Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2009 Aug 1.
Published in final edited form as: J Invest Dermatol. 2008 Nov 27;129(5):1203–1207. doi: 10.1038/jid.2008.364

Transient Anti-CD40L Co-stimulation Blockade Prevents Immune Responses Against Human Bullous Pemphigoid Antigen 2

Implications For Gene Therapy

Christoph M Lanschuetzer 1, Edit B Olasz 1, Zelmira Lazarova 1, Kim B Yancey 1
PMCID: PMC2681490  NIHMSID: NIHMS95532  PMID: 19037236

Abstract

Skin grafts from mice expressing hBPAG2 in epidermal basement membrane elicit hBPAG2-specific IgG and graft loss in wild type (Wt) recipients. Graft loss was dependent upon CD4+ T cells and correlated with the production and tissue deposition of hBPAG2-specific IgG. To explore the role of CD40/CD40 ligand (CD40L) interaction in this model, Wt mice grafted with Tg skin were treated with hamster anti-CD40L monoclonal antibody MR1. In contrast to grafted Wt mice treated with equivalent doses of control IgG, 22 of 23 MR1-treated Wt mice did not develop hBPAG2-specific IgG or graft loss for ≥60 days. MR1-treated mice also accepted a second Tg skin graft without durable production of hBPAG2-specific IgG or graft loss. Moreover, splenocytes and enriched CD4+ T cells from MR1-treated graft recipients transferred un- or hyporesponsiveness to hBPAG2 to other mice and demonstrated a dominant tolerant effect over co-transferred naïve splenocytes following adoptive transfer to Rag2 -/- mice. Successful inhibition of hBPAG2-specific IgG production and Tg graft loss following CD40:CD40L co-stimulatory blockade in this model provides opportunities to study mechanisms of peripheral tolerance and generate antigen-specific regulatory CD4+ cells - issues of relevance to patients with pemphigoid as well as individuals undergoing gene replacement therapy for epidermolyis bullosa.

Keywords: animal model, autoimmunity, bullous disease, gene therapy, CD40L

INTRODUCTION

To study experimental immune responses against human bullous pemphigoid antigen 2 (hBPAG2) (Franzke et al., 2005; Van den Bergh and Giudice, 2003) our laboratory recently developed C57BL/6 transgenic (Tg) mice expressing hBPAG2 in murine epidermal basement membrane (BM). Full thickness grafts of hBPAG2 Tg skin placed on the flanks of gender-matched, syngeneic wild type (Wt) mice consistently elicited prompt (detectable within 16±2 days), robust (titer ≥ 1280), and durable (present >270 days) IgG that bound 1) human epidermal BM, 2) BPAG2 extracted from human keratinocytes and 3) the NC16A domain of hBPAG2. In vivo deposits of IgG in epidermal BMs of grafts prompted complement activation, neutrophil-rich leukocytic infiltrates, subepidermal blister formation, and loss of Tg skin grafts within 28-30 days. Major histocompatibility class (MHC) II -/- mice grafted with Tg skin developed neither specific IgG nor graft loss, indicating that MHC II:CD4+ T cell interactions are crucial for these responses and that CD8+ T cells do not mediate graft loss in this model (Olasz et al., 2007). These studies have direct relevance to patients with the pemphigoid group of autoimmune blistering diseases as well as individuals with generalized atrophic benign epidermolysis bullosa (GABEB, OMIM 226650), (Bauer and Lanschuetzer, 2003; Darling et al., 1997; Hintner and Wolff, 1982) who are at risk of developing unwanted immune responses to hBPAG2 as a consequence of successful gene therapy (Chen and Woodley, 2006; Dellambra et al., 2000; Khavari, 1998; Mavilio et al., 2006; Ohyama et al., 2003; Woodley et al., 2007). The consistent experimental responses seen in Wt mice grafted with Tg skin indicate that this animal model has utility for the study of interventions that may block, attenuate, and/or silence the production of IgG directed against hBPAG2 in epidermal BM. Given the central role that CD40:CD40 Ligand (L) interactions play in T cell-dependent humoral immune responses, we sought to determine if blockade of CD40L by monoclonal antibody MR1 could impair the development (or attenuate the titer) of anti-hBPAG2 IgG in Wt mice bearing Tg skin grafts (Noelle et al., 1992; Roy et al., 1993).

RESULTS

CD40L engagement is necessary for anti-BM IgG production following hBPAG2 Tg skin graft placement

Wt mice (n=4) grafted with gender-matched, syngeneic hBPAG2 Tg skin developed IgG that bound human epidermal BM with high and durable titers within 17 days (specifically, titers = 640 to 5120 vs. the epidermal side of 1M NaCl split skin ≥ day 17 after grafting [observation period, 90 days]). Conversely, CD40L -/- mice (n=4) grafted with Tg skin did not develop anti-BM IgG. Moreover, while Wt recipients lost Tg skin grafts within 28 to 30 days, CD40L -/- mice retained Tg skin grafts for their entire period of study (i.e., 235 days). These studies showed that CD40:CD40L interactions are crucial for generation of anti-hBPAG2 IgG in this animal model and that blockade of this interplay may impair or avert such humoral immune responses.

Transient co-stimulatory blockade with anti-murine CD40L mAb (MR1) prevented anti-BM IgG production and hBPAG2 Tg skin graft loss in Wt mice

To assess the effect of CD40:CD40L blockade in this animal model, Wt mice (n=23) were grafted with Tg skin and treated with MR1 as described. Sera from 22 of 23 mice showed no evidence of IgG reactive with human epidermal BM for ≥ 60 days following graft placement. (Figure 1a). Tg skin grafts placed on MR1-treated Wt mice remained viable and intact for as long as 60 to 305 days (i.e., their entire period of study). Interestingly, the one mouse in this series that developed anti-BM IgG displayed graft loss within 10 days of becoming seropositive on day 30. Conversely, Wt mice grafted with Tg skin and treated with equivalent amounts of hamster IgG (controls, n=14) developed IgG that bound human epidermal BM (Figure 1a). All controls developed anti-BM IgG with kinetics and titers like those seen in untreated Wt mice grafted with Tg skin. Nine of 14 hamster IgG-treated controls lost Tg grafts within 30 days like untreated Wt graft recipients; for unknown reasons, five controls displayed a delay in the involution and/or loss of Tg skin grafts. Immunoblot studies showed that sera from three representative controls contained IgG that bound hBPAG2 in HK extracts as well as a bacterial recombinant corresponding to the NC16A immunodominant portion of this protein (Figures 1b and 1c, respectively). Conversely, sera from five representative MR1-treated mice showed no evidence of IgG reactive with hBPAG2 or NC16A by immunoblot (Figures 1b and 1c, respectively).

Figure 1.

Figure 1

Figure 1

Figure 1

Open in a new tab

Transient CD40L blockade prevented anti-BM IgG production and loss of Tg skin grafts on Wt mice: a. Anti-BM IgG and graft loss did not develop in 22 of 23 MR1-treated Wt mice grafted with hBPAG2 Tg skin. Interestingly, the one mouse in this series that developed anti-BM IgG (day 30, *) lost its graft within 20 days of the appearance of this specific antibody. In contrast, Wt mice grafted with Tg skin and treated with equivalent amounts of control hamster IgG developed anti-BM IgG with titers, kinetics, and durations equivalent to those seen in untreated Wt mice grafted with Tg skin. Immunoblot studies of HK extracts (b) and a bacterial recombinant corresponding to the NC16A immunodominant portion of hBPAG2 (c) show that sera from three representative controls treated with hamster IgG contained IgG reactive with hBPAG2 as well as NC16A, whereas no such IgG was found in sera from five representative MR1-treated mice (all samples obtained 60 days after graft placement). Positive controls in these studies included sera from patients with bullous pemphigoid (BP1 and BP2) and a murine mAb specific for NC16A (HD18); negative controls included normal human serum (NHS) and normal mouse serum (NMS).

Transient anti-CD40L blockade did not diminish serum IgM and IgG levels

To determine if MR1 administration selectively blocked elicitation of anti-hBPAG2 IgG or caused an overall reduction in serum immunoglobulin levels, IgG and IgM in the sera of mice treated with either MR1 (n=8) or control hamster IgG (n=2) were quantitated at various time points within 60 days of graft placement. These studies found no reductions in levels of IgG and IgM at any time point in experimental subjects or controls. These findings are in accord with prior studies showing that anti-CD40L antibody treatment does not elicit generalized immunosuppression in mice. (Durie et al., 1993; Early et al., 1996; Gerritse et al., 1996; Mohan et al., 1995)

MR1-treatment did not affect established anti- human BM antibody titers in grafted Wt mice

To investigate the effect of MR1 treatment on an established and robust hBPAG2-specific humoral immune response, two seropositive Wt mice previously grafted with Tg skin (i.e., immune Wt mice) were treated with 1 mg of MR1 on day 20 post grafting and with 0.5 mg of MR1 on days 22, 24, 27, and 34. As shown in Figure 2, administration of MR1 to immune Wt mice did not diminish existing levels of anti-BM IgG.

Figure 2.

Figure 2

Open in a new tab

Administration of MR1 to immune Wt mice did not diminish existing levels of anti-BM IgG. Administration of MR1 mAb (total doses equivalent to those used in experiments shown in Figure 2) administered 20 to 34 days after placement of Tg skin grafts did not abrogate established titers of anti-BM IgG (n = 2; triangles represent kinetics of anti-BM IgG titers in two separate mice).

CD40L blockade rendered Wt mice hypo- or non-responsive to a second Tg skin graft

To assess further the long-term non responsiveness of MR1-treated mice to Tg skin, sequential graft experiments were performed in seronegative Wt mice (n = 5) that had retained an initial Tg skin graft for 60 days (a time point when MR1 concentrations in the circulation were greatly diminished [calculated half-life of MR1 in C57BL/6 mice=10.4 days] (Pearson et al., 2003)). Two of 5 mice in this series did not develop anti-BM IgG after placement of a second Tg skin graft. Of three mice that seroconverted following placement of a second Tg skin graft, all displayed diminished, delayed, or transient titers of specific IgG in contrast to high titers of specific IgG seen in controls initially treated with hamster IgG and subjected to a second Tg skin graft on day 60 (Table 1). Moreover, all five MR1-treated mice accepted both the initial and the second Tg skin grafts for the full observation period (i.e., 300 days).

Table 1.

CD40L Blockade Rendered Wt Mice Hypo- or Non-Responsive to a Second Tg Skin Graft

Titers of Anti-BM IgG in MR1-Treated Mice
Days (d) After 1st Graft Days (d) After 2nd Graft
Mouse d 20 d 40 d 60 d 14 d 20 d 40 d 120 d 160
1 0 0 0 80 320 320 5120 640
2 0 0 0 0 0 0 160 0
3 0 0 0 0 0 0 0 0
4 0 0 0 0 0 0 0 0
5 0 0 0 320 320 320 0 0
Titers of Anti-BM IgG in Hamster IgG-Treated Mice (Controls)
Days (d) After 1st Graft Days (d) After 2nd Graft
Mouse d 20 d 40 d 60 d 14 d 20 d 40 d 120 d 160
1 2560 5120 5120 5120 5120 5120 5120 5120
2 640 640 1280 2560 5120 5120 5120 5120
Open in a new tab

Lymphocytes from MR1-treated mice prevented or diminished anti-BM IgG production by naive lymphocytes following co-transfer to Rag2 -/- mice

To determine if lymphocytes from MR1-treated mice had a “dominant” or “infectious” (Qin et al., 1993) tolerogenic effect over lymphocytes from naïve Wt mice, a series of adoptive transfer studies were performed. Initial studies showed that adoptive transfer of 2 × 107 lymphocytes from naïve Wt mice to Rag2 -/- mice endowed the latter (n=5) with the ability to develop high and durable titers of anti-BM IgG following placement of hBPAG2 Tg skin grafts. (Figure 3a). Moreover, Tg skin grafts on such seropositive Rag2 -/- mice involuted and disappeared within 30 to 55 days. Thus, “reconstituted” Rag2 -/- mice grafted with Tg skin were able to generate hBPAG2-specific IgG and lose grafts like Wt mice grafted with Tg skin. Interestingly, when Rag2 -/- mice (n=4) received a mixture of 2 × 107 lymphocytes from MR1-treated mice and an equivalent number of lymphocytes from naïve, Wt mice, they were rendered hyporesponsive to hBPAG2 Tg skin grafts. More specifically, one mouse in this series failed to develop anti-hBPAG2 IgG, while three others displayed minimal and/or delayed development of specific antibody (Figure 3a). Moreover, the only mouse in this series that lost its graft did so after eventually developing the highest titer of anti-BM IgG (titer, 2560). All other mice in this series displayed relatively low and transient levels of hBPAG2-specific IgG and accepted skin grafts for the entire period of study (i.e., as long as 235 days).

Figure 3.

Figure 3

Figure 3

Open in a new tab

Lymphocytes from MR1-treated mice diminished or blocked anti-BM IgG production by naive lymphocytes following co-transfer to Rag2 -/- mice: a. Adoptive transfer of 2 × 107 lymphocytes from naïve Wt mice to Rag2 -/- mice endowed the latter (n=5, circles) with the ability to develop high and durable titers of anti-BM IgG following placement of hBPAG2 Tg skin grafts; Tg skin grafts were lost in all mice within 10 to 20 days of the appearance of such IgG. In contrast, when Rag2 -/- mice received 2 × 107 lymphocytes from MR1-treated mice mixed with an equivalent number of lymphocytes from naïve, Wt mice, they were hypo- or nonresponsive to a subsequent hBPAG2 Tg skin graft (n = 4, triangles represent individual animals). Only one such Rag2 -/- mouse lost its graft (again, soon after developing high titers of anti-BM IgG [*]). b. Rag2 -/- mice (n = 2) that received 5 × 106 CD4+ T cells from MR1-treated mice mixed with 2 × 107 naïve Wt lymphocytes showed either low and transient, or greatly delayed anti-BM IgG production following placement of Tg skin grafts (triangles represent separate mice; controls represent the same shown in Figure 3a). Again, the one mouse in this set that developed high titers of anti-BM IgG (*, day 80), lost its graft within the subsequent 25 days. The dominant tolerogenic effect of CD4+ T cells from MR1-treated mice appeared to be dose-dependent in that 2 × 107 of such CD4+ cells completely blocked anti-BM IgG production by an equal number of Wt lymphocytes in Rag2 -/-mice grafted with Tg skin (n = 2, small diamonds represent separate mice with identical results).

In an analogous set of adoptive transfer experiments, CD4+ T cells from MR1-treated Wt mice were mixed with lymphocytes from naïve, Wt mice and transferred to Rag 2-/- mice that were then grafted with hBPAG2 Tg skin. These studies found that 5 × 106 CD4+ T cells from MR-1 treated mice delayed or averted the immune responses of co-transferred 2 × 107 naïve, Wt lymphocytes to hBPAG2 Tg skin. Again, the only mouse in this experiment that lost its Tg skin graft did so within 25 days of developing a relatively high titer of anti-BM IgG on day 80 (titer, 5120). (Figure 3b). Anti-BM IgG did not develop in Rag 2-/- Tg graft recipients that had been similarly reconstituted with a mix of 2 × 107 CD4+ T cells from MR1-treated mice and an equal number of naïve, Wt splenocytes (Figure 3b). These experiments demonstrated that CD4+ T cells from mice that were tolerant to hBPAG2 could transfer such hyporesponsiveness to other mice and that this effect appeared to be dose-dependent. Of note, 1.8 to 2.0 × 107 B cells from MR1-treated mice did not diminish immune responses to hBPAG2 by 2 × 107 co-transferred, naïve, Wt lymphocytes in Rag 2-/- graft recipients (n = 5) (data not shown).

DISCUSSION

The demonstration that Tg skin grafts elicited hBPAG2-specific IgG (and graft loss) in Wt but not CD40L -/- recipients illustrates the importance of CD40L signaling in the generation of experimental immune responses in this defined animal model. This conclusion was substantiated by the demonstration that CD40L blockade with MR1 mAb consistently prevented specific IgG production in this model. Transient MR1 administration not only blocked anti-BM IgG production, but also fostered long-term acceptance of Tg skin grafts and rendered such mice nonresponsive to a second hBPAG2 Tg skin graft. Since CD40L blockade by monotherapy has been perceived to inhibit humoral-based diseases only (Ferrant et al., 2004), the efficacy of this intervention supports the premise that graft loss in this model is antibody-mediated. Of note, anti-CD40L monotherapy has not been successful in controlling allograft rejection by CD8+ (Honey et al., 1999) or CD4+ effector T cells (Jarvinen et al., 2003).

MR1 treatment is thought to block the initiation of humoral immune responses since the expression of CD40L is tightly regulated and because it is only transiently expressed on activated T-cells (i.e., 1 to 2 hours after activation, peak expression at 6 to 8 hours, rapid disappearance thereafter) (van Kooten and Banchereau, 2000). In contrast, CD40:CD40L blockade does not interfere with established humoral immune responses exerted by long-lived CD40:CD40L independent, antibody-secreting plasma cells. In accord with these prior observations, our studies found that MR1 administration did not reduce serum immunoglobulin levels or lower established titers of hBPAG2-specific IgG in immune Wt mice. These findings suggest that CD40L blockade may have utility for prevention of unwanted humoral immune responses to hBPAG2 in GABEB patients undergoing gene replacement therapy without causing systemic immunosuppression, but not abrogate established autoimmune responses in patients with bullous pemphigoid. Our studies highlight the complexities of immunomodulatory effects resulting from transient CD40L blockade. They also suggest that an active immune regulatory mechanism may underlie tolerance to hBPAG2 in MR1-treated mice and account for the dominant and tolerogenic responses observed in our adoptive transfer studies. Interestingly, this tolerance appeared to be mediated by CD4+ T cells that exerted a dominant effect over larger numbers of cotransferred naïve Wt lymphocytes.

Our results are in accord with those developed in analogous murine animal models exploring experimental immune responses to Dsg3. MR1 (but not hamster IgG, control) administration delayed and/or abrogated development of anti-Dsg3 IgG in Dsg3 -/- mice grafted with Wt skin that expressed this desmosomal cadherin (Ohyama et al., 2003). Similarly, CD40:CD40L blockade inhibited production of anti-Dsg3 IgG in Dsg3-positive Rag 2 -/- mice carrying lymphocytes from Dsg3 -/- mice (Aoki-Ota et al., 2006). Hence, studies in two separate experimental animal models indicate that CD40:CD40L signaling plays an important role in the generation of humoral immune responses to target antigens in skin in vivo.

MATERIALS AND METHODS

Mice

The following strains of mice were used in these studies: C57BL/6 Tg mice expressing hBPAG2 in murine epidermal BM (Olasz et al., 2007); C57BL/6 wild-type (Wt) and CD40L -/- mice (Jackson Laboratory, Bar Harbor, ME); Rag2 -/- mice (Taconic Farms, Germantown, NY). Mice were housed under sterile conditions in the Biomedical Resource Center at the Medical College of Wisconsin and were typically used between six and twelve weeks of age. All studies in animals were approved by the Medical College of Wisconsin’s Animal Care and Use Committee.

Skin Grafting Studies

Tail skin was harvested from hBPAG2 Tg mice and grafted onto the backs of age- and gender-matched Wt, CD40L -/-, and Rag2 -/- recipients on day 0 as described previously (Olasz et al., 2007; Rosenberg and Singer, 1988). In sequential graft experiments, five MR1-treated Wt mice (all seronegative for anti-hBPAG2 IgG and retaining initial Tg skin grafts) received a second Tg skin graft on their alternate flanks on day 60. Bandages were removed 7 days post surgery and grafts were evaluated for viability and size twice weekly. Grafts were said to be lost if they became 20% or less of their original size.

MR1/hamster IgG treatment regimen

Wt mice were grafted with Tg skin and injected i.p. with 1 mg hamster anti-CD40L MR1 mAb (Taconic Farms) or an equivalent amount of control hamster IgG (Cappel Products, Aurora, OH) on day 0 and with 0.5 mg of these respective reagents on days 2, 4, 7, and 14.

Adoptive transfer studies

2 × 107 lymphocytes from lymph nodes and spleens of MR1-treated Wt mice bearing intact and viable hBPAG2 Tg skin grafts for 60 days were mixed with a equal number of lymphocytes from naïve, Wt mice and adoptively transferred to Rag2 -/- mice. In an analogous set of experiments, 5 to 20 × 106 CD4+ T cells (purity >90% by flow cytometry [CD4 subset column kit, R & D Systems, Minneapolis, MN]) from MR1-treated Tg graft recipients were co-transferred with 2 × 107 lymphocytes from naive Wt mice to Rag2 -/- mice. As controls, Rag2 -/- mice were constituted with 2 × 107 lymphocytes from naïve Wt mice alone. These sets of Rag2-/- mice were subsequently grafted with Tg skin and followed for anti-BM IgG production and graft survival.

Indirect Immunofluorescence (IF) Microscopy

Serial dilutions of sera from mice grafted with hBPAG2 Tg skin were tested for anti-BM IgG by indirect IF microscopy studies of 1 M NaCl split skin (Olasz et al., 2007).

Immunoblotting

IgG in murine sera were characterized for their reactivity to HK extracts as well as a bacterial recombinant corresponding to the NC16A domain of hBPAG2 by immunoblotting (Olasz et al., 2007). Sera from bullous pemphigoid patients and a murine mAb directed against NC16A (HD-18) were used as positive controls; normal mouse serum and normal human serum served as negative controls.

Total IgG and IgM serum levels

Serum levels of murine IgG and IgM were quantitated with IgG or IgM ELISA kits (Bethyl Laboratories, Montgomery, TX).

Acknowledgments

This work was supported in part by NIH Grant RO1 AR048982 (KBY) and the Max Kade Foundation (CML)

Abbreviations

BM

Basement membrane

CD40L

CD40 ligand

hBPAG2

human bullous pemphigoid antigen2

hIgG

hamster IgG

mAb

monoclonal antibody

Tg

transgene/transgenic

Wt

wildtype

REFERENCES

  1. Aoki-Ota M, Kinoshita M, Ota T, Tsunoda K, Iwasaki T, Tanaka S, et al. Tolerance induction by the blockade of CD40/CD154 interaction in pemphigus vulgaris mouse model. J Invest Dermatol. 2006;126:105–113. doi: 10.1038/sj.jid.5700016. [DOI] [PubMed] [Google Scholar]
  2. Bauer JW, Lanschuetzer C. Type XVII collagen gene mutations in junctional epidermolysis bullosa and prospects for gene therapy. Clin Exp Dermatol. 2003;28:53–60. doi: 10.1046/j.1365-2230.2003.01192.x. [DOI] [PubMed] [Google Scholar]
  3. Chen M, Woodley DT. Fibroblasts as target cells for DEB gene therapy. J Invest Dermatol. 2006;126:708–710. doi: 10.1038/sj.jid.5700216. [DOI] [PubMed] [Google Scholar]
  4. Darling TN, Bauer JW, Hintner H, Yancey KB. Generalized atrophic benign epidermolysis bullosa. Adv Dermatol. 1997;13:87–119. discussion 120. [PubMed] [Google Scholar]
  5. Dellambra E, Pellegrini G, Guerra L, Ferrari G, Zambruno G, Mavilio F, et al. Toward epidermal stem cell-mediated ex vivo gene therapy of junctional epidermolysis bullosa. Hum Gene Ther. 2000;11:2283–2287. doi: 10.1089/104303400750035825. [DOI] [PubMed] [Google Scholar]
  6. Durie FH, Fava RA, Foy TM, Aruffo A, Ledbetter JA, Noelle RJ. Prevention of collagen-induced arthritis with an antibody to gp39, the ligand for CD40. Science. 1993;261:1328–1330. doi: 10.1126/science.7689748. [DOI] [PubMed] [Google Scholar]
  7. Early GS, Zhao W, Burns CM. Anti-CD40 ligand antibody treatment prevents the development of lupus-like nephritis in a subset of New Zealand black x New Zealand white mice. Response correlates with the absence of an anti-antibody response. J Immunol. 1996;157:3159–3164. [PubMed] [Google Scholar]
  8. Ferrant JL, Benjamin CD, Cutler AH, Kalled SL, Hsu YM, Garber EA, et al. The contribution of Fc effector mechanisms in the efficacy of anti-CD154 immunotherapy depends on the nature of the immune challenge. Int Immunol. 2004;16:1583–1594. doi: 10.1093/intimm/dxh162. [DOI] [PubMed] [Google Scholar]
  9. Franzke CW, Bruckner P, Bruckner-Tuderman L. Collagenous transmembrane proteins: recent insights into biology and pathology. J Biol Chem. 2005;280:4005–4008. doi: 10.1074/jbc.R400034200. [DOI] [PubMed] [Google Scholar]
  10. Gerritse K, Laman JD, Noelle RJ, Aruffo A, Ledbetter JA, Boersma WJ, et al. CD40-CD40 ligand interactions in experimental allergic encephalomyelitis and multiple sclerosis. Proc Natl Acad Sci U S A. 1996;93:2499–2504. doi: 10.1073/pnas.93.6.2499. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hintner H, Wolff K. Generalized atrophic benign epidermolysis bullosa. Arch Dermatol. 1982;118:375–384. [PubMed] [Google Scholar]
  12. Honey K, Cobbold SP, Waldmann H. CD40 ligand blockade induces CD4+ T cell tolerance and linked suppression. J Immunol. 1999;163:4805–4810. [PubMed] [Google Scholar]
  13. Jarvinen LZ, Blazar BR, Adeyi OA, Strom TB, Noelle RJ. CD154 on the surface of CD4+CD25+ regulatory T cells contributes to skin transplant tolerance. Transplantation. 2003;76:1375–1379. doi: 10.1097/01.TP.0000093462.16309.73. [DOI] [PubMed] [Google Scholar]
  14. Khavari PA. Gene therapy for genetic skin disease. J Invest Dermatol. 1998;110:462–467. doi: 10.1038/jid.1998.3. [DOI] [PubMed] [Google Scholar]
  15. Mavilio F, Pellegrini G, Ferrari S, Di Nunzio F, Di Iorio E, Recchia A, et al. Correction of junctional epidermolysis bullosa by transplantation of genetically modified epidermal stem cells. Nat Med. 2006;12:1397–1402. doi: 10.1038/nm1504. [DOI] [PubMed] [Google Scholar]
  16. Mohan C, Shi Y, Laman JD, Datta SK. Interaction between CD40 and its ligand gp39 in the development of murine lupus nephritis. J Immunol. 1995;154:1470–1480. [PubMed] [Google Scholar]
  17. Noelle RJ, Roy M, Shepherd DM, Stamenkovic I, Ledbetter JA, Aruffo A. A 39-kDa protein on activated helper T cells binds CD40 and transduces the signal for cognate activation of B cells. Proc Natl Acad Sci U S A. 1992;89:6550–6554. doi: 10.1073/pnas.89.14.6550. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Ohyama M, Ota T, Aoki M, Tsunoda K, Harada R, Koyasu S, et al. Suppression of the immune response against exogenous desmoglein 3 in desmoglein 3 knockout mice: an implication for gene therapy. J Invest Dermatol. 2003;120:610–615. doi: 10.1046/j.1523-1747.2003.12090.x. [DOI] [PubMed] [Google Scholar]
  19. Olasz EB, Roh J, Yee CL, Arita K, Akiyama M, Shimizu H, et al. Human bullous pemphigoid antigen 2 transgenic skin elicits specific IgG in wild-type mice. J Invest Dermatol. 2007;127:2807–2817. doi: 10.1038/sj.jid.5700970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Pearson T, Markees TG, Wicker LS, Serreze DV, Peterson LB, Mordes JP, et al. NOD congenic mice genetically protected from autoimmune diabetes remain resistant to transplantation tolerance induction. Diabetes. 2003;52:321–326. doi: 10.2337/diabetes.52.2.321. [DOI] [PubMed] [Google Scholar]
  21. Qin S, Cobbold SP, Pope H, Elliott J, Kioussis D, Davies J, et al. “Infectious” transplantation tolerance. Science. 1993;259:974–977. doi: 10.1126/science.8094901. [DOI] [PubMed] [Google Scholar]
  22. Rosenberg AS, Singer A. Evidence that the effector mechanism of skin allograft rejection is antigen-specific. Proc Natl Acad Sci U S A. 1988;85:7739–7742. doi: 10.1073/pnas.85.20.7739. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Roy M, Waldschmidt T, Aruffo A, Ledbetter JA, Noelle RJ. The regulation of the expression of gp39, the CD40 ligand, on normal and cloned CD4+ T cells. J Immunol. 1993;151:2497–2510. [PubMed] [Google Scholar]
  24. Van den Bergh F, Giudice GJ. BP180 (type XVII collagen) and its role in cutaneous biology and disease. Adv Dermatol. 2003;19:37–71. [PubMed] [Google Scholar]
  25. van Kooten C, Banchereau J. CD40-CD40 ligand. J Leukoc Biol. 2000;67:2–17. doi: 10.1002/jlb.67.1.2. [DOI] [PubMed] [Google Scholar]
  26. Woodley DT, Remington J, Huang Y, Hou Y, Li W, Keene DR, et al. Intravenously Injected Human Fibroblasts Home to Skin Wounds, Deliver Type VII Collagen, and Promote Wound Healing. Mol Ther. 2007;15:628–635. doi: 10.1038/sj.mt.6300041. [DOI] [PubMed] [Google Scholar]

RESOURCES