weeks later, mice were bled for the measurement of serum primary antibody responses. The mice were then either euthanized and cellular immune responses in lymph nodes and spleen determined, or boosted i.p. with 25 µg of p1102 protein, p1226 or 1226BSA and 25 µg of OVA in PBS. The secondary antibody responses were measured from sera collected 1 week after the boosting. Serum p7388-specific, p1226-specific or OVA-specific IgG responses were determined by ELISA. Plates were coated with 1 µg/ml of synthetic peptide p1226 or p7388 conjugated to BSA (with synthetic peptide only when 1226BSA was used), or 1 µg/ml of OVA or p1102. Plates were subsequently probed with alkaline phosphatase-conjugated goat anti-mouse IgG as a secondary reagent (Southern Biotechnology Associates, Birmingham, AL). Total anti-1226 IgG concentrations were calculated using a standard curve with known concentrations of target antigen and mAb B3.11, specific for the p1226 epitope of p1102, as standard. Anti-p7388 of p1102 and anti-OVA (specificity control) were also determined by ELISA. Lymph node T cell responses were measured in vitro using [3H]thymidine incorporation, as described previously (8).
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Results
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Fc
RII deletion has no effect on tolerance induction by IgGAg molecules
We originally expected that the excellent tolerogenicity of IgG carriers was due in part to their persistence and to their interaction with a variety of APC by Fc receptors. However, the tolerogenicity of gene-transferred Ig fusion proteins did not correlate with the amount of circulating IgGAg (5). Moreover, use of MHC class II knockouts as B cell donors suggested an important role for presentation by the transduced B cells as paramount in tolerance induction (8). This result did not eliminate the possibility that these B cell APC secrete and efficiently present Ig-Ag after FcR uptake. To test the hypothesis that IgG tolerogens may be secreted, taken up and presented on MHC class II molecules by B cell APC, we used knockouts for the three major classes of FcR. Because Fc
RII is present mainly on lymphoid and myeloid lineages (10,11) and is of low affinity to IgG, we transduced activated splenocytes from Fc
RII/ or wild-type with either p1226IgG or OVAIgG and injected them into naive (+/+) recipients in our standard gene therapy protocol (5,6,8). After challenge and boost with 1226 and OVA, the levels of anti-1226 antibodies in the recipients of transduced p1226IgG Fc
RII/ and the p1226IgG B cells were significantly reduced (i.e. tolerant) compared to control regardless of the source of the B cell APC (Fig. 1A, top). As previously reported (5,8), tolerance was specific in that the antibody response to OVA was normal in 1226IgG B cell-treated mice, but reduced in recipients of OVAIgG-treated B cells (Fig. 1A, bottom). These results show that humoral tolerance was induced in recipients of p1226IgG Fc
RII/ and that its induction is not influenced by the removal of Fc
RII. Similar results were obtained with FcR/ knockout recipients, thus eliminating the role of host cells in tolerance (Fig. 1B; see also Fig. 2)
Fc
RI and Fc
RIII do not influence humoral tolerance.
Knowing that Fc
RI-binding affinity to IgG is highest among the other FcR (10,11), we used splenocytes from Fc
R/ lacking the expression of Fc
RI, Fc
RIII and Fc
RI. LPS-activated spleen cells from Fc
R/ or wild-type donors were transduced with p1102IgG or OVAIgG were injected into naive mice. The data in Fig. 2 show that Fc
R on the tolerogenic APC do not influence the ability to induce B cell tolerance, as measured by the humoral response to p1226. This suggests that secretion of the IgGAg and its uptake by Fc
RI is not involved in this pathway of B cell tolerance induction.
Total FcR deletion does not affect IgGAg induction of B or T cell tolerance
In order to further test the role of FcR in tolerance induction by IgGAg tolerogens, we used splenocytes from double-knockout mice lacking all FcR and transferred them into syngeneic naive mice after retroviral transduction with p1102IgG and OVAIgG. We used p1102IgG in order to follow the response to epitopes recognized by the background strains (6); thus, tolerance, presumably in helper T and B cells, was measured as antibody secondary responses to p7388 or full-length p1102. We found a significant decrease in the levels of total IgG anti-7388 and anti-1102 in groups that received the tolerogen-transduced splenocyte blasts from wild-type or total FcR/ as compared to control (Fig. 3A). In addition, T cell responsiveness, measured by lymph node T cell proliferation from mice receiving transduced knockout or wild-type blasts, showed that T cell tolerance was not affected in this experiment against p7388 (Fig. 3B). These data also suggest that the involvement of FcR in T or B cell tolerance induced by IgGAg tolerogens is minimal. Again, tolerance was specific as measured by responsiveness to OVA in p1102IgG-treated B cell recipients (data not shown).


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Fig. 3. FcR do not influence cellular or humoral tolerance. (A) B6 x 129 mice were injected with 2 x 107 p1102IgG or OVAIgG gene-transferred B cell blasts from naive B6 x 129 or FcR/ mice. Five days later, mice were immunized with 25 µg of p1226 and 25 µg of OVA emulsified 1:1 in CFA. Two weeks later, mice were boosted with the same antigens in PBS. A week after boost, all mice were bled for determination of anti-p7388 and anti-OVA (data not shown) titers by ELISA. Antibody IgG total serum levels against p7388 in recipient mice of p1102IgG transduced B cell blasts are represented by open columns, while those in OVA controls are represented by solid columns (total IgG P < 0.005). Data are presented as means ± SE. One of two representative experiments is shown, with five mice per group. (B) B6 x 129 mice were infused with 2 x 107 p1102IgG or OVAIgG gene-transferred B cell blasts from naive B6 x 129 or FcR/ mice. After immunization, lymph node T cell proliferation (day 1012) was measured against p7388, the immunodominant epitope in H-2b mice. Cultures were pulsed with [3H]thymidine. One set of two representative experiments is shown, with pooled LN cells from five animals per group. Data are presented as means ± SE. OVAIgG represents the control group retrovirally transduced with an unrelated construct, whereas p1102IgG represents the experimental group and knockout represents recipients of FcR double knockout B cell blasts. Responsiveness to OVA was normal in 1226IgG tolerant mice (data not shown).
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Mutation of the Fc portion of IgG1 at position 297 has no effect on tolerance induction
In an attempt to further understand the role of the Fc portion of murine IgG in the mechanisms of tolerance induction using IgGAg tolerogens, we mutated the C region of the heavy chain at position 297 of the p1102IgG construct. This particular amino acid is known to be a conserved glycosylation site that was found to be critical for binding and activation of IgG molecules to all three Fc
R and in complement-mediated lysis (1015). Thus, normal mice can be used as donors and recipients to eliminate any influence of knockout background genes. Mice were injected with transduced lymphocytes with mock cells, p1102IgG or the mutated construct
p1102IgG. Tolerance (B cell) was assessed by measuring the levels of circulating anti-1102 or anti-1226 antibodies and cellular tolerance was assessed using standard lymph node T cell proliferation to immunodominant peptides. Tolerance to p1102 or its immunodominant peptide 1226 was no different in groups receiving p1102IgG or
p1102IgG as compared to mice receiving mock injection (Fig. 4A and B). Although presence of the heavy chain influences the maintenance of tolerance with IgG chimeras (6), these results suggest that the Fc fragment of the IgG is not involved because the mutated construct did not alter the tolerance induction to 1102 or its immunodominant peptide 1226. These data also suggest that secretion and uptake is probably not the preferential way by which IgGAg fusion protein induces tolerance.


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Fig. 4. The mutation of position 297 in the Fc portion of the IgG does not affect the induction of tolerance by p1102IgG. (A) CB6F1/J mice were injected with 2 x 107 p1102IgG, p1102IgG or untransduced (Mock) gene-transferred B cell blasts from naive CB6F1/J mice. Five days later, mice were immunized with 25 µg of p1226 and 25 µg of OVA emulsified 1:1 in CFA. Two weeks later, mice were boosted with the same antigens in PBS. A week after boost, all mice were bled for determination of anti-p1102, anti-p1226 and anti-OVA (data not shown) titers by ELISA. Antibody IgG total serum levels against p1102, p1226 in recipient mice of untransduced B cell blasts are represented by solid columns, while those in p1102IgG and the mutant counterpart are in open and hatched columns respectively (total IgG P < 0.005). Data are presented as means ± SE. One of two representative experiments is shown, with five mice per group. (B) CB6F1/J mice were infused with 2 x 107 p1102IgG, p1102IgG or untransduced (Mock) gene-transferred B cell blasts from naive CB6F1/J. After immunization, lymph node T cell proliferation (day 1012) was measured against p1226, the immunodominant epitope in H-2d mice, and p1102. Cultures were pulsed with [3H]thymidine. One set of two representative experiments is shown, with pooled lymph node cells from five animals per group. Data are presented as means ± SE.
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Humoral tolerance in gene-transferred recipients is due in part to specific B cell tolerance
The fact that humoral tolerance to p1102, p1226 and p7388 was reduced significantly in the above experiments (Figs 14) suggested that both B and T cell tolerance was being induced, as previously reported (5). However, we need to formally test that the reduced IgG titers against those peptides was due to B cell [in addition to Th (9)] tolerance induction by the transferred IgGAg fusion protein construct. Therefore, recipients of B cell blasts transduced with p1226IgG were challenged with p1226 coupled to BSA in CFA to provide BSA-specific T cell help (3,6,16). They were boosted with 1226BSA in saline 2 weeks later. IgG titers of anti-1226 were significantly lower in the 1226BSA-challenged group of treated mice. However, the titers were significantly higher than the tolerant control group challenged with 1226 alone (195 µg/ml in the p1226IgG group challenged with 1226BSA versus 56 µg/ml in the p1226IgG group challenged with 1226 alone). This may be due to the presence of both B and partial T cell tolerance in mice challenged with 1226 (which has both a T cell and a B cell epitope), whereas only B cell tolerance is revealed with 1226BSA, in which BSA-specific T cells can provide help to partially overcome tolerance.
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Discussion
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FcR are known to balance the immune response, and the lack of any of them can lead to autoimmunity and inflammation mediated by T cells (1015). However, the role that FcR play in tolerance is poorly understood. Based on the excellent tolerogenicity of IgG carriers (15,9), it was assumed that the Fc portion of the IgG might be important in tolerance. In fact, IgG is more tolerogenic than F(ab)2 fragments (16) and clearly leads to greater persistence in vivo. Moreover, deletion of the IgG carrier reduces both the efficacy and persistence of gene-therapy-induced tolerance (6). As noted above, however, the tolerogenicity of gene-transferred Ig fusion proteins does not correlate with the amount of circulating IgGAg (5). That result, coupled with the failure of B cells from MHC class II knockouts to be tolerogenic APC (8), suggested that presentation by transfected B cells was most critical in tolerance induction. However, this did not rule out a role for uptake and processing of the expressed chimeric protein by these cells. This necessitated further study of the role of FcR in this tolerance model. The results presented herein, however, suggest that FcR are not important in tolerance induced by IgGAg in this model.
In this study, we directly tested the hypothesis that the IgGAg tolerogen enters the presenting B cells via the FcR and is then presented on B cell APCs MHC class II for tolerance induction. Using our gene therapy model for tolerance, our results showed that humoral tolerance is not affected by the deletion of FcR nor by the mutation of the Fc portion of the murine Ig. This suggests that secretion of the IgG and its uptake by FcR is not a major part of the mechanism by which tolerance is induced using the IgGAg chimeras. In addition, it has been reported that tolerance induction in FcR knockout mice can be induced at both cellular and humoral levels (17). Our results support those findings despite differences in the systems, especially the dose of antigen. Despite the fact that FcR may play a role in enhancing antigen presentation (18,19), the lack of those molecules did not attenuate cellular tolerance in our system. In addition, in vivo anti-FcR (2.4G2) treatment of transgenic mice expressing and secreting as much as 10 µg/ml of 1226IgG by B cells (20) showed no effect on tolerance induction at the cellular and humoral levels, and in terms of cytokine levels (El-Amine and Scott, unpublished data). Thus, the secretion and uptake hypothesis also was not supported by the results from the mutation in the Fc portion of the Ig that showed both humoral and cellular tolerance (Fig. 4) in all groups. These data, in addition to an MHC class II knockout result from our previous studies (8) showing reversal of tolerance in the absence of B cell presentation, clearly shows that docking of the IgGAg fusion protein on the FcR is not necessary for tolerance induction. Nonetheless, while our data support the hypothesis of presentation rather than secretion and uptake as paramount in this model of tolerance, additional work with constructs lacking a signal sequence to prevent secretion is needed to prove that endogenous presentation is sufficient for tolerance.
Finally, tolerance induction using IgGAg is a powerful approach for tolerance induction that can be used in human clinical trials and is currently used in clinical animal models (7,21,22) for treatment of autoimmune diseases. The mechanisms behind this tolerance induction are important to elucidate better manipulation of the system in the future.
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Acknowledgements
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The authors would like to thank Dragana Jankovi
(NIAID, NIH, Bethesda, MD) for providing us with Fc
R knockout mice. We would like to thank Ms Hao Nguyen for her technical assistance. This project was supported by NIH grants AI35622 and JDFI #196110.
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Abbreviations
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Agantigen
APCantigen-presenting cell
CFAcomplete Freunds adjuvant
LPSlipopolysaccharide
OVAovalbumin

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Fig. 5. Gene therapy induces specific B cell tolerance. BALB/c mice were injected with 2 x 107 p1226IgG or OVAIgG gene-transferred B cell blasts from naive BALB/c. Five days later, mice were immunized with 25 µg of p1226 or p1226 coupled to BSA as a carrier to provide T cell help (8), as well as with 25 µg of OVA as a specificity control, all emulsified 1:1 in CFA. Two weeks later, mice groups were boosted with the same antigens in PBS. A week after boost, all mice were bled for determination of anti-p1226 and anti-OVA (data not shown) titers by ELISA. Antibody IgG total serum levels against p1226 in recipient mice of p1226IgG-transduced B cell blasts are represented by open columns, while those in OVA controls are in solid columns (total IgG P < 0.001). Data are presented as means ± SE. One of two representative experiments is shown, with five mice per group.
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