A swift humoral response, usually mediated by preexisting antibodies or memory B cells specific for transplant antigens, can lead to rapid rejection of allografts and xenografts. This response — known as hyperacute rejection (1) — was first observed in human renal grafts and extensively studied in more recent models of xenotransplantation (6–12). Clinically, the transplant rapidly (within minutes and often during the surgery) blackens from extensive coagulation and complement mediated tissue destruction. The organ has to be quickly removed to prevent a subsequent systemic inflammatory response syndrome–like sequelae from developing. Hyperacute rejection has been most debilitating to prospects for xenotransplantation (2).
The mechanistic insights gained from early studies have led to the development of several therapeutic strategies to prevent or ameliorate antibody-mediated hyperacute responses. These include treatment with intravenous immunoglobulin (Ig), B-cell depletion with rituximab (anti-CD20), complement inhibitors such as eculizumab, chemicals that directly target plasma cells (bortezomib), and others (3–10). However, because most of these approaches broadly target the antibody response in the host, there are considerable adverse effects. The transplant recipient is severely immunocompromised and must be supported by antibiotics and other strategies. In the case of xenotransplantation, a more focused effort has been made — by removing potential antibody targets on the xenografts. This includes the generation of genetically modified pigs that lack the α-galactoside (α-gal) carbohydrate groups predominantly eliciting the hyperacute response (11) and animals expressing negative regulators of human complement cascade activity on their cell surface (12). Unfortunately, these strategies have not been as widely successful as initially hoped. The α-gal knockout grafts, for example, continue to elicit accelerated rejection responses involving antibodies targeting alternate antigens on the surface (13). Therefore, it is clear that further efforts are required to develop more effective strategies capable of suppressing antibody-mediated rejection responses, preferably in a donor-specific fashion.
In this study, we describe a new mouse model that uses a simple adoptive transfer strategy to sensitize a hyperacute response. Monoclonal helper T cells from the B10.A(H-2a),5C.C7,RAG2−/− T-cell receptor (TCR) transgenic mouse (5C.C7) were found to be alloreactive against the B10.S(9R) strain of the major histocompatibility complex (MHC) haplotype H-2t4. Soon after the initial transfer, the host displayed a hyperacute antibody response against subsequent H-2a grafts. Interestingly, chronic antigen stimulation rendered the 5C.C7 T cells somewhat resistant to this rejection. Finally, we were able to validate a form of donor-specific tolerization against the hyperacute response in this model, by adoptively transferring a small number of H-2a B cells to the host, a few days before the first graft of 5C.C7 T cells.
5C.C7 TCR Transgenic T Cells Are Alloreactive Against B10.S(9R) Splenocytes
The B10.S(9R) strain was originally obtained as a result of a cross between the B10.S (H-2s) and B10.A (H-2a) strains of mice. A recombination in the H-2 locus resulted in a novel MHC class II molecule with a crossover within the IEβ gene, after the β1 exon (16). The resulting H-2 locus (Fig. 1A) consists of the 5′ elements from the H-2s chromosome and the 3′ elements from the H-2a chromosome and was named H-2t4. Because this allele had been reported to allogeneically stimulate certain pigeon cytochrome c (PCC)–specific T-cell clones, we asked if T cells from the MCC(88–103)-specific TCR-5C.C7 transgenic mouse (on a RAG2-deficient background) were also alloreactive against H-2t4. 5C.C7 T cells showed a clear proliferative response, in vitro, to as few as 10,000 B10.S(9R),CD3[Latin Small Letter Open E]−/− splenocytes (Fig. 1B). In contrast, they showed no significant proliferative response to the syngeneic B10.A splenocytes (Fig. 1B).
The in vitro alloreactivity we discovered allowed us to consider the B10.S(9R) mouse as an in vivo model for studying GVH responses using the 5C.C7 T cells. Adoptively transferred 5C.C7 (Ly5.1+) T cells expanded rapidly in B10.S(9R) (Ly5.2+) mice for up to 3 days after transfer (Fig. 1C) but not in a B10.A host, which does not express any stimulatory antigen for the 5C.C7 TCR (Fig. 1C). Subsequently, the number of T cells dropped precipitously. Such a pattern is similar to the behavior of 5C.C7 T cells in hosts that express their cognate antigen – PCC (15). However, we have previously reported that if such PCC transgenic hosts were devoid of endogenous T cells, the deletional phase could be largely eliminated. To examine that in this model, B10.S(9R),CD3[Latin Small Letter Open E]−/− mice were generated wherein endogenous T-cell development is abrogated. Although adoptive transfer of 5C.C7 T cells into these mice resulted in a more robust T-cell expansion (Fig. 1D) than observed in the intact B10.S(9R) host, the recovery of T cells still declined after the fifth day and was below detection beyond 30 to 35 days. As previously reported, 5C.C7 T cells in syngeneic B10.A,CD3[Latin Small Letter Open E]−/− hosts persisted, with a characteristic “homeostatic” expansion (Fig. 1D).
Deletion of 5C.C7 T Cells Is Accompanied by the Development of an H-2a–Specific Hyperacute Response
The deletion of the alloreactive 5C.C7 T-cell population in the B10.S(9R),CD3[Latin Small Letter Open E]−/− host could be due to T-cell–autonomous changes over the course of their response or due to changes in the allogeneic environment, induced by the T-cell response. We attempted to distinguish the two, by transferring a fresh cohort of Carboxy Flourescein Succinimidyl Ester (CFSE)-labeled naive 5C.C7 T cells into T-cell–experienced B10.S(9R),CD3[Latin Small Letter Open E]−/− recipients (that had begun to delete an initial cohort of 5C.C7 T cells administered 14 days previously). Surprisingly, even 1 day after the second transfer, we could not recover the fresh cohort of 5C.C7 T cells from the T-cell–experienced B10.S(9R),CD3[Latin Small Letter Open E]−/− mice (Fig. 2A). A similar transfer to a PCC transgenic host (Fig. 2A) resulted in successful engraftment. This rapid deletion of a second cohort was evident as early as 6 days after sensitization by an initial transfer of 5C.C7 T cells into a B10.S(9R),CD3[Latin Small Letter Open E]−/− mouse (day 6; Fig. 2B) and persisted as long as 61 days afterward. The transferred T cells do reach the lymphoid organs of T-cell–experienced B10.S(9R),CD3[Latin Small Letter Open E]−/− mice because a small number could be seen 2 hours after transfer (Fig. 2C), but this number further reduces over the next 6 hours (Fig. 2C). Therefore, the rejection process is quite acute, starting as early as 2 hours after grafting (Fig. 2D).
Polyclonal T cells from B10.A mice (Fig. 2E), but not the ones from B10.S(9R) mice (Fig. 2E) were also rejected in day 12 B10.S(9R),CD3[Latin Small Letter Open E]−/− mice (Fig. 2E) but not in naive B10.S(9R),CD3[Latin Small Letter Open E]−/− (Fig. 2E). Thus, the rejection process was neither TCR transgene-specific nor required antigen-specific interactions mediated through the 5C.C7 TCR on the second graft of T cells. In fact, it also applied to B10.A B cells and CD8+ T cells as well (data not shown), suggesting that it is a rejection of H-2a tissue in general.
Antibodies Mediate Hyperacute Rejection of Secondary Grafts
We examined the production of antibodies against the H-2a–derived T cells, using serum from B10.S(9R),CD3[Latin Small Letter Open E]−/− recipients of 5C.C7 T cells (Fig. 3). Serum from B10.S(9R),CD3[Latin Small Letter Open E]−/− mice that received 5C.C7 T cells 6 days before (Fig. 3A) clearly stained H-2a T cells and did not label the H-2b CD4+ T cells, whereas sera from naive B10.S(9R), CD3[Latin Small Letter Open E]−/− mice (Fig. 3A) stained neither. We also tested the ability of these antibodies to target cytotoxicity to H-2a T cells by incubating naive 5C.C7 T cells with serum from 5C.C7-experienced mice, at 4°C, before transfer into a naive B10.S(9R),CD3[Latin Small Letter Open E]−/− host (Fig. 3B). Incubation with serum from B10.S(9R),CD3[Latin Small Letter Open E]−/− mice (13 or 22 days after the first T-cell transfer) resulted in deletion confirming the ability of H-2a-specific antisera produced by these hosts to acutely reject H-2a cells in vivo.
To further validate the role of the B cells, we generated a RAG2-deficient B10.S(9R) strain lacking B and T cells. As in the B10.S(9R),CD3[Latin Small Letter Open E]−/− hosts, the 5C.C7 T cells expanded quickly in the B10.S(9R).Rag2−/− hosts, peaking by day 4 (Fig. 3C). Although the T-cell numbers did subsequently undergo a significant decline (80% loss between days 4 and 15), complete deletion of T cells did not occur. In fact, T cells could be recovered from this host even 2 months after transfer. The B10.S(9R),Rag2−/− hosts also did not develop a hyperacute response. Interestingly, we recovered a greater number of the newly transferred T-cell cohort from the 12-day T-cell–experienced B10.S(9R),Rag2−/− recipients, relative to naive B10.S(9R),Rag2−/−, after the second transfer (Fig. 3D).
Finally, we asked whether serum isolated from 5C.C7 experienced B10.S(9R),CD3[Latin Small Letter Open E]−/− mice can transfer deletion to the RAG2−/− host. B10.S(9R),Rag2−/− mice were treated with T-cell–experienced B10.S(9R),CD3[Latin Small Letter Open E]−/− serum for two consecutive days (Fig. 3E) before injecting CFSE-labeled naive 5C.C7 T cells. There was a marked decrease in the number of T cells recovered from the Rag2−/− mice treated with hyperacute serum compared with those that received only serum from naive B10.S(9R),CD3[Latin Small Letter Open E]−/− mice.
The development of a robust B-cell–mediated anti–H-2a hyperacute response in the B10.S(9R),CD3[Latin Small Letter Open E]−/− mice can account for the rapid deletion of the 5C.C7 T cells in this model. However, although the secondary cohorts of H-2a tissues are rapidly deleted (within 24 hours), the primary 5C.C7 graft is actually detectable for up to 2 weeks after, presenting a paradox. This could be due to the primary graft 1) occupying niche(s) not accessible to the toxic antibodies, 2) being in an antigen-experienced state, or 3) being under chronic stimulation by the persistent stimulus. To distinguish between these possibilities, we adoptively transferred 5C.C7 T cells that were either naive (Fig. 3F), activated in vitro to a memory phenotype (17) or chronically stimulated in B10.A,PCC+,CD3[Latin Small Letter Open E]−/− mice as previously reported (17), into B10.S(9R),CD3[Latin Small Letter Open E]−/− mice after the development of the hyperacute phase. Interestingly, although both the naive and antigen-experienced 5C.C7 T cells were rapidly deleted, a significant proportion of the chronically stimulated 5C.C7 T cells survived the onslaught of rejecting antibodies. These cells did indeed succumb to rejection during a 72-hour period (Fig. 3G), but this was considerably delayed compared with the rejection of naive T cells (Fig. 3H). This suggests that long-term TCR stimulation affords a partial protection to T cells from antibody-mediated hyperacute rejection.
Modeling Therapeutic Strategies to Prevent the Development of a Hyperacute Rejection
A transgenic mouse model for the hyperacute response is valuable not only for studying the cellular processes underlying a hyperacute response but also for developing and validating potential therapeutic strategies for circumventing the process. First, we validated a reputed therapeutic regimen in such transplantation settings—using the blockade of CD40L. Both the blockade of this molecule using antibody treatment (Fig. 4A) and the abolition of its role in the primary 5C.C7 response by using a CD40L-deficient T cell resulted in the elimination of the subsequent hyperacute response (Fig. 4A).
We then attempted to develop a rational cellular therapy that would eliminate the hyperacute response in this model from first principles. The hyperacute response is likely initiated when 5C.C7 T cells first meet alloantigen presenting B cells, irrespective of the antigen specificity of the B-cell receptor. However, if some B cells are also capable of recognizing the H-2a molecule on the 5C.C7 T cell, the combination of Ig signaling and T cell help will rapidly drive an anti–H-2a B-cell response. We therefore hypothesized that any strategy that would tolerize the H-2a-specific B cells before their encounter with the 5C.C7 T cell would prevent the onset of the hyperacute response. To achieve this end, we developed a simple strategy that involved adoptive transfer of purified H-2a B cells (donor haplotype) to the B10.S(9R),CD3[Latin Small Letter Open E]−/− mice 4 to 7 days before the infusion of 5C.C7 T cells. These B cells would be expected to migrate to the B-cell areas of the recipient mice and allow host B cells to interact with the H-2a antigen. Because these interactions take place in the absence of T-cell help, the recipient’s B cell would be expected to be tolerized by the H-2a antigen.
Adoptive transfer of 5 million purified B10.A B cells, 4 days before the transfer of 5C.C7 T cells, demonstrates the viability of this strategy. The transferred B cells engraft and can be detected even 32 days after transfer (Fig. 4B). 5C.C7 T cells in the donor B-cell–treated mice (Fig. 4C) expanded similarly to control, untreated mice (Fig. 4C). However, after the expansion phase, although the 5C.C7 cells were rapidly deleted in the untreated mice, no appreciable deletion was observed in the B-cell–treated animals. This prolonged survival of the alloreactive T cell is similar to previous observations in PCC transgenic CD3[Latin Small Letter Open E]−/− hosts. Finally, serum samples from B-cell–treated mice did not have significant levels of antibodies to H-2a (Figs. 4D and E) confirming the complete tolerization of the hyperacute response.
The hyperacute rejection response presents a major hurdle to successful transplantation of allografts and xenografts. We describe a new mouse model system that is highly amenable to the study of a hyperacute response. Interestingly, it also incorporates the phenomenon of adaptation, where the chronically activated T cells are resistant to antibody attack (18). We further demonstrate the utility of this model in formulating new therapeutic strategies for preventing hyperacute rejection in a donor-specific fashion.
The 5C.C7 TCR transgenic mouse generates a population of naive CD4+ T cells specific for a peptide from PCC presented on IEk. These T cells have no known cross-reactivity with self antigens and retain a naive phenotype in intact B10.A mice. The alloreactivity that we now describe against B10.S(9R) cells adds an important extension to this model system. We have previously reported the behavior of 5C.C7 T cells in an adoptive transfer model where they encounter their cognate antigen (PCC) as a self protein. In this model, PCC is expressed in the hematopoietic system and presented by antigen-presenting cells including B cells. Interestingly, in otherwise T-cell–deficient animals (CD3[Latin Small Letter Open E]−/−), the strong anti-PCC response is followed by prolonged survival of the T cells with no appreciable deletional component. This had led us to propose that “clonal deletion” in peripheral T cells is not determined by cell-intrinsic processes but rather by regulatory interactions originating from neighboring T cells. A prediction of this hypothesis was that deletion would be either absent or severely blunted in T-cell–deficient hosts. The development of the alloreactive model has allowed us to examine this hypothesis in a different context. Surprisingly, the 5C.C7 T cells were deleted (albeit slowly) in this new model. Our analysis of the mechanism involved, however, revealed a B cell response that actively eliminates the antigen-specific T cell. Similar rejection of lymphocytes by CD8+ T cells has been reported before (19, 20). Thus, this further validates the idea that non–T-cell–autonomous mechanisms dominate in peripheral clonal deletion/elimination. Furthermore, when we are able to tolerize the B-cell response against 5C.C7 MHC molecules in the CD3[Latin Small Letter Open E]−/− host or to eliminate B cells completely in the RAG2−/− host, the responding T cells did persist.
The strategy we developed to tolerize the B-cell response is a modification of the concept of microchimerism developed by Sharabi et al. (21) and Yang et al. (22). Although this has largely been developed using stem-cell transplants, our approach makes use of mature B lymphocytes. The strategy has several advantages: 1) The donor B cells are most likely to migrate to the B cell areas of the host — thereby effectively targeting the very B cells that generate a hyperacute response against the donor. 2) Naive B cells are relatively poor activators of de novo T-cell responses and are therefore likely to tolerize rather than prime antidonor responses (23). 3) From a practical standpoint, B cells can be obtained from donors (even under clinical settings) in relatively large numbers compared with stem cells.
A potential caveat in the application of this strategy more broadly is the presence of alloreactive T cells in the host. In the model we use, the lack of host T cells (CD3[Latin Small Letter Open E]−/−) eliminates this difficulty. It also allows the H-2a–reactive B cells to be easily tolerized (by engaging antigens on the donor B cells in the absence of T-cell help). In a more complete in vivo milieu, the host T cells would probably have to be suppressed during the initial engraftment of the donor B cells. Although several options are available for this (cyclosporine, sirolimus, abatacept, etc.), this is still a major limitation of the current study as presented. Nevertheless, a reductionist yet refined TCR transgenic model will facilitate molecular genetic dissection of hyperacute rejection in a very sensitive fashion. However, those observations should be validated in widely available polyclonal models before further application.
MATERIALS AND METHODS
All mice were bred in the National Institute of Allergy and Infectious Diseases (NIAID) contract barriers at Taconic Farms, Inc (Germantown, NY) and housed at the Association for Assessment and Accreditation of Laboratory Animal Care International–certified specific pathogen-free facilities maintained by NIAID, National Institutes of Health. The B10.S(9R)/SgSnAi stain was originally obtained from Dr. Jack Stimpfling in 1975. The CD3[Latin Small Letter Open E]−/− and RAG2−/− backgrounds were crossed onto the strain from the B10.A,CD3[Latin Small Letter Open E]−/− and B10.A,RAG2−/− lines of mice. The resulting progeny were intercrossed and then selected for homozygosity at the H-2t4 MHC locus and the respective knockout alleles. The B10.A,5C.C7,RAG2−/− (14) and the B10.A,5C.C7,RAG2−/−,Ly5.1+/+ mice have been previously described (15). All animal protocols were approved by the NIAID/DIR Animal Care and Use Committee. CD40L deficiency was bred on the B10.A,5C.C7,RAG2-/- background from an original B6 strain.
Lymphocyte Isolation and Adoptive Transfer
T cells were isolated from the lymph nodes of 5C.C7 mice and single-cell suspensions prepared in phosphate-buffered saline supplemented with 5% fetal calf serum and antibiotics. Typically, these preparations were 93% to 95% CD4+ and were not further purified. B cells were purified from the spleens of B10.A,CD3[Latin Small Letter Open E]−/− mice using the magnetically activated cell sorter B-cell isolation kit (Miltenyi Biotec, Gladbach, Germany) as per the manufacturer’s recommendations and using an AutoMACS separator (Miltenyi Biotec). In some experiments, the enriched cells (already >98% purity) were further purified by fluorescence-activated cell sorter (FACS) for CD19+ and negative for CD11c, CD11b, DX5, NK1.1, and GR-1. All antibodies were purchased from BD Biosciences (San Jose, CA). Isolated cells were resuspended in phosphate-buffered saline, and 1 to 5 million were injected intravenously by the suborbital route. The expansion of T cells in vivo was monitored by recovering the lymph nodes and spleen from transfer recipients, preparing single-cell suspensions, counting viable cells, and staining for the TCR (Vβ3), CD4, and Ly5.1 among the 7AAD− cells. Data were collected either on a BD FACS Calibur or on a BD FACS Sort upgraded by Cytek Inc (Fremont, CA) and analyzed with CellQuest (BD Immunocytometry) or FlowJo software (Treestar, Ashland, OR).
The authors thank Chuan Chen and Andrew Medina-Marino for assistance with experiments, the NIAID flow cytometry facility (Carol Henry and Calvin Eigsti) for flow sorting, and members of Laboratory of Cellular and Molecular Immunology, NIAID for discussions.
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