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*
Department of Immunology, American Red Cross, J. Holland Laboratory, Rockville, MD 20855; and
Department of Immunology, George Washington University Medical Center, Washington DC 20037
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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repressor cI sequence p1-102 or its
immunodominant epitopes (p12-26, p73-88) can be elicited when bone
marrow (BM) or LPS blasts are transduced and injected into naive or
even primed recipients. To explore the mechanism of tolerance, class
II-/- (knockout, KO) BM cells were transduced with
p1-102-IgG and transferred to irradiated recipients. These cells failed
to induce tolerance to challenge with p1-102 epitopes, whereas
transduced +/+ BM cells did. This supports the importance of class II
MHC on the tolerogenic APC rather than secretion and representation in
tolerogenesis. When BM cells from µMT KO mice were transfected with
p12-26-IgG and injected into irradiated mice, these transduced BM cells
also failed to induce tolerance to an immunodominant epitope. These
results suggest the direct involvement of B cells in tolerance to
p1-102 epitopes. IL-10 KO BM cells infected with a p12-26-IgG construct
were still tolerogenic. Importantly, anti-CTLA-4 injections
reversed tolerance in primed, but not in naive, recipients of
transduced LPS blasts. These data emphasize the importance of MHC class
II presentation, B cell involvement, and CTLA-4 engagement in induction
and/or maintenance of tolerance. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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repressor cI molecule; Ref. 5) in-frame
at the N terminus of an IgG1 heavy chain and expressed this in two
systems for the induction of tolerance. In one system, we used a
Moloney leukemia retroviral vector (6) to express the
p12-26-IgG fusion protein (or the full-length cI repressor as
p1-102-IgG) in bone marrow
(BM)4-derived cells or
in LPS blasts (2, 3, 4, 7). Alternatively, we used B cells
from transgenic mice expressing (soluble) the p12-26-IgG molecule as a
source of APC (3). Both systems lead to potent
unresponsiveness to the epitopes associated with the IgG carrier and
provide model systems for eventual clinical applicability via gene
therapy. We have proposed that the efficacy of the IgG fusion protein
approach may be based on the tolerogenicity of soluble IgG as
tolerogenic carriers, as well as on preferential B cell Ag presentation
(8, 9, 10, 11). Although B cells were efficient tolerogenic APC
in the two models, previous results using scid mice as BM
donors suggested that B cells may be sufficient but are not necessarily
required for the success of this gene therapy protocol
(4). Thus, the role of the secreted IgG fusion protein and
the precise mechanisms of tolerance in this system needed further
evaluation. To clarify the mechanisms of tolerance by IgG fusion proteins expressed by B lineage APC, we used a series of knockout mice (lacking class II, IL-10, or mature B cells (µMT)) as well as treatment with anti-CTLA-4. Our results suggest that MHC class II molecules and B cell Ag presentation are critical for tolerance induction. Furthermore, CTLA-4 appears to play a regulatory role in this process, especially in the case of primed animals, in that anti-CTLA-4 treatment ablated the induction and/or maintenance of hyporesponsiveness. These data suggest that B cells presenting IgG fusion-derived peptides in their MHC groove deliver a tolerogenic signal, which is mediated in part by CTLA-4.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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BALB/c, C57BL/6 and CB6F1 mice were
purchased from The Jackson Laboratory (Bar Harbor, ME), while
A
b N5 (MHC class II KO),
Ab N6 (wild type), and
BALB/c IL-10 KO mice were purchased from Taconic Farms (Germantown,
NY). C57BL/6-Igh-6tmlCgn B cell-deficient
mice (µMT) were purchased from The Jackson Laboratory. µMT mice
were bred and backcrossed to BALB/c (8–10 backcrosses) at the Holland
Laboratory. In some experiments, transgenic mice expressing a
p12-26-IgG fusion protein driven by the IgH promoter/enhancer (referred
to as transgenic line 17 or L17) were used as a source of B cells. All
animals were initially used at 6–8 wk of age and housed in
pathogen-free microisolator cages in our animal facility.
Antibodies
Monoclonal hamster anti-CTLA-4 (UC10-4F10-11) and control hamster IgG were purchased from PharMingen (San Diego, CA); goat anti-mouse IgG, IgG1, and IgG2a with alkaline phosphatase conjugate were purchased from Southern Biotechnology Associates (Birmingham, AL).
Peptides
The major antigenic peptides of p1-102 in
H-2d and H-2b mice,
residues 12–26 (LEDARRLKAIYEKKK) and residues 73–88
(VEEFSPSIAREIYEMY), respectively (5, 12), were
synthesized in the Molecular Biology Core of the Holland Laboratory
using a solid-phase method and were purified to 
95% homogeneity
by HPLC.
Retroviral constructs encoding cI p1-102-IgG, p12-26-IgG or
OVA-IgG and virus-producer cell lines
A 320-bp DNA fragment encoding p1-102 was amplified by PCR (30
cycles: 94°C, 15 s; 55°C, 15 s; 72°C, 1. 5 min) from
pRB104 (a kind gift from Richard M. Breyer, Vanderbilt University
Medical Center, Nashville TN). The 5' primer, GCG GGC CGC ATG AGC ACA
AAA AAG AAA CC, contained a NotI restriction site and
N-terminal sequences of 1-102; the 3' primer, CGC GTC GAC CTA CTA CTC
ATA CTC ACT TCT AAG TGA, contained a SalI restriction site
and translational stop codon sequences. The p1-102 DNA was subsequently
inserted into the VH sequence of a murine IgG1
heavy chain between the 5' first framework region (framework region 1)
and framework region 1 repeat. The resulting p1-102-IgG fragment, which
included the leader sequence, VH region inserted
with p1-102, DJC region, and stop codon sequences, was then subcloned
into MBAE downstream of a -actin promoter/enhancer (4, 6). This is referred to as p1-102-IgG.
The p12-26-IgG construct contains only the major
I-Ad-restricted 12-26 peptide in-frame in the
IgG1 heavy chain, as described previously (2, 3, 4). In
addition, a construct containing OVA in-frame with IgG1 heavy chain was
also engineered and used as a specificity control in some experiments.
Since the original murine IgG1 heavy chain binds with high affinity to
the nitroiodophenyl (NIP) hapten when assembling with the light
chain, the recombinant p1-102-IgG fusion protein can be detected with a
NIP-gelatin-binding ELISA, although this is an underestimate of
secreted IgG fusion protein (4).
Virus-producer cell lines were prepared by lipofection of 
-2
packaging cell lines with p12-26-IgG MBAE, p1-102-IgG-MBAE, or
OVA-IgG-MBAE retroviral constructs, respectively, and were found to be
helper virus free and to contain
105-106
neomycin-resistant NIH 3T3 CFU/ml, using methods as described
previously (4, 7). In some experiments, -2 parental
cells without retroviral vectors were used for control transfection
(see "mock" below).
Retroviral-mediated gene transfer to BM and LPS-stimulated B cell blasts
Retroviral-mediated gene transfer into BM and bacterial LPS (Escherichia coli 055:B5; Sigma, St. Louis, MO)-stimulated splenic B cells has been described elsewhere (4, 7). Briefly, cells were cultured (3 x 106 cell/ml) for 48 h with irradiated (2000 rad) p12-26-IgG, p1-102-IgG, OVA-IgG, or packaging cell lines without retroviral vectors (mock) in the presence of 6 µg/ml polybrene and either 50 µg/ml LPS (for B cell blasts) or 200 U/ml of IL-3, IL-6, and IL-7 (Genzyme Genetics, Framingham, MA) for BM. For adoptive transfer of BM, adult mice exposed to 300–600 rad of irradiation were injected i.v. with 2–3 x 106 gene-transferred or mock-transduced BM cells. Primed or naive BALB/c mice were also adoptively transferred by tail i.v. injection of 2 x 107 B cell blasts from BALB/c or L17 mice.
Serum NIP binding IgG1 results from mice receiving BM vary and do not correlate with the extent of tolerance (cf Ref. 4). For example, recipients of normal BM with F12.7: mouse 1, <0.1 ng/ml; mice 2 and 3, <4 ng/ml. Groups receiving MHC class II KO BM with F12.7: mouse 1, <15 ng/ml; mice 2–4, <4 ng/ml. Control groups receiving BM not gene transferred had serum NIP binding Ig under the detection levels.
Immunologic protocols
Mice were s.c. immunized in one footpad and at the base of the tail with 25 µg of recombinant p1-102 protein (MHC class II KO experiment) or 20 µg of p12-26 emulsified 1:1 with 25 µg of hen egg lysozyme (HEL) in CFA as a specificity control. Two weeks later (1 wk later for primed animals in the anti-CTLA-4 experiment), mice were bled for the measurement of serum primary Ab responses. The mice were then either sacrificed and cellular immune responses in lymph nodes and spleen determined or boosted i.p. with 25 µg of p1-102 protein or 20 µg of p12-26 and 25 µg of HEL in PBS. The secondary Ab responses were measured from sera collected 1 wk after the boosting. Serum p1-102-specific, p12-26-specific, or HEL-specific IgG responses were determined by ELISA using coating plates with 50 µg/ml synthetic peptide or 1 µg/ml HEL or p1-102 and subsequently probed with alkaline phosphatase-conjugated goat anti-mouse IgG, IgG1, and IgG2a as a secondary reagent. Total or isotype-specific IgG concentrations were calculated using a standard curve with known concentrations of target Ag and mAb B3.11, specific for the p12-26 epitope of p1-102, as standard. Lymph node T cell responses were measured in vitro using [3H]thymidine incorporation, as described previously (7).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It was previously demonstrated that retrovirally mediated gene
therapy can be successfully employed for the induction of tolerance to
peptide IgG constructs (4, 7). However, the precise
mechanisms involved in this process were unknown. We knew that BM cells
from scid donors could be transfected and employed for
tolerance induction (4), suggesting that B cell secretion
of the fusion peptide-IgG was not necessary for tolerance. Because the
scid mutation is known to be leaky, we repeated this
experiment with defined KO strains. First, we determined the role of
MHC class II in gene-transferred tolerance, using BM cells from MHC
class II KO (H-2b) and normal control C57BL/6
mice (H-2b). These BM cells were transduced with
IgG-p1-102 and injected into sublethally irradiated (300 rad) syngeneic
normal mice with functional MHC class II. Under these conditions, B
lymphocytes or other potential tolerogenic APC that develop from the KO
BM will have the capacity of secreting but not presenting the
engineered tolerogen, as they lack MHC class II molecules
(13). When these animals were immunized 6 wk later with
p1-102 in CFA, we found that recipients of MHC class II KO were not
tolerant to p1-102 compared with recipients of transduced normal (class
II+/+) marrow (Fig. 1
). These data support a role for cells
expressing both the transduced fusion protein and MHC class II through
the presentation of epitopes in their MHC groove. Moreover, our results
indicate that secretion of the IgG fusion protein and its
"re-presentation" on host APC either is not required or does not
play a critical role in this tolerogenic process.
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To formally test the hypothesis that B cell Ag presentation is
required for the induction of tolerance, we injected sublethally
irradiated BALB/c mice with p12-26-IgG gene-transferred BM cells
from µMT (B cell KO) mice or +/+ donors. Tolerance was assessed
using standard lymph node T cell proliferation to immunodominant
peptides and to HEL, as a specificity control. Tolerance to the 12–26
peptide was observed in normal BM chimeras (Fig. 2, left panel). Interestingly,
there was no T cell tolerance to p12-26 in recipients of µMT BM (Fig. 2, right panel). These data support the hypothesis that B
cells are crucial APC for tolerance induction.
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We have reported previously (3, 4, 7) that tolerance
induction with IgG tolerogens is effective on both Th1 and Th2
responses. Nonetheless, it was possible in our gene therapy model that
Th2-type cytokines may be produced which inhibit Th1 cell development.
IL-10 is a potent stimulator of B cell differentiation (14, 15) and Ig synthesis (15, 16, 17), and a potent
Th1-immunosuppressive Th2 cytokine. To test the involvement of IL-10 in
our gene therapy model, we constructed radiation chimeras in normal
BALB/c (control) or IL-10 KO mice injected with BM cells transduced
with either p12-26-IgG or mock retrovirus. When challenged with p12-26
and HEL, we found that similar levels of tolerance were induced in all
groups (Fig. 3), suggesting that IL-10 is
not involved in this protocol for tolerance induction.
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According to the two-signal hypothesis, B and T cell activation
requires mutual recognition, engagement, and recall of different
signaling molecules on the surface of both cells. Thus, CTLA-4 vs CD28
engagement with B7 molecules either enhances or abrogates T cell
proliferation (18, 19, 20, 21, 22). Furthermore, reversing an ongoing
immune response is challenging because of the complex nature of the
signaling between APC and T lymphocytes. Therefore, to understand the
role of CTLA-4 in tolerance induction and its possible role in
modulating primary and secondary immune responses, we employed
anti-CTLA-4 to block B7 interactions in two models for tolerance
induction. As reported previously, B cell blasts from transgenic mice
expressing the p12-26-IgG fusion protein (L17), or LPS blasts
transduced with the p12-26-IgG retrovirus, were capable of inducing
profound humoral tolerance even in already primed animals
(3, 4). When p12-26-IgG L17-transgenic B cell blasts were
transferred to primed or unprimed syngeneic recipients, who were
treated with anti-CTLA-4, we found that anti-CTLA-4 reversed
tolerance induction in primed animals (as measured by humoral
responsiveness) and returned the immune response to control levels
(Fig. 4). This observation was
reproducible whether total IgGs or isotypes such as IgG1 (Fig. 4) or
IgG2a (data not shown) were measured. This suggests the direct
involvement of CTLA-4 in tolerance induction on activated lymphocytes
in support of the two-signal hypothesis.
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, anti-CTLA-4 did not reverse tolerance to p12-26 in unprimed
animals, in terms of the humoral responses (Fig. 5A),
regardless of isotype measured (IgG2a; data not shown), or
cell-mediated responses (Fig. 5B). These results suggest
that CTLA-4 plays a role in down-regulating primed but not naive T
cells, which do not express CTLA-4 on their surface.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It is not surprising that MHC class II presentation by tolerogenic APC plays a role in tolerance. Moreover, these results and data from B cell KO mice clearly favor the presentation hypothesis over secretion and representation by the host. Our results suggest that B cells are the most effective tolerogenic APC (4, 7, 8, 9, 10, 11). It is possible that B cells are required because they can provide light chain for potential assembly and secretion. MHC class II is necessary for the transduced cell to present, and these cells have a selective advantage to present their own IgG. It is possible that in lieu of class II, the small amount of IgG fusion protein that is secreted after gene therapy and taken up by other cells is insufficient to induce tolerance. However, using H-2b-transgenic B cells expressing p12-26-IgG to induce tolerance, we have recently obtained evidence for cross-presentation to host cells (M. El-Amine, J. Hinshaw, H. Nguyen, and D. W. Scott, manuscript in preparation). This is likely to be due to the higher levels of expression in the transgenic vs the gene therapy systems.
Additional experiments to examine a role for FcR, perhaps by binding
locally secreted IgG tolerogens by the B cell APC, have now been
performed. These data, using FcR
RII-/- cells
as donor APC or 2.4G2 anti-FcR treatment, suggest that Fc receptors
do not play a major role in tolerogenic presentation, although an
involvement in the tolerization of Th1 cells cannot be eliminated (M.
El-Amine, J. Hinshaw, H. Nguyen, and D. W. Scott, manuscript in
preparation). This is not consistent with the importance of the IgG
carrier, long appreciated in tolerance literature, and reaffirmed in
our gene therapy model as important for both the efficacy and duration
of tolerance (7). Further studies on the role of
Fc-dependent mechanisms are in progress using mutated IgG constructs
and other FcR KO mice.
Because BALB/c mice express high levels of IL-10, we also wished to
test whether tolerance by IgG carriers was due to their high level of
production by IL-10 stimulation. Indeed, Zaghouani and coworkers
(24) reported that tolerance induced by peptide IgG
conjugates may be optimized by aggregation of the IgG and the induction
of IL-10 synthesis by activated T cells. Although there are many
differences between their system and ours (IgG2
vs IgG1 carrier; neonatal vs adult treatment;
aggregated Ag is often immunogenic), the observation that IL-10 is a
powerful suppressor to Th1 responses led us to test a possible role of
this cytokine in gene-transferred tolerance. Since BM cells from IL-10
KO mice were equally tolerogenic as normal transduced BM cells, this
rules out the involvement of this cytokine in this model of tolerance
induction (Fig. 3
). However, this does not exclude the possible
involvement of other cytokines such as IL-4 or TGF- in
down-regulating responsiveness. This is rendered unlikely by the lack
of differential Th1 and Th2 tolerance.
The fact that activated B cells express high levels of B7 molecules (23) and that they induce a profound T cell unresponsiveness (3, 4, 7) indicates that lack of costimulation is not a mechanism for tolerance in our model. Therefore, we hypothesized that CTLA-4 down-regulation of T cell responsiveness could be involved in our model of tolerance. Using two different strategies (injection of gene-transferred blasted B cells or LPS blasts from a peptide-IgG-transgenic mouse), we have clearly shown that in primed adult animals, CTLA-4 is required for induction and maintenance of the tolerance state, as measured by humoral responsiveness. Notably, the administration of anti-CTLA-4 to tolerized animals did not enhance the immune response nonspecifically because the response to an unrelated Ag (HEL) was comparable to control levels. Interestingly, anti-CTLA-4 Ab treatment failed to reverse tolerance in unprimed "tolerant" mice, presumably due to the lack of strong costimulatory interactions via CTLA-4. Although this contrasts with the results of Abbas and coworkers (18), significant differences exist between these systems.
Although the delivery via gene therapy of peptides-IgG fusion proteins as tolerogens is still in its early stage, this system offers a platform technology for application in autoimmunity and gene therapy for genetic diseases due to a lack of protein (such as hemophilia). Importantly, very few activated spleen cells or B cells are needed to induce the state of tolerance since 2 x 105 activated B cells or 5 x 105 activated spleen cells were effective, and tolerance is maintained for at least 6 mo (M. El-Amine and D. W. Scott, unpublished data). This indicates that very few B cells need to be transduced for potential clinical efficacy. In summary, our data suggest that the presentation of the fusion IgG epitope by B cell APC on MHC class II surface molecules and that CTLA-4 down-regulation of responsiveness are occurring, at least in primed recipients. Further studies will focus on the fate of tolerized cells and the signals involved in their down-regulation by this gene therapy approach for tolerance and its application in models of autoimmune diseases. Recent results show clinical efficacy in both experimental allergic encephalomyelitis (M. Melo and D. W. Scott, unpublished data) and uveitis (25).
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Acknowledgments |
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Footnotes |
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2 This is publication number 66 from the Department of Immunology, American Red Cross Holland Laboratory.
3 Address correspondence and reprint requests to Dr. David W. Scott, Department of Immunology, American Red Cross Holland Laboratory, 15601 Crabbs Branch Way, Rockville, MD 20855.
4 Abbreviations used in this paper: BM, bone marrow; KO, knockout; L17, transgenic mice, line 17; HEL, hen egg lysozyme.
Received for publication December 20, 1999. Accepted for publication August 28, 2000.
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References |
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cooperate to induce anti-CD40-activated naive human B cells to secrete immunoglobulin A. J. Exp. Med. 175:671.-mediated splenic anergy. J. Exp. Med. 188:2007.This article has been cited by other articles:
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  A. S. De Groot, L. Moise, J. A. McMurry, E. Wambre, L. Van Overtvelt, P. Moingeon, D. W. Scott, and W. Martin Activation of natural regulatory T cells by IgG Fc-derived peptide "Tregitopes" Blood, October 15, 2008; 112(8): 3303 - 3311. [Abstract] [Full Text] [PDF]
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 Y. Su, G. Carey, M. Maric, and D. W. Scott B Cells Induce Tolerance by Presenting Endogenous Peptide-IgG on MHC Class II Molecules via an IFN-{gamma}-Inducible Lysosomal Thiol Reductase-Dependent Pathway J. Immunol., July 15, 2008; 181(2): 1153 - 1160. [Abstract] [Full Text] [PDF]
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P. B. Silver, R. K. Agarwal, S.-B. Su, I. Suffia, R. S. Grajewski, D. Luger, C.-C. Chan, R. M. Mahdi, J. M. Nickerson, and R. R. Caspi Hydrodynamic Vaccination with DNA Encoding an Immunologically Privileged Retinal Antigen Protects from Autoimmunity through Induction of Regulatory T Cells J. Immunol., October 15, 2007; 179(8): 5146 - 5158. [Abstract] [Full Text] [PDF]
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M. Werner-Klein, C. Dresch, P. Marconi, and T. Brocker Transcriptional Targeting of B Cells for Induction of Peripheral CD8 T Cell Tolerance J. Immunol., June 15, 2007; 178(12): 7738 - 7746. [Abstract] [Full Text] [PDF]
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M. T. Litzinger, Y. Su, T. C. Lei, N. Soukhareva, and D. W. Scott Mechanisms of Gene Therapy for Tolerance: B7 Signaling Is Required for Peptide-IgG Gene-Transferred Tolerance Induction J. Immunol., July 15, 2005; 175(2): 780 - 787. [Abstract] [Full Text] [PDF]
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T. C. Lei and D. W. Scott Induction of tolerance to factor VIII inhibitors by gene therapy with immunodominant A2 and C2 domains presented by B cells as Ig fusion proteins Blood, June 15, 2005; 105(12): 4865 - 4870. [Abstract] [Full Text] [PDF]
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 C. M. Snyder, K. Aviszus, R. A. Heiser, D. R. Tonkin, A. M. Guth, and L. J. Wysocki Activation and Tolerance in CD4+ T Cells Reactive to an Immunoglobulin Variable Region J. Exp. Med., November 8, 2004; (2004) jem.20031234. [Abstract] [Full Text] [PDF]
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E. Dobrzynski, F. Mingozzi, Y.-L. Liu, E. Bendo, O. Cao, L. Wang, and R. W. Herzog Induction of antigen-specific CD4+ T-cell anergy and deletion by in vivo viral gene transfer Blood, August 15, 2004; 104(4): 969 - 977. [Abstract] [Full Text] [PDF]
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 V C Kyttaris, Y-T Juang, and G C Tsokos Gene therapy in systemic lupus erythematosus Lupus, May 1, 2004; 13(5): 353 - 358. [Abstract] [PDF]
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 M. El-Amine, J. A. Hinshaw, and D. W. Scott In vivo induction of tolerance by an Ig peptide is not affected by the deletion of FcR or a mutated IgG Fc fragment Int. Immunol., July 1, 2002; 14(7): 761 - 766. [Abstract] [Full Text] [PDF]
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M. E. F. Melo, J. Qian, M. El-Amine, R. K. Agarwal, N. Soukhareva, Y. Kang, and D. W. Scott Gene Transfer of Ig-Fusion Proteins Into B Cells Prevents and Treats Autoimmune Diseases J. Immunol., May 1, 2002; 168(9): 4788 - 4795. [Abstract] [Full Text] [PDF]
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