Up to 40% of heart transplant (HTx) recipients demonstrate allograft dysfunction due to acute antibody- mediated rejection (AMR) during early post-HTx period (1–5). Histopathologic assessment of AMR is characterized by capillary injury, positive immunofluorescence for C4d, CD68 in endomyocardial biopsies, and detection of donor-specific antibodies (DSA) to mismatched human leukocyte antigens (HLA) class I/II (6, 7). Pretransplant sensitization to mismatched HLA has also been identified as an independent risk factor for development of AMR. Several studies have demonstrated a significant association between development of DSA and both acute and chronic cardiac allograft rejection (5, 7–9). Patients with AMR who develop antibodies (Abs) to donor HLA often progress to transplant-associated cardiac allograft vasculopathy (CAV) early when compared with patients without anti-HLA (10, 11). A growing body of evidence suggests that increase in proinflammatory mediators including interferon (IFN)-γ, interleukin (IL)-1, -12, and -17 during early posttransplant period is associated with development of DSA that subsequently leads to chronic allograft rejection (10, 12–14). Additionally, immune responses to non-HLA antigens have also been implicated in immunopathogenesis of acute and chronic allograft rejection (15–19).
Both immune and nonimmune factors contribute to chronic endothelial inflammation and fibroproliferation resulting in CAV (14, 15, 20). Recently, alloimmune responses to mismatched donor HLA have also been implicated in induction of immune responses to self-antigens (15, 19, 21). A significant number of HTx recipients with histologic evidence of rejection develop anti-skeletal muscle glycolipid, anti-muscle protein, and anti-intracellular adhesion molecule-1 (17, 18, 22). Studies from our laboratory have shown immune responses to self-antigens, collagen-V (Col-V), an extracellular matrix protein, and K-α1-Tubulin (KAT), a gap junction intermediate filament cytoskeletal protein in lung transplant recipients undergoing chronic rejection (23, 24). We tested the possibility that these proteins may be antigenic targets in other transplanted organs besides the lung allograft. In cardiac tissue, endothelial cells have a large number of gap junctions (25) and given the increased levels of cytoskeletal KAT expression in gap junctions (26) and the demonstrated mutations of α-1-Tubulin in the pathogenesis of postcardiac transplant fatal cardiomyopathy, we studied KAT as an antigen target in HTx recipients. On the other hand, Col-V is a protein that is selectively expressed in the human body and comprises up to 2% of the entire extracellular matrix protein in heart (27). Given that Col-V is found in interstitial connective tissue and has been shown to play an integral role in the structure and function of cardiac tissue, we examined Col-V as an antigenic target in HTx recipients (28). The objective of this study was to evaluate the role of DSA to mismatched HLA and serum levels of Abs against two novel cardiac self-antigens, Col-V, and KAT in post-HTx patients who were diagnosed with AMR and CAV. To define the mechanism for development of Abs, CD4+ T lymphocyte responses specific to individual self-antigens and their cytokine secretion pattern were also determined.
The characteristics of 137 HTx recipients in study cohort are detailed in Table 1, Supplemental Digital Content 1 (http://links.lww.com/TP/A395). Of 137 patients, 60 patients were monitored for development of acute AMR in early post-HTx period (early period [EP]≤12 months), whereas 77 patients were followed up for development of CAV in late post-HTx period (late period [LP]>12 months). Mean (±SD) follow-up after HTx was 3.6±2.6 years while median follow-up was 3.4 years. Nine patients had diagnostic criteria consistent with acute AMR (AMR[+]) and 14 patients had an evidence of moderate or severe CAV (CAV[+]). Although patients with active infection at the time of study enrollment met exclusion criteria, five patients developed systemic infections (three with bacterial and two with viral) after study enrollment. Among the nine patients who were AMR+, six were C4d+. Four AMR+ patients had hemodynamic compromise. There was a mean 5.6±8.8 Units of blood transfusion to patients before heart transplant. Seven of the nine patients were treated for AMR. All patients diagnosed with DSA were treated with intravenous immunoglobulin and rituximab.
Development of Abs to Self-Antigens, Col-V, and KAT Are Significantly Increased in AMR(+) Recipients
We analyzed EP HTx recipient sera for development of Abs to Col-V and KAT using ELISA. Patients with AMR develop increased Abs to Col-V (Fig. 1; control: 85±35 μg/mL, AMR(−): 172±42 μg/mL, AMR(+): 383±72 μg/mL) compared with stable HTx recipients without AMR (P=0.033). Antibodies to Col-V were detected 2.5±2.3 months before acute AMR was diagnosed. Donor-specific Abs in AMR(+) patients was identified 3.3±2.1 months before detection of anti-Col-V (Fig. 5). Similarly, patients with AMR develop increased Abs to KAT (Fig. 1; control:49±23 μg/mL, AMR(−): 61±23 μg/mL, AMR(+): 252±57 μg/mL) compared with stable HTx recipients without AMR (P=0.014). Antibodies to KAT were detected 1.2±2.3 months before acute AMR was diagnosed. DSA in AMR(+) patients was identified 4.6±2.1 months before detection of anti-KAT (Fig. 2). Antibodies to Col-I, -II, and -IV were not significantly increased in AMR(+) patients compared with AMR(−) recipients (Fig. 1).
AMR(+) Recipients Demonstrated Increased Frequencies of IL-5, IFN-γ, and Decreased Frequencies of IL-10 Secreting CD4+ T Cells Specific to Col-V and KAT
To identify immune responses that contribute to development of Abs to self-antigens, frequency of CD4+ T cells secreting IFN-γ, IL-5, -17, and -10 specific to Col-V or KAT were determined using Enzyme Linked Immunosorbent Spot (ELISPOT) assay. Patients with AMR demonstrated increased frequencies of IL-5 and IFN-γ against Col-V (Fig. 3A; IFN-γ: AMR[−]:28±9 spots per million cells [spm], AMR[+]:64±19 spm, P=0.008; IL-5: AMR[−]: 42±14 spm, AMR[+]: 134±42 spm, P=0.003) and KAT (Fig. 3B; IFN-γ: AMR[−]: 21±7 spm, AMR[+]: 68±18 spm, P=0.03; IL-5: AMR[−]: 35±10 spm, AMR[+]: 138±36 spm, P=0.004). AMR(+) recipients demonstrated decreased frequencies of IL-10 secreting CD4+ T cells against Col-V (Fig. 3A; AMR[−]: 354±45 spm, AMR[+]: 184±38 spm, P=0.009) and KAT (Fig. 3B; AMR[−]: 445±56 spm, AMR[+]: 296±43 spm, P=0.03). There was no significant difference in frequencies of CD4+ T cells secreting IL-17 specific to Col-V (Fig. 3A; AMR[−]: 28±12 spm, AMR[+]: 42±12 spm, P=0.35) and KAT (Fig. 3B: AMR[−]: 28±10 spm, AMR[+]: 38±12 spm, P=0.29) between AMR(−) and AMR(+) recipients. Patients with AMR demonstrate high frequencies of CD4+ T helper cells specific to self-antigens that predominantly secrete IL-5 and IFN-γ. Induction of IL-5 and strong IFN-γ response by self-antigen reactive CD4+T cells in AMR(+) recipients provides a mechanism that may contribute to activation and differentiation of B cells involved in autoantibody production.
Development of Abs to Col-V and KAT Are Significantly Associated With Development of DSA in AMR(+) Recipients
Patients who developed AMR and had DSA (DSA[+] AMR[+]) demonstrated increased Abs to Col-V (see Table 2, Supplemental Digital Content 2, http://links.lww.com/TP/A396: DSA[−]AMR[+]: 176±65 μg/mL, DSA[+]AMR[+]: 344±94 μg/mL, P=0.03) and KAT (see Table 2, Supplemental Digital Content 2, http://links.lww.com/TP/A396: DSA[−] AMR[+]: 91±42 μg/mL, DSA[+]AMR[+]: 296±71 μg/mL, P=0.003). There was no significant difference in Abs to Col-V (see Table 2, Supplemental Digital Content 2, http://links.lww.com/TP/A396: DSA[−]AMR[−]: 56±21 μg/mL, DSA[+]AMR[−]: 61±28 μg/mL, P=0.92) and KAT (see Table 2, Supplemental Digital Content 2, http://links.lww.com/TP/A396: DSA[−]AMR[−]: 59±18 μg/mL, DSA[+]AMR[−]: 67±24 μg/mL, P=0.89) in AMR(−) patients regardless of whether they were DSA(+) or DSA(−). These results demonstrate that development of DSA to mismatched HLA and alloimmune responses may play an important role in development of Abs to self-antigens in AMR(+) recipients.
Development of Abs to Self-Antigens, Col-V, and KAT Are Significantly Increased in CAV(+) Recipients
We tested LP HTx recipient sera for development of Abs to Col-V and KAT using ELISA. Patients with CAV develop increased Abs to Col-V (Fig. 4; control: 85±35 μg/mL, CAV[−]: 242±68 μg/mL, CAV[+]: 848±132 μg/mL) compared with stable HTx recipients without CAV (P=0.025). Similarly, patients with CAV develop increased Abs to KAT (Fig. 3; control: 49±13 μg/mL, CAV[−]: 196±62 μg/mL, CAV[+]: 768±206 μg/mL) compared with stable HTx recipients without CAV (P=0.001). Antibodies to Col-I, -II, and -IV were not significantly increased in CAV(+) patients compared with CAV(−) recipients (Fig. 3). These results demonstrate that patients with moderate/severe CAV develop significant increased Abs to self-antigens, Col-V, and KAT compared with patients with none/minimal CAV.
CAV(+) Recipients Demonstrated Increased Frequencies of IL-17 and Decreased Frequencies of IL-10 Secreting CD4+ T Cells Specific to Col-V and KAT
To identify immune responses that contribute to development of Abs to self-antigens, frequency of CD4+ T cells secreting IFN-γ, IL-5, -17, and -10 specific to Col-V or KAT were determined using ELISPOT. Patients with CAV demonstrated increased frequencies of IL-17 secreting CD4+ T cells against Col-V (Fig. 5A; CAV[−]: 28±12 spm, CAV[+]: 108±21 spm, P=0.002) and KAT (Fig. 5B; CAV[−]: 28±10 spm, CAV[+]: 121±31 spm, P=0.003). CAV(+) recipients demonstrated decreased frequencies of IL-10 secreting CD4+ T cells against Col-V (Fig. 5A; CAV[−]: 284±45 spm, CAV[+]: 146±38 spm, P=0.008) and KAT (Fig. 5B; CAV[−]: 345±56 spm, CAV[+]: 180±43 spm, P=0.005). There was no significant difference in frequencies of CD4+ T cells secreting IFN-γ and IL-5 specific to Col-V (Fig. 5A; IFN-γ: CAV[−]: 28±9 spm, CAV[+]: 44±19 spm, P=0.55; IL-5: CAV[−]: 42±14 spm, CAV[+]: 48±12 spm, P=0.54) and KAT (Fig. 5B; IFN-γ: CAV[−]: 21±19 spm, CAV[+]: 36±18 spm, P=0.64; IL-5: CAV[−]: 35±10 spm, CAV[+]: 44±11 spm, P=0.24) between CAV(−) and CAV(+) recipients. Patients with CAV demonstrate high frequencies of CD4+ T helper cells specific to self-antigens that predominantly secrete the Th17 cytokine, IL-17. Induction of IL-17 by self-reactive CD4+T cells in CAV(+) recipients indicate that IL-17 can facilitate germinal center formation and activation of B lymphocytes that can contribute to autoantibody development.
Development of Abs to Col-V and KAT Are Significantly Associated With Development of DSA in CAV(+) Recipients
Patients who developed CAV and had DSA (DSA[+]CAV[+]) demonstrated increased Abs to Col-V (see Table 3, Supplemental Digital Content 3, http://links.lww.com/TP/A397: DSA[−]CAV[+]: 313±134 μg/ mL, DSA[+]CAV[+]: 812±142 μg/mL, P=0.001) and KAT (see Table 3, Supplemental Digital Content 3, http://links.lww.com/TP/A397: DSA[−]CAV[+]: 220±78 μg/mL, DSA[+]CAV[+]: 796±210 μg/mL, P=0.02). There was no significant difference in Abs to Col-V (see Table 3, Supplemental Digital Content 3, http://links.lww.com/TP/A397: DSA[−]CAV[−]: 211±87 μg/mL, DSA[+]CAV[−]: 264± 76 μg/mL, P=0.63) and KAT (see Table 3, Supplemental Digital Content 3, http://links.lww.com/TP/A397: DSA[−]CAV[−]: 134±104 μg/mL, DSA[+]CAV[−]: 196±67 μg/mL, P=0.78) in CAV(−) patients regardless of whether they were DSA(+) or DSA(−). As in AMR, DSA to mismatched HLA are significantly associated with development of Abs to self-antigens in CAV(+) recipients supporting the conclusion that alloimmune responses can facilitate induction of immune responses to self-antigens.
The development of AMR and CAV after HTx is likely due to both cellular and humoral immune responses to mismatched donor HLA and/or cardiac-specific self-antigens myosin and vimentin (29–31). Circulating DSA to mismatched HLA and non-HLA antigens including ABO, MICA, and cardiac self-antigens such as myosin and vimentin have been considered as major risk factors for development of AMR and CAV after HTx (29–31). After transplantation, inflammation, alloimmune responses, and subsequent tissue remodeling expose cryptic self-antigens that lead to activation of self-reactive immune responses (32–34). Regulatory T cells (Tregs) are known to inhibit both autoreactive and alloreactive T cells (35). However, in the context of potent posttransplant immunosuppression including use of calcineurin inhibitors that are not conducive to Treg proliferation, there is loss of peripheral tolerance to self-antigens resulting in immune responses to self-antigens (36, 37). We assessed the temporal relationship between development of DSA and Abs to cardiac self-antigens, Col-V and KAT, and the concomitant T-cell responses to these self-antigens, in pathogenesis of AMR and CAV in post-HTx recipients. A significant association for development of Abs to mismatched HLA as well as to Col-V and KAT were identified in patients with AMR and CAV. Furthermore, induction of IL-5 and IFN-γ secreting self-antigen-reactive CD4+T cells with concomitant reduction in IL-10 leads to AMR, whereas a cytokine switch to IL-17 pathway with a similar reduction in IL-10 leads to CAV.
In this study, AMR(+) patients developed Abs to both Col-V and KAT (Fig. 1) and increased frequencies of IFN-γ and IL-5 secreting CD4+ T cells specific to these self-antigens (Fig. 2A,B). Concurrently, AMR+ patients demonstrated decreased frequency of IL-10 secreting CD4+ T cells, indicating a loss of peripheral tolerance to these self-antigens (Fig. 2A,B). These results provide evidence for the first time as an important role for IL-5 in proliferation of B cells together with strong IFN-γ responses to self-antigens that facilitate autoantibody production. A concomitant decline in IL-10 may also assist in induction of autoimmune response in AMR patients. Patients with AMR and DSA (AMR[+]DSA[+]) demonstrated significantly increased Abs to both Col-V and KAT compared with patients with AMR and without DSA (AMR[+]DSA[−], see Table 2, Supplemental Digital Content 2, http://links.lww.com/TP/A396). These results underline an important role for cross-talk between alloimmune responses and autoimmune responses after HTx leading to AMR. The ligation of class I molecules on allograft epithelium or endothelium by anti-HLA class I initiates an inflammatory state characterized by neutrophil and macrophage infiltration as well as concomitant release of inflammatory cytokines, chemokines, and fibrogenic growth factors (38, 39). The ensuring proinflammatory microenvironment can lead to development of immune responses against exposed domains of sequestered self-antigens. Serial monitoring of DSA, anti-Col-V, and anti-KAT in patients with AMR revealed that DSA precede development of anti-Col-V by 3.3±2.1 months, anti-KAT by 4.6±2.1 months, and clinical diagnosis of AMR by 5.8±2.0 months. Taken together, these results strongly suggest an important role for alloimmune response as evidenced by development of DSA to mismatched HLA in development of autoimmunity to cardiac self-antigens.
Antibodies to cardiac self-antigens such as vimentin have been demonstrated to cause lesions consistent with coronary artery vasculopathy in a murine model and thus, provide preclinical evidence for role of autoimmune response in accelerating CAV (40). In this study, CAV(+) patients demonstrated development of Abs to Col-V and KAT (Fig. 3). Furthermore, CAV(+) patients demonstrated increased frequency of IL-17 secreting CD4+ T cells specific to Col-V and KAT (Fig. 4A,B). Similar to AMR(+) patients, CAV(+) recipients demonstrated a decreased frequency of IL-10 secreting CD4+ T cells specific to self-antigens. It has been shown that IFN-γ plays a significant role in alloimmune response (41, 42), whereas IL-17 plays a critical role in autoimmune response (43). Enhanced IFN-γ in AMR setting indicates a predominant role of IFN-γ and alloimmune responses in the AMR in the early period. However, in the late period, in the development of CAV, we propose an important role for IL-17-mediated autoimmune process and development of immune responses to self-antigens. Studies have provided evidence for cross-talk between alloimmunity and autoimmunity in solid organ transplantation (44). However, the relatively low sample size of AMR(+) (n=9) and CAV(+) (n=14) might warrant further randomized multicenter studies to conclusively support this finding. Furthermore, although we demonstrate enhanced IFN-γ levels in AMR and IL-17 levels in CAV to self-antigens, the mechanisms for the immunopathogenesis of AMR and CAV needs further studies.
Recent reports have suggested that the mechanisms contributing to the immunogenicity of Col-V leading to chronic rejection pathology after lung transplantation is predominantly mediated by the IL-17-dependent CD4+ T-cell pathway (19). It has been reported that self-antigen-reactive T cells secreting IL-17 can induce extracellular matrix remodeling by modulating the MMP/TIMP and RANKL/osteoprotegerin complex (45). On the other hand, KAT as a gap junction protein is involved in signal transduction affecting cardiac contractility and autoimmune responses to this protein has the potential to significantly alter function of the transplanted heart. Previous studies from our laboratory have demonstrated that incubation of airway epithelial cells from bronchiolitis obliterans syndrome (BOS)(+) patients who developed KAT Abs resulted in the increased expression of transcription factors (TCF5 and c-Myc), leading to increased expression of fibrogenic growth factors and fibroproliferation (24). Put together, these data suggest a signaling mechanism for anti-KAT playing a crucial role in pathogenesis of chronic rejection.
Therefore, we propose that early detection and abrogation of alloimmune response by serial monitoring of development of Abs to mismatched donor HLA antigens can identify patients who are at high risk for development of immune responses to self-antigens. It is likely that prevention of development of immune responses to self-antigens by early intervention of alloimmune responses will not only prevent AMR but also can prevent and delay onset of CAV after human cardiac transplantation.
MATERIALS AND METHODS
We enrolled 137 patients who underwent HTx at Barnes-Jewish Hospital/Washington University in an Institutional Review Board approved research study. Serum and peripheral blood mononuclear cells (PBMCs) were isolated from whole blood and stored at −135°C (46, 47). Endomyocardial biopsies were performed in all patients in early period (EP≤12 months) while coronary angiograms were performed in all patients in late period (LP>12 months) post-HTx. At the time of endomyocardial biopsies or coronary angiograms, blood samples were collected from each patient. In accordance with International Society of Heart and Lung Transplantation recommendations, a diagnosis of AMR was reached by assessment of clinical, histologic, and serologic findings by the attending transplant cardiologist as described in our previous study (7, 31). Based on International Society of Heart and Lung Transplantation guidelines, CAV was assessed based on the extent of allograft dysfunction and coronary artery stenosis detected by angiography (48). No detectable disease, left main disease less than 50% or primary coronary vessel/branch stenosis less than 70% without allograft dysfunction was termed none/mild (CAV[−]). Left main disease more than 50%, single or multiple primary coronary vessel/branch stenosis more than 70% with or without allograft dysfunction was termed moderate/severe (CAV[+]).
Detection of Abs to HLA
Presence of DSA in sera from HTx recipients was determined using Luminex technology (Biosource International Inc., CA) (46, 49). Before transplantation, all patients were screened for preformed anti-HLA antibodies using the LABScreen Single Antigen assay (One Lambda Inc., Canoga Park, CA). Donor hearts were accepted only if a virtual crossmatch with all previously identified antibodies was compatible. At transplantation, retrospectively, all recipients had a direct cytotoxicity crossmatch using serum obtained at the day of surgery, and the results were available postoperatively. Thus, none of the patients who underwent transplant had DSA and all the transplants were cytotoxicity crossmatch negative with the donor. Mean fluorescence intensity (MFI) and ratio of MFI-to-positive control bead (MFI ratio) were recorded for all anti-HLA specificities. Anti-HLAs were defined as donor specific (DSA) if donor for HTx shared same HLA allele. In addition, we performed follow-up testing for DSA every month during early period (≤1 year) in patients with AMR who developed DSA. Because of fluctuations in MFI between positive controls, our center's HLA laboratory calculates a ratio of sample MFI-to-positive control MFI and defines a ratio of 0.2 or higher as positive. Most of the patients who developed DSA were positive within 4 months of transplant.
Detection of Abs to Self Antigens, Col-I, -II, -IV, -V, and KAT
Patient sera were tested for development of Abs to Col-I, -II, -IV, -V, and KAT by enzyme-linked immunosorbent assay (ELISA) (15, 24). Sera were tested at dilutions of 1:500 for Abs against Col-I, -II, -IV, -V, and 1:1250 for Abs against KAT (24). To determine positive titers of Abs to self-antigens, 2 SD from mean concentration of Abs to Col-I, -II, -IV, -V, and KAT in healthy age-matched (n=11, age 47.8±12.4, male=5 and female=6) control subjects were used as cutoff. The development of auto-Abs was monitored in the serum every 1 month in patients with AMR and 6 months for patients with CAV.
Frozen PBMC collected from patients were cultured overnight in complete Roswell Park Memorial Institute (culture medium)-1640 and viability was determined by trypan blue exclusion. PBMCs with viability of a minimum of 90% were used for ELISPOT assays. The CD4+T cells were enriched by negative selection using immunomagnetic separation cocktails (Stem cell Technologies, Canada). Enriched CD4+ T cells (3×105) with approximately 95% purity were cultured in triplicate in the presence of Col-V (20 μg/mL) or KAT (20 μg/mL) on the precoated ELISPOT plates (Multiscreen IP plate, Millipore, MA) with autologous irradiated CD4 depleted PBMCs as antigen presenting cells (APCs) (3×104) in complete Roswell Park Memorial Institute (culture medium)-1640 medium. Cultures were placed for 48 to 72 hr in humidified 5% CO2 incubator at 37°C and the plates were washed and developed to detect IFN-γ, IL-5, -10, and -17 using an ImmunoSpot analyzer (Cellular Technology, Shaker Heights, OH). CD4+ T cells plus autologous APCs cultured in medium without antigens was a negative control, whereas CD4+ T cells plus autologous APCs cultured with phytohemagglutinin (5 μg/mL) was a positive control. Number of spots in negative control subtracted from spots in experimental wells was reported as results in spm. The AMR cohort was serially monitored once a month and CAV cohort every 6 months.
Graphpad Prism version 4.03 software (GraphPad Software Inc., CA) was used. Mann–Whitney test assessed differences in CD4+ T-cell responses specific to individual antigens between two groups (AMR[+] vs. AMR[−]; CAV[+] vs. CAV[−]). Correlation analysis was performed using Spearman rank test. Two-sided level of significance was set at P less than 0.05 and results were expressed in mean±SD.
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