Secondary Logo

Journal Logo

mTOR Inhibition Suppresses Posttransplant Alloantibody Production Through Direct Inhibition of Alloprimed B Cells and Sparing of CD8+ Antibody-Suppressing T cells

Avila, Christina L. MPH; Zimmerer, Jason M. PhD; Elzein, Steven M. BS; Pham, Thomas A. MD, MS; Abdel-Rasoul, Mahmoud MS; Bumgardner, Ginny L. MD, PhD

doi: 10.1097/TP.0000000000001291
Original Basic Science—Liver
Free
SDC

Background De novo alloantibodies (donor-specific antibody) contribute to antibody-mediated rejection and poor long-term graft survival. Because the development of donor-specific antibody is associated with early graft loss of cell transplants and reduced long-term survival of solid organ transplants, we hypothesized that conventional immunosuppressives, calcineurin inhibitors (CNi), and mammalian target of rapamycin inhibitors (mTORi), may not be as effective for suppression of humoral alloimmunity as for cell-mediated immunity.

Methods Wild-type or CD8-depleted mice were transplanted with allogeneic hepatocytes. Recipients were treated with mTORi and/or CNi and serially monitored for alloantibody and graft survival. The direct effect of mTORi and CNi on alloprimed B cell function was investigated in Rag1−/− mice adoptively transferred with alloprimed IgG1+ B cells. The efficacy of mTORi and/or CNi to suppress CD8-mediated cytotoxicity of IgG1+ B cells was evaluated in in vitro and in vivo cytotoxicity assays.

Results Mammalian target of rapamycin inhibitors, but not CNi, reduced alloantibody production in transplant recipients, directly suppressed alloantibody production by alloprimed IgG1+ B cells and delayed graft rejection in both low and high alloantibody producers. Combination treatment with mTORi and CNi resulted in loss of the inhibitory effect observed for mTORi monotherapy in part due to CNi suppression of CD8+ T cells which downregulate alloantibody production (CD8+ TAb-supp cells).

Conclusions Our data support that mTORi is a potent inhibitor of humoral immunity through suppression of alloprimed B cells and preservation of CD8+ TAb-supp cells. In contrast, alloantibody is readily detected in CNi-treated recipients because CNi does not suppress alloprimed B cells and interferes with downregulatory CD8+ TAb-supp cells.

The authors demonstrate that mTOR inhibitor, not calcineurin inhibitor, directly suppresses alloantibody production by alloprimed IgG1+ B cells and delays graft rejection in a mice model with hepatocytes transplantation.

1 Department of Surgery, Comprehensive Transplant Center, The Ohio State University, Columbus, OH.

2 Center for Biostatistics, The Ohio State University, Columbus, OH.

Received 21 December 2015. Revision received 4 April 2016.

Accepted 19 April 2016.

This work was supported in part by grants from the ASTS-NKF (National Kidney Foundation) Folkert Belzer, MD, Research Award (to T.A.P.), and National Institutes of Health grants F32 DK082148 (NIDDK; to J.M.Z.) and AI083456 (to G.L.B.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

The authors declare no conflicts of interest.

C.L.A. participated in research design, writing of the paper, performance of the research, and data analysis. J.M.Z. participated in research design, writing of the paper, performance of the research, and data analysis. S.M.E. participated in research design, writing of the paper, and performance of the research. T.A.P., participated in research design, performance of the research, and data analysis. M.A.-R. participated in statistical analysis. G.L.B. participated in research design, data analysis, and writing of the article.

Correspondence: Ginny L. Bumgardner, MD, PhD, FACS, Division of Transplant, Department of Surgery, The Ohio State University Wexner Medical Center, 395W. 12th Avenue, 166 Faculty Tower, Columbus, OH 43210-1250. (ginny.bumgardner@osumc.edu).

Supplemental digital content (SDC) is available for this article. Direct URL citations appear in the printed text, and links to the digital files are provided in the HTML text of this article on the journal’s Web site (www.transplantjournal.com).

Antibody-mediated rejection (AMR), caused by preformed or de novo donor-specific alloantibodies (DSA), is an important cause of graft rejection,1-3 and DSA is associated with reduced long-term allograft survival.4 De novo DSA are particularly detrimental to cellular transplants, which have relatively smaller parenchymal cell mass and increased exposure to circulating antibodies.5 Development of humoral alloimmunity after islet6-8 and hepatocyte transplant9 is associated with deterioration of graft function and is a barrier to long-term graft survival. Current therapies available for treatment of AMR include removal of deleterious alloantibodies, targeting IgG+ cells, cellular depletion, or a combination of these strategies.10,11 However, these therapies, initiated after the development of AMR, have produced unpredictable and often suboptimal results.10,12 Optimal maintenance immunosuppressive strategies to prevent posttransplant alloantibody production would mitigate the acute and long-term consequences of AMR.

In vitro data support the suppressive effects of mammalian target of rapamycin inhibitors (mTORi) on both murine and human B cell proliferation and maturation into antibody secreting cells.13-16 When mTORi and calcineurin inhibitors (CNi) were compared, proliferation of LPS-stimulated mouse B cells in vitro was suppressed after mTORi (but not CNi) treatment.17 In contrast, other studies suggest CNi under select conditions inhibits B cell responses.17,18 Despite the fact that in vitro studies have shown efficacy of mTORi, and in some circumstances CNi, for suppression of human B cells, the clinical literature demonstrates a considerable number of recipients treated with these immunosuppressives continue to develop alloantibodies.19-22 Surprisingly, there is a relative paucity of published studies investigating the in vivo effects of these immunosuppressives on the humoral response after transplant.

Our group is the first to report that a population of CD8+ T cells, which we will refer to as CD8+ antibody-suppressing T (CD8+ TAb-supp) cells, negatively regulate humoral responses by killing allospecific IgG1+ B cells through the use of both Fas-FasL interactions and perforin.23 These studies were published in a well-validated model of hepatocyte transplant, characterized by a specific, Th2-driven IgG1-dominant pathway of alloantibody production24-29 which not only causes cell transplant rejection but is also known to result in graft rejection in vascularized cardiac transplant mouse models30,31 Thus, this CD8-dependent regulatory pathway applies to posttransplant alloantibody production after both cell and vascularized organ transplants.

The current studies were undertaken to address the relative efficacy of mTORi and CNi for suppression of in vivo humoral alloimmunity. We further determined whether combination CNi and mTORi produced additive or synergistic effects on humoral alloimmunity, and the effects on CD8+ TAb-supp cell and alloprimed B cell function.

Back to Top | Article Outline

MATERIALS AND METHODS

Experimental Animals

FVB/N (H-2q MHC haplotype; Taconic, Hudson, NY) mice were used as allogeneic donors and C57BL/6, CD8 KO, and Rag1 KO (all H-2b; Jackson Labs, Bar Harbor, ME) mouse strains were used as transplant and adoptive transfer (AT) recipients (6-10 weeks of age). Transgenic FVB/N mice expressing human α-1 antitrypsin (hA1AT) served as the source of “donor” hepatocytes, as previously described.24 All experiments were performed in compliance with the guidelines of the Institutional Laboratory Animal Care and Use Committee of The Ohio State University (Protocol 2008A0068-R2).

Back to Top | Article Outline

Hepatocyte Isolation, Purification, and Transplantation

Hepatocyte isolation, purification, and transplantation were performed, as reported.24 Graft survival was determined by detection of secreted hA1AT in serial recipient serum samples by enzyme-linked immunosorbent assay.24,28 The reporter protein hA1AT does not elicit an immune response and syngeneic, hA1AT-expressing hepatocytes survive long term.24

Back to Top | Article Outline

Immunosuppressive Treatments

Recipient mice were treated with in vivo doses of mTORi (Rapamycin, Rapamune) and/or CNi (FK506, Tacrolimus) (R-5000 and F-4900, respectively; LC laboratories, Woburn, MA) via intraperitoneal (i.p.) injection at concentrations of 0.001 to 5.0 mg/kg dissolved in PBS with 5% dimethyl sulfoxide (DMSO). In vitro assays used mTORi or CNi at concentrations of 1, 10, and 50 nM in 4-hour incubations with cells.

Back to Top | Article Outline

CD8+ T Cell Depletion

Recipients were depleted of circulating CD8+ T cells by i.p. injection of 100 mg of mAb (clone 53.6.72; days −2, −1), as described.32 Depletion was confirmed through flow cytometric analysis of recipient peripheral blood lymphocytes.

Back to Top | Article Outline

CD8+ T Cell Isolation

CD8+ T cells were isolated from mouse splenocytes by negative selection columns as per manufacturer's recommendations (MCD8C-1000, R&D Systems, Minneapolis, MN; purity routinely >90%). For AT, 10 × 106 CD8+ T cells per mouse were suspended in serum-free media.

Back to Top | Article Outline

B Cell Isolation

B cells were purified from splenocytes using antimouse B220+ magnetic beads (130-049-501) to isolate from untransplanted mice (naive B cells) or antimouse IgG+ magnetic beads (130-048-401) to isolate from transplanted mice (alloprimed B cells) following the manufacturer's instructions (Miltenyi Biotech, Auburn, CA; purity routinely >95%).

Back to Top | Article Outline

Donor-Reactive Antibody

To quantify alloantibody titer, we analyzed recipient serum using published methods.33

Back to Top | Article Outline

Alloprimed B Cell Reconstitution in Rag1 KO Recipients

Naive Rag1 KO mice underwent AT of 10 × 106 alloprimed IgG1+ B cells (isolated on day 5 posttransplant from high alloantibody-producing CD8 KO recipients). After AT on day 0, Rag1 KO received FVB/N splenocytes (20 × 106 cells; i.p.) as a source of alloantigen. On the day after AT, mice were treated with IL-4 (0.625 μg, 14-8041-80; eBioscience, San Diego, CA) and anti-CD40 (200 μg, BE0016-2; Bio X Cell, West Lebanon, NH).

Back to Top | Article Outline

In Vitro CD8/B Cell Cytotoxicity Assay

Measurement of in vitro cytolytic elimination of alloprimed IgG1+ B cells was performed as previously published.23 The level of B cell-induced apoptosis was determined by propidium iodide uptake (LIVE/DEAD cell-mediated cytotoxicity kit, L-7010; ThermoFisher Scientific) and analyzed by flow cytometry, as previously published.23

Back to Top | Article Outline

In Vivo CD8/B Cell Cytotoxicity Assay

Measurement of in vivo cytolytic elimination of alloprimed IgG1+ B cells was performed as previously published.23

Back to Top | Article Outline

Statistical Analysis

General linear models were fit for each continuous outcome and contrasts used to compare relevant groups to test the primary hypothesis/hypotheses in each experiment. Model assumptions were assessed, and violations to the normality assumption were addressed by transforming data to the natural log scale. Log rank tests were used to compare time to graft rejection. For experiments where all groups were compared with a control, Dunnett method was used to adjust for multiple comparisons, otherwise, Holm stepdown procedure was used to maintain the overall type 1 error rate at 5%. All analyses were conducted using SAS statistical Software Version 9.3 (SAS Institute, Inc., Cary, NC). To demonstrate the distribution of the data, alloantibody titer and in vitro and in vivo cytotoxicity results are listed as the mean plus or minus the standard error; graft survival results are listed as the median survival time (MST).

Back to Top | Article Outline

RESULTS

Superior Efficacy of mTORi Compared With CNi for Suppression of Posttransplant Alloantibody Production and Prolongation of Allograft Survival

C57BL/6 mice (wild-type [WT]; H-2b) were transplanted with allogeneic FVB/N hepatocytes (H-2q) on day 0. On days 0 to 14 posttransplant mice were treated with a vehicle control (5% DMSO), mTORi (1 mg/kg per day), or CNi (1 mg/kg per day). Alloantibody titer was determined from recipient serum on day 14 posttransplant as mice exhibit peak antibody production at this time.32 Recipients treated with CNi did not exhibit reduced alloantibody (titer = 94 ± 21) compared with DMSO-treated recipients (titer = 113 ± 31). mTORi-treated recipients had significantly reduced alloantibody (titer = 15 ± 3) compared with DMSO-treated (P < 0.001) and CNi-treated recipients (P = 0.001) (Figure 1A). Both mTORi-treated (MST = 24.5 days, P = 0.005) and CNi-treated recipients (MST = 26 days, P = 0.006) had prolonged allograft survival compared with DMSO-treated controls (MST = 12 days) (Figure 1B).

FIGURE 1

FIGURE 1

In previous studies, we found that CD8-depletion of wild-type recipients results in high alloantibody titer and graft rejection occurs primarily by an antibody-mediated (rather than cell-mediated) mechanism.34 To investigate the efficacy of mTORi and CNi for suppression of alloantibody production and prevention of AMR, we treated CD8-depleted recipients with mTORi and CNi. C57BL/6 mice were CD8-depleted, transplanted with FVB/N hepatocytes, and treated with the DMSO control, mTORi (1 mg/kg per day), or CNi (1 mg/kg per day) on days 0 to 14 posttransplant. Alloantibody titer was determined in recipient serum on day 14. High alloantibody titer occurred in DMSO-treated (titer = 263 ± 55) and CNi-treated recipients (titer = 220 ± 49). In contrast, mTORi treatment significantly inhibited alloantibody production (titer = 28 ± 8) compared with DMSO-treated (P < 0.001) and CNi-treated recipients (P < 0.001) (Figure 1C). This inhibition of alloantibody was associated with significant prolongation of graft survival in mTORi-treated recipients (MST = 25.5 days) compared with controls (MST = 14 days, P = 0.02). Rejection in mTORi-treated recipients occurred approximately 2 weeks after cessation of mTORi therapy and 3 weeks after cessation of anti-CD8 mAb treatment. In these conditions, which focus on AMR without the contribution of CD8-mediated rejection, CNi-treated recipients (MST = 12 days) had no survival advantage compared with controls (Figure 1D). Alloantibody production was inversely related to mTORi dosage (SDC Figure 1A,http://links.lww.com/TP/B291) and high doses led to prolonged graft survival (SDC Figure 1B,http://links.lww.com/TP/B291). Inhibition of alloantibody production and enhanced survival was not observed with high doses of CNi.

Back to Top | Article Outline

mTORi (But Not CNi) Treatment Suppresses In Vivo Alloantibody Production by Alloprimed IgG1+ B Cells

We developed a new model using immunoincompetent Rag1−/− mice to directly investigate the effect of mTORi on alloprimed B cell effector function (alloantibody production) in vivo. IgG1+ B cells were isolated from CD8 KO hepatocyte transplant recipients on day 5 posttransplant and AT into naive Rag1 KO mice. These mice received FVB/N splenocytes on day 0 as a source of alloantigen. On day 1 post-AT αCD40 mAb and IL-4 were injected i.p. as a surrogate for endogenous CD4+ T cell “help.” Transferred B cells produce high levels of alloantibody, which peaks on day 7 relative to AT, as can be observed in the DMSO-treated controls (titer = 500 ± 100) (Figure 2). Control Rag1 KO mice which received AT of naive B cells (without allosplenocytes as an antigen source) did not produce alloantibody (data not shown). Cohorts of the mice reconstituted with IgG1+ B cells were treated with mTORi or CNi on days 1 to 7 relative to AT. Calcineurin inhibitor treatment did not suppress alloprimed B cell alloantibody production in reconstituted Rag1 KO mice (titer = 500 ± 100) compared with DMSO-treated controls. However, mTORi-treated mice had significantly reduced levels of alloantibody (titer = 70 ± 12) compared with control (P < 0.001) and CNi-treated mice (P < 0.001).

FIGURE 2

FIGURE 2

Back to Top | Article Outline

mTORi-Mediated Suppression of In Vivo Alloantibody Production Is Negated When Combined With CNi Treatment

Given that many clinical protocols use combinations of therapies including mTORi and CNi, we investigated whether combination therapy might show additive or synergistic effects on alloantibody production or prolongation of allograft survival. C57BL/6 recipients underwent hepatocyte transplant on day 0 and were treated with the DMSO control, mTORi (0.25 mg/kg per day), CNi (1 mg/kg per day), combined mTORi (0.25 mg/kg per day), and CNi (1 mg/kg per day) on days 0 to 14 posttransplant. Additional recipients were CD8-depleted and treated with DMSO control, mTORi (1 mg/kg per day), CNi (1 mg/kg per day), combined mTORi (1 mg/kg per day), and CNi (1 mg/kg per day). Alloantibody titer in recipient serum was quantified on day 14 posttransplant. Unexpectedly, we found dual treatment with mTORi and CNi together did not result in additive or synergistic effects on alloantibody production but instead resulted in a loss of the inhibitory effects of mTORi monotherapy in both low and high alloantibody-producing recipients. Combination-treated WT recipients had higher levels of alloantibody (titer = 75 ± 11) compared to recipients treated with mTORi alone (titer = 25 ± 5, P = 0.005) (Figure 3A). Combination-treated CD8-depleted recipients also had higher levels of alloantibody (titer = 133 ± 33) compared with recipients treated with mTORi alone (titer = 28 ± 8, P = 0.003). (Figure 3A). Furthermore, whereas mTORi prolonged graft survival in WT recipients (MST = 24.5), graft survival in combination-treated WT recipients (MST = 12.5 days) was equivalent to DMSO-treated controls (MST = 12 days, P = ns) (Figure 3B). On the other hand, graft survival was prolonged to a similar extent in CD8-depleted (high alloantibody producers) recipients treated with mTORi alone (MST = 25.5) or with combined mTORi and CNi (MST = 35.5 days, P = ns) compared with DMSO-treated recipients (MST = 14 days, P = 0.02, P = 0.002 respectively) (Figure 3C). These unexpected results of immunosuppressants on in vivo alloantibody production prompted us to consider the effects of conventional mTORi and CNi immunosuppressants on other pathways, which regulate humoral alloimmunity.

FIGURE 3

FIGURE 3

Back to Top | Article Outline

CNi (But Not mTORi) Inhibits CD8-Mediated Killing of Alloprimed B Cells In Vitro

We have previously reported a novel mechanism by which recipient CD8+ TAb-supp cells mediate in vivo cytotoxic killing of alloprimed B cells and downregulate posttransplant alloantibody production.23 If CNi treatment interfered with this downregulatory mechanism, this might explain why combination therapy adversely affected mTORi monotherapy in WT recipients. We used an in vitro cytotoxicity assay to determine if CNi treatment inhibits CD8-mediated killing of alloantibody-producing IgG1+ B cells. Coculturing alloprimed CD8+ T cells with alloprimed IgG1+ B cells resulted in significant B cell apoptosis (11.4 ± 1.2%). CNi treatment resulted in significant suppression of CD8-mediated killing of B cells (3.5 ± 0.8%) compared with control (P = 0.01) and mTORi-treated (11.1 ± 2.2%, P = 0.02) cocultures. Mammalian target of rapamycin inhibitor treatment had no effect on cytotoxicity compared with control cocultures. (Figures 4A and B) Neither mTORi nor CNi at any doses used (1, 10, 50 nM) was found to be directly cytotoxic to IgG1+/B220+ B cells or alloprimed/naive CD8+ T cells (data not shown).23 Taken together, these findings indicate that mTORi and CNi not only demonstrate differential effects on posttransplant alloantibody production, but they demonstrate distinct effects on at least 1 inhibitory immune pathway which downregulates humoral alloimmunity. Consequently, when these agents are combined, unexpected and deleterious consequences on humoral alloimmunity occur.

FIGURE 4

FIGURE 4

Back to Top | Article Outline

Cni (But Not Mtori) Inhibits Cd8-Mediated Killing of Alloprimed B Cells In Vivo

To investigate whether CNi inhibits CD8-mediated killing of alloantibody producing IgG1+ B cells in vivo, we used an in vivo cytotoxicity assay as previously described.23 A cohort of WT mice was transplanted with FVB/N hepatocytes and treated with the DMSO control, mTORi (1 mg/kg per day), CNi (1 mg/kg per day), combined mTORi (1 mg/kg per day), and CNi (1 mg/kg per day) on days 1 to 7 posttransplant. In vivo killing of alloprimed B cells (IgG1+ B cells) was determined on day 7 posttransplant. Similar to the in vitro results, CNi treatment interfered with CD8-mediated cytotoxic clearance of alloprimed B cells (27.5 ± 5.4%) compared with DMSO-treated (61.5 ± 5.3%, P = 0.009) and mTORi-treated (65.2 ± 7.4%, P = 0.005) recipients. In vivo CD8-mediated B cell killing remained intact in mTORi-treated recipients. CD8-mediated B cell killing in combination-treated recipients was similar to CNi monotherapy-treated recipients (31.3 ± 6.1%) (Figures 5A and B). These findings support the interpretation that mTORi and CNi exert markedly different effects on de novo posttransplant alloantibody production, on alloprimed B cell effector function and CD8+ T cell antibody-regulating pathways.

FIGURE 5

FIGURE 5

Back to Top | Article Outline

DISCUSSION

Given the importance of AMR in causing acute graft dysfunction11 and the negative correlation of DSA with long-term allograft survival for both cell and organ transplants,7-9,35-38 it is of great interest to devise optimal maintenance immunosuppression to suppress the emergence of DSA posttransplant. The clinical literature includes publications which report that no current immunosuppressive regimens effectively prevent the development of DSA,10,11 whereas others infer relative efficacy of CNi by reporting that conversion of kidney transplant recipients from CNi to mTORi-based therapy is associated with a negative impact on DSA production and graft survival.19,39-41 These clinical data are difficult to interpret in aggregate and generally flawed due to the retrospective nature of the studies, the variability in treatment regimens (doses, timing, combination therapies, use of induction agents), and variability in reporting isotype, specificity and timing of DSA measurement. Investigation in animal models permits investigators to focus on specific immune pathways with rigorous controls. In the current studies, our aim was to investigate the efficacy of commonly used immunosuppressive agents mTORi and CNi on posttransplant alloantibody production and allograft survival.

Results from these studies demonstrate for the first time that mTORi is a powerful suppressant of alloantibody production in part through inhibition of alloprimed B cell effector function. We found that mTORi's lack of interference with CD8+ TAb-supp cells is an added advantage, which contributes to reduced alloantibody titers in mTORi-treated recipients. Human and murine studies published by other groups suggest mTORi suppresses humoral alloimmunity through inhibition of CD40-mediated B cell activation, proliferation, and antibody production.13-15,17,42 In vitro studies also show that mTORi suppress human B cell maturation and isotype switching in both IL-2–dependent and IL-2–independent humoral responses.14,17,18 Collectively, these published human studies and our current experimental studies in mice support the efficacy of mTORi for suppression of humoral alloimmunity.

The effects of CNi treatment on human B cells are less clear. Calcineurin inhibitor has been shown to inhibit B cell responses to anti-IgM, low-dose IgM and IL-4, but not to Staphylococcus aureus and CD40L stimulation.14,17 Heidt et al43 additionally showed that, in contrast to mTORi, CNi failed to inhibit B cell antibody production after coculture with preactivated T cells. Interestingly, a recent study shows that CNi inhibits naive IL-2 and anti–CD40-stimulated human B cell proliferation and differentiation but fails to inhibit total human B cell responses under similar conditions.18 Thus, the existing in vitro data support CNi-mediated suppression of human B cells only under specific conditions.

The experimental model used in these studies focuses on Th2-driven IgG1 alloantibody production. There is some correlation between mouse and human IgG isotypes' biological functions and antigen-binding specificity. For example, murine IgG1 binds mast cells similar to human IgG4 and murine IgG2a and IgG2b isotypes fix complement and bind protein antigens similar to human IgG1 and IgG3.44 However, a better link between mouse and human alloantibody isotypes may be to consider the cytokines which stimulate their production. Our studies show that mTORi inhibits IL-4–dependent murine IgG1 alloantibody production32,45 which mediates graft rejection in both hepatocyte and vascularized cardiac transplant mouse models.30,31,34 In contrast to murine studies, where IL-4 drives primarily IgG1 and IgE,46 the human response to IL-4 is more complex. Kotowicz and Callard47,48 found that low doses of IL-4 drive IgG1 (and also IgG2 and IgG3 but not IgG4) production, whereas only high doses of IL-4 drive IgG4 production.48 Human IgG1 alloantibody is of particular importance due to reports correlating IgG1 with kidney allograft rejection and reduced allograft function.49 In addition, other reports suggest IL-4 drives human IgG4 and IgE production.50-53 Extrapolation from the current rodent studies to humans would predict mTORi-mediated inhibition of multiple IL-4-driven alloantibody isotypes.

Calcineurin inhibitors are powerful immunosuppressives that inhibit the protein phosphatase calcineurin, thereby blocking T cell IL-2 production, thus inhibiting T cell activation and downstream effector functions.54,55 Treatment with CNi after renal and other solid organ allograft transplantation is associated with reduced episodes of acute rejection and enhanced allograft survival. Consequently, CNi-based therapies are widely used for maintenance immunosuppression after transplantation.56,57 Calcineurin inhibitor-based immunotherapies are used after clinical cell transplantation including hepatocyte transplantation but have been associated with limited success for sustained hepatocellular allograft survival9,58-60 Although early studies have not sufficiently explored alloantibody responses after clinical hepatocyte transplant, recently, Jorns et al9 reported the development of de novo donor-specific HLA antibody in 2 patients who underwent hepatocyte transplantation. The onset of humoral alloimmunity and graft loss occurred in the context of lowering and eventual discontinuation of immunosuppression in 1 patient and despite the use of maintenance immunosuppression with CNi and low-dose steroids in the second case. The current experimental studies which demonstrate the lack of efficacy of CNi for suppression of alloantibody production provide insight into clinical studies which demonstrate detection of DSA in CNi-treated recipients.

The current studies clearly show that treatment with CNi does not effectively suppress humoral alloimmunity after transplantation. In our previous studies, we discovered an important regulatory pathway which decreases alloantibody production; we reported that CD8+ T cells kill IgG1+ B cells in an allospecific manner and that when CD8+ T cells are depleted, significant augmentation of alloantibody levels occurs.23 In the current studies, we found that CNi suppresses CD8+ TAb-supp cell-mediated killing of IgG1+ B cells in vitro and in vivo. These studies do not exclude other potential causes for B cell death; however, the CD8-mediated cytotoxic killing of antibody-producing IgG1+ B cells is a dominant pathway, as recipients depleted of CD8+ T cells have a marked increase in alloprimed B cell survival and higher alloantibody production following hepatocyte,23 skin, or islet transplant (unpublished observations). To our knowledge, this report provides first evidence for the failure of CNi to suppress alloprimed B cell production of alloantibody in vivo and for CNi-mediated suppression of CD8+ T cells with downregulatory functions (CD8+ TAb-supp) on humoral alloimmunity. However, these antibody-promoting effects of CNi therapy are counterbalanced by CNi-mediated immunosuppression of CD4+ T cells, which facilitate antibody production.18,43 This would account for minimal reduction of alloantibody production in CNi monotherapy and adverse effect on alloantibody production when CNi is combined with mTORi. However, this does not exclude other potential explanations for the unexpected effects of combination therapy with CNi and mTORi. For example, dose-dependent CNi-mediated antagonism of mTORi effects has been reported for mouse T cells61 and for mouse and human alloprimed B cells.14,17 The competitive inhibition between mTORi and CNi has been attributed to saturation of FK506-binding protein 12.62 There is some evidence for this antagonism in our data. Because we observed that mTORi-mediated suppression of alloantibody production is reversed in both WT and CD8-depleted recipients after combination therapy, the antagonistic effects of CNi may occur independent of CD8+ T cells. Although this CNi-mediated antagonism on alloantibody production (higher alloantibody titers) was associated with shortened graft survival in combination-treated WT recipients, this was not the case in combination-treated CD8-depleted recipients which had prolonged graft survival despite the higher alloantibody titers. The latter results may reflect the effect of higher doses of mTORi (1.0 mg/kg/d) used in the CD8-depleted recipients compared with the wild-type recipients (0.25 mg/kg per day) on macrophages, which contribute to antibody-dependent cell-mediated cytotoxicity (ADCC).45 Mammalian target of rapamycin inhibitor, but not CNi, dampens macrophage-mediated ADCC in a dose-dependent fashion (unpublished observations). Thus, we speculate that in combination with CNi, the low dose of mTORi (0.25 mg/kg per day) did not suppress macrophage-mediated ADCC and graft rejection (MST = 12.5 days), whereas the higher mTORi dose (1.0 mg/kg per day) did and led to prolonged hepatocyte survival (MST = 35.5 days) despite the presence of alloantibody.

Mammalian target of rapamycin inhibitor is known to inhibit T cell proliferation and cytokine production,43 which is the basis for use in transplantation to prevent cellular rejection; however, accumulating evidence shows that T cell subsets demonstrate differential susceptibility to inhibition by mTORi. Mouse and human studies show that although mTORi mediates suppression of conventional alloreactive CD4+ T cells, it does not suppress CD4+ regulatory T (Treg) cells.63 Our results extend these findings to CD8+ T cell populations and show that mTORi selectively spares CD8+TAb-supp cell function but not CD8+ T cells mediating rejection. These results demonstrate a differential effect of mTORi and CNi on different CD8+ T cell subsets under the same circumstances. Our results have similarity to those published by Ferrer et al64 in which treatment with mTORi did not suppress the number and effector function of CD8+ T cells which recognize ovalbumin-peptide conjugated to Listeria monocytogenes, whereas it did reduce the number and effector function of graft-specific CD8+ T cells which recognize ovalbumin expressed on grafted skin. Alternatively, mTORi may have differential effects on T cells based on their utilization of the mTOR signaling pathway. The mTOR signaling pathway is a central regulator of T cell development and promotes Th1, Th2, and Th17 cells while inhibiting Treg cells.65 Indeed, Zeiser et al63 found that Treg mTOR insensitivity is due to minimal usage of the mTOR-signaling pathway.

Collectively, these studies support the efficacy of mTORi for suppression of humoral alloimmunity in part through direct effects on alloprimed B cell function and by sparing of CD8+ TAb-supp cells. These studies also highlight the need to monitor not only the production of DSA but to include assessment of alloantibody isotypes and quantity. Our dose-response studies emphasize the importance of considering the quantitative exposure to immunosuppressants for interpretation of mTORi and CNi effects on T- and B cell responses in future clinical cell and solid organ transplant studies. Because our results conflict with some studies in humans some caution is warranted regarding direct application of these findings to humans. Nevertheless, our studies provide a strong rationale for future translational studies to determine how and under what conditions these apply to humoral immunity in humans.

Back to Top | Article Outline

REFERENCES

1. Einecke G, Sis B, Reeve J, et al. Antibody-mediated microcirculation injury is the major cause of late kidney transplant failure. Am J Transplant. 2009;9:2520–2531.
2. Sellares J, de Freitas DG, Mengel M, et al. Understanding the causes of kidney transplant failure: the dominant role of antibody-mediated rejection and nonadherence. Am J Transplant. 2012;12:388–399.
3. Gaston RS, Cecka JM, Kasiske BL, et al. Evidence for antibody-mediated injury as a major determinant of late kidney allograft failure. Transplantation. 2010;90:68–74.
4. Sun Q, Yang Y. Late and chronic antibody-mediated rejection: main barrier to long term graft survival. Clin Dev Immunol. 2013;2013:859761.
5. Walker JP, Bumgardner GL. Hepatocyte immunology and transplantation: current status and future potential. Curr Opin Organ Transplant. 2005;10:67–76.
6. Campbell PM, Salam A, Ryan EA, et al. Pretransplant HLA antibodies are associated with reduced graft survival after clinical islet transplantation. Am J Transplant. 2007;7:1242–1248.
7. Brooks AM, Carter V, Liew A, et al. De novo donor-specific HLA antibodies are associated with rapid loss of graft function following islet transplantation in type 1 diabetes. Am J Transplant. 2015;15:3239–46.
8. Piemonti L, Everly MJ, Maffi P, et al. Alloantibody and autoantibody monitoring predicts islet transplantation outcome in human type 1 diabetes. Diabetes. 2013;62:1656–1664.
9. Jorns C, Nowak G, Nemeth A, et al. De novo donor-specific HLA antibody formation in two patients with Crigler-Najjar syndrome type I following human hepatocyte transplantation with partial hepatectomy preconditioning. Am J Transplant. 2016;16:1021–30.
10. Bartel G, Schwaiger E, Bohmig GA. Prevention and treatment of alloantibody-mediated kidney transplant rejection. Transplant Int. 2011;24:1142–1155.
11. Puttarajappa C, Shapiro R, Tan HP. Antibody-mediated rejection in kidney transplantation: a review. J Transplant. 2012;2012:193724.
12. Clatworthy MR. B cell modulation in transplantation. Clin Exp Immunol. 2014;178(1 Suppl):61–63.
13. Sakata A, Kuwahara K, Ohmura T, et al. Involvement of a rapamycin-sensitive pathway in CD40-mediated activation of murine B cells in vitro. Immunology Letters. 1999;68:301–309.
14. Aagaard-Tillery KM, Jelinek DF. Inhibition of human B lymphocyte cell cycle progression and differentiation by rapamycin. Cell Immunol. 1994;156:493–507.
15. Heidt S, Roelen DL, Eijsink C, et al. Effects of immunosuppressive drugs on purified human B cells: evidence supporting the use of MMF and rapamycin. Transplantation. 2008;86:1292–1300.
16. Hornung N, Raskova J, Raska K Jr, et al. Responsiveness of preactivated B cells to IL-2 and IL-6. Effect of cyclosporine and rapamycin. Transplantation. 1993;56:985–990.
17. Wicker LS, Boltz RC Jr, Matt V, et al. Suppression of B cell activation by cyclosporin A, FK506 and rapamycin. Eur J Immunol. 1990;20:2277–2283.
18. De Bruyne R, Bogaert D, De Ruyck N, et al. Calcineurin inhibitors dampen humoral immunity by acting directly on naive B cells. Clin Exp Immunol. 2015;180:542–550.
19. Kamar N, Del Bello A, Congy-Jolivet N, et al. Incidence of donor-specific antibodies in kidney transplant patients following conversion to an everolimus-based calcineurin inhibitor-free regimen. Clin Transplant. 2013;27:455–462.
20. Del Bello A. Prevalence, incidence and risk factors for donor-specific anti-HLA antibodies in maintenance liver transplant patients. Am J Transplant. 2014;14:867–875.
21. Everly MJ, Rebellato LM, Haisch CE, et al. Incidence and impact of de novo donor-specific alloantibody in primary renal allografts. Transplantation. 2013;95:410–417.
22. Wiebe C, Gibson IW, Blydt-Hansen TD, et al. Evolution and clinical pathologic correlations of de novo donor-specific HLA antibody post kidney transplant. Am J Transplant. 2012;12:1157–1167.
23. Zimmerer JM, Pham TA, Wright CL, et al. Alloprimed CD8(+) T cells regulate alloantibody and eliminate alloprimed B cells through perforin- and FasL-dependent mechanisms. Am J Transplant. 2014;14:295–304.
24. Bumgardner GL, Heininger M, Li J, et al. A functional model of hepatocyte transplantation for in vivo immunologic studies. Transplantation. 1998;65:53–61.
25. Bumgardner GL, Li J, Heininger M, et al. In vivo immunogenicity of purified allogeneic hepatocytes in a murine hepatocyte transplant model. Transplantation. 1998;65:47–52.
26. Bumgardner GL, Li J, Prologo JD, et al. Patterns of immune responses evoked by allogeneic hepatocytes: evidence for independent co-dominant roles for CD4+ and CD8+ T-cell responses in acute rejection. Transplantation. 1999;68:555–562.
27. Bumgardner GL, Orosz CG. Unusual patterns of alloimmunity evoked by allogeneic liver parenchymal cells. Immunol Rev. 2000;174:260–279.
28. Bumgardner GL, Gao D, Li J, et al. Rejection responses to allogeneic hepatocytes by reconstituted SCID mice, CD4, KO, and CD8 KO mice. Transplantation. 2000;70:1771–1780.
29. Bumgardner GL. Evidence for multiple allograft rejection mechanisms within the same experimental system. Curr Opin Organ Transplant. 2005;10:20–27.
30. Hirohashi T, Uehara S, Chase CM, et al. Complement independent antibody-mediated endarteritis and transplant arteriopathy in mice. Am J Transplant. 2010;10:510–517.
31. Yin D, Zeng H, Ma L, et al. Cutting Edge: NK cells mediate IgG1-dependent hyperacute rejection of xenografts. J Immunol. 2004;172:7235–7238.
32. Zimmerer JM, Pham TA, Sanders VM, et al. CD8+ T cells negatively regulate IL-4-dependent, IgG1-dominant posttransplant alloantibody production. J Immunol. 2010;185:7285–7292.
33. Bickerstaff A, Nozaki T, Wang JJ, et al. Acute humoral rejection of renal allografts in CCR5(−/−) recipients. Am J Transplant. 2008;8:557–566.
34. Horne PH, Lunsford KE, Walker JP, et al. Recipient immune repertoire and engraftment site influence the immune pathway effecting acute hepatocellular allograft rejection. Cell Transplant. 2008;17:829–844.
35. Morath C, Opelz G, Zeier M, et al. Clinical relevance of HLA antibody monitoring after kidney transplantation. J Immunol Res. 2014;2014:845040.
36. Kaneku H, O'Leary JG, Banuelos N, et al. De novo donor-specific HLA antibodies decrease patient and graft survival in liver transplant recipients. Am J Transplant. 2013;13:1541–1548.
37. Grabhorn E, Binder TM, Obrecht D, et al. Long-term clinical relevance of de novo donor-specific antibodies after pediatric liver transplantation. Transplantation. 2015;99:1876–1881.
38. Campbell PM, Senior PA, Salam A, et al. High risk of sensitization after failed islet transplantation. Am J Transplant. 2007;7:2311–2317.
39. Ruiz San Millan JC, Lopez-Hoyos M, Segundo DS, et al. Predictive factors of allosensitization in renal transplant patients switched from calcineurin to mTOR inhibitors. Transplant Int. 2014;27:847–856.
40. Croze LE, Tetaz R, Roustit M, et al. Conversion to mammalian target of rapamycin inhibitors increases risk of de novo donor-specific antibodies. Transplant Int. 2014;27:775–783.
41. Liefeldt L, Brakemeier S, Glander P, et al. Donor-specific HLA antibodies in a cohort comparing everolimus with cyclosporine after kidney transplantation. Am J Transplant. 2012;12:1192–1198.
42. Kay JE, Kromwel L, Doe SE, et al. Inhibition of T and B lymphocyte proliferation by rapamycin. Immunology. 1991;72:544–549.
43. Heidt S, Roelen DL, Eijsink C, et al. Calcineurin inhibitors affect B cell antibody responses indirectly by interfering with T cell help. Clin Exp Immunol. 2010;159:199–207.
44. Scott MG, Briles DE, Nahm MH. Selective IgG subclass expression: biologic, clinical and functional aspects. The Human IgG Subclasses. 1990.
45. Horne PH, Zimmerer JM, Fisher MG, et al. Critical role of effector macrophages in mediating CD4-dependent alloimmune injury of transplanted liver parenchymal cells. J Immunol. 2008;181:1224–1231.
46. Snapper CM, Finkelman FD, Paul WE. Differential regulation of IgG1 and IgE synthesis by interleukin 4. J Exp Med. 1988;167:183–196.
47. Avery DT, Bryant VL, Ma CS, et al. IL-21-induced isotype switching to IgG and IgA by human naive B cells is differentially regulated by IL-4. J Immunol. 2008;181:1767–1779.
48. Kotowicz K, Callard RE. Human immunoglobulin class and IgG subclass regulation: dual action of interleukin-4. Eur J Immunol. 1993;23:2250–2256.
49. Griffiths EJ, Nelson RE, Dupont PJ, et al. Skewing of pretransplant anti-HLA class I antibodies of immunoglobulin G isotype solely toward immunoglobulin G1 subclass is associated with poorer renal allograft survival. Transplantation. 2004;77:1771–1773.
50. Ishizaka A, Sakiyama Y, Nakanishi M, et al. The inductive effect of interleukin-4 on IgG4 and IgE synthesis in human peripheral blood lymphocytes. Clin Exp Immunol. 1990;79:392–396.
51. Aalberse RC, Stapel SO, Schuurman J, et al. Immunoglobulin G4: an odd antibody. Clin Exp Allergy. 2009;39:469–477.
52. Lundgren M, Persson U, Larsson P, et al. Interleukin 4 induces synthesis of IgE and IgG4 in human B cells. Eur J Immunol. 1989;19:1311–1315.
53. Spiegelberg HL, Falkoff RJ, O'Connor RD, et al. Interleukin-2 inhibits the interleukin-4-induced human IgE and IgG4 secretion in vivo. Clin Exp Immunol. 1991;84:400–405.
54. Ho S, Clipstone N, Timmermann L, et al. The mechanism of action of cyclosporin A and FK506. Immunol Today. 1996;80(3 Pt 2):S40–45.
55. Fruman DA, Klee CB, Bierer BE, et al. Calcineurin phosphatase activity in T lymphocytes is inhibited by FK 506 and cyclosporin A. Proc Natl Acad Sci U S A. 1992;89:3686–3690.
56. Scott LJ, McKeage K, Keam SJ, et al. Tacrolimus: a further update of its use in the management of organ transplantation. Drugs. 2003;63:1247–1297.
57. Matas AJ, Smith JM, Skeans MA, et al. OPTN/SRTR 2012 Annual Data Report: kidney. Am J Transplant. 2014;14(1 Suppl):11–44.
58. Puppi J, Tan N, Mitry RR, et al. Hepatocyte transplantation followed by auxiliary liver transplantation—a novel treatment for ornithine transcarbamylase deficiency. Am J Transplant. 2008;8:452–457.
59. Ambrosino G, Varotto S, Strom SC, et al. Isolated hepatocyte transplantation for Crigler-Najjar syndrome type 1. Cell Transplant. 2005;14:151–157.
60. Horslen SP, McCowan TC, Goertzen TC, et al. Isolated hepatocyte transplantation in an infant with a severe urea cycle disorder. Pediatrics. 2003;111(6 Pt 1):1262–1267.
61. Dumont FJ, Melino MR, Staruch MJ, et al. The immunosuppressive macrolides FK-506 and rapamycin act as reciprocal antagonists in murine T cells. J Immunol. 1990;144:1418–1424.
62. van Rossum HH, Romijn FP, Smit NP, et al. Everolimus and sirolimus antagonize tacrolimus based calcineurin inhibition via competition for FK-binding protein 12. Biochem Pharmacol. 2009;77:1206–1212.
63. Zeiser R, Nguyen VH, Hou JZ, et al. Early CD30 signaling is critical for adoptively transferred CD4 + CD25+ regulatory T cells in prevention of acute graft-versus-host disease. Blood. 2007;109:2225–2233.
64. Ferrer IR, Wagener ME, Robertson JM, et al. Cutting edge: rapamycin augments pathogen-specific but not graft-reactive CD8+ T cell responses. J Immunol. 2010;185:2004–2008.
65. Chi H. Regulation and function of mTOR signalling in T cell fate decisions. Nat Rev Immunol. 2012;12:325–338.

Supplemental Digital Content

Back to Top | Article Outline
Copyright © 2016 Wolters Kluwer Health, Inc. All rights reserved.