American Journal of Transplantation 2014; 14: 685–693 Wiley Periodicals Inc.
C
Copyright 2014 The American Society of Transplantation and the American Society of Transplant Surgeons doi: 10.1111/ajt.12592
Brief Communication
Immune Responses to Collagen-IV and Fibronectin in Renal Transplant Recipients With Transplant Glomerulopathy y
y
N. Angaswamy1, , C. Klein2, , V. Tiriveedhi1,z, J. Gaut3, S. Anwar2, A. Rossi2, D. Phelan4, J. R. Wellen1, S. Shenoy1, W. C. Chapman1 and T. Mohanakumar1,3,* 1
Department of Surgery, Washington University School of Medicine, St. Louis, MO 2 Department of Medicine, Washington University School of Medicine, St. Louis, MO 3 Department of Pathology & Immunology, Washington University School of Medicine, St. Louis, MO 4 HLA Laboratory—Barnes-Jewish Hospital, St. Louis, MO Corresponding author: Thalachallour Mohanakumar, [email protected] y Both authors contributed equally. z Present address: Department of Biology, Tennessee State University, Nashville, TN
Antibodies (Abs) to donor HLA (donor-specific antibodies [DSA]) have been associated with transplant glomerulopathy (TG) following kidney transplantation (KTx). Immune responses to tissue-restricted selfantigens (self-Ags) have been proposed to play a role in chronic rejection. We determined whether KTx with TG have immune responses to self-Ags, Collagen-IV (Col-IV) and fibronectin (FN). DSA were determined by solid phase assay, Abs against Col-IV and FN by enzyme-linked immunosorbent assay and CD4þ T cells secreting interferon gamma (IFN-g), IL-17 or IL-10 by ELISPOT. Development of Abs to self-Ags following KTx increased the risk for TG with an odds ratio of 22 (pvalue ¼ 0.001). Abs to self-Ags were IgG and IgM isotypes. Pretransplant Abs to self-Ags increased the risk of TG (22% vs. 10%, p < 0.05). Abs to self-Ags were identified frequently in KTx with DSA. TG patients demonstrated increased Col-IV and FN specific CD4þ T cells secreting IFN-g and IL-17 with reduction in IL-10. We conclude that development of Abs to self-Ags is a risk factor and having both DSA and Abs to self-Ags increases the risk for TG. The increased frequency of self-Ag-specific IFN-g and IL-17 cells with reduction in IL-10 demonstrate tolerance breakdown to self-Ags which we propose play a role in the pathogenesis of TG. Keywords: Alloimmunity, autoimmunity, Collagen-IV, fibronectin, kidney transplantation, transplant glomerulopathy
Abbreviations: Abs, antibodies; AMR, antibody-mediated; AT1R, angiotensin II type 1 receptor; CAN, chronic allograft nephropathy; Col-IV, Collagen-IVDSA, donorspecific antibodies; ELISA, enzyme-linked immunosorbent assay; FN, fibronectin; GBM, glomerular basement membrane; HSPG, heparan sulfate proteoglycan; IFN-g, interferon gamma; KAT-1, K-alpha-1 tubulin; KTx, kidney transplantation; MFI, mean fluorescence intensity; PBMC, peripheral blood mononuclear cell; selfAgs, self-antigens; spm, spots per million cells; TG, transplant glomerulopathy Received 27 June 2013, revised 08 November 2013 and accepted for publication 11 November 2013
Introduction Chronic rejection remains a major impediment to the longterm survival of renal allografts following human kidney transplantation (KTx). Transplant glomerulopathy (TG) is associated with chronic renal allograft rejection and is characterized by duplication of the glomerular basement membrane (GBM) and peritubular capillary suggesting endothelial injury (1). Although advances in the virtual crossmatch have significantly decreased the incidence of antibody mediated rejection, chronic rejection remains a major problem. A role for immune responses to donor HLA and tissue-restricted self-antigens (self-Ags) has been proposed in the pathogenesis of chronic rejection (2). Recently, immune responses to angiotensin II type 1 receptor (AT1R) have been shown to be detrimental for renal allograft function (3,4). To determine the correlation between the immune responses to kidney-restricted self-Ags (Collagen-IV [Col-IV] and fibronectin [FN]) in the development of chronic rejection following KTx, we analyzed a group of biopsy proven TG in KTx recipients at our institute. Biopsy proven stable transplant recipients were utilized as control. Immune responses to Col-IV have been documented in animal models of renal allograft rejection (5). Increased expression of FN has been reported in glomerulopathy (6). Studies have reported an association between TG and development of antibodies (Abs) to donor HLA, and/or C4d deposition suggesting an important role for Ab-mediated damage to the kidney parenchyma leading to TG (7–9). Studies have also shown that patients with TG often develop chronic allograft nephropathy (CAN) (10). Therefore, we postulated 685
Angaswamy et al
that TG patients will be an ideal candidate to determine the development of immune responses not only to HLA but also to kidney-restricted self-Ags. Col-IV and FN were chosen as renal tissue-restricted selfAgs. In this report, we confirm not only the published reports demonstrating a correlation between immune responses to HLA to development of TG, but also provide compelling evidence for immune responses to kidneyrestricted self-Ags in the pathogenesis of TG.
Materials and Methods Study design and patients Sera from KTx recipients (2000–2011) with biopsy proven TG (n ¼ 26) and with ‘‘for cause’’ biopsy showing no histopathologic abnormality (stable patients) (n ¼ 10) were analyzed. The mean age of TG and stable patients was 50 14.3 and 45.8 9.8 years (p ¼ 0.19) respectively. The two cohorts contained 16, five male and 10, five female, respectively. The major indications for KTx in both the groups included focal segmental glomerulosclerosis, hereditary nephritis, diabetes mellitus, hypertension, obstruction, polycystic kidney diseases, and hemolytic uremic syndrome. Immunosuppression treatment was similar for TG and stable groups. The statistical analyses were performed by comparison to the normal cohort of 33 subjects with no kidney diseases (17 male, 16 female; mean age: 49.3 13.6). All patients were included in the study following approval by the Washington Universal human studies committee.
CD4þ T cells plus autologous antigen presenting cells was negative control while CD4þ T cells plus autologous antigen presenting cells with phytohemagglutinin (5 mg/mL) was positive control. Number of spots in negative control subtracted from spots in experimental wells was reported as results in spots per million cells (spm).
Histopathologic scoring Original biopsy slides were available for detailed Banff scoring (14) in 23 of 26 TG patients. Scoring was performed by a transplant pathologist blinded to the DSA and clinical status of the patient.
Statistical analysis Continuous data were checked for normality using the Shapiro–Wilk test. Nonnormal data were transformed with a log transformation. Type 1 error was controlled when performing multiple t-tests using the Dunn–Sidak correction. Tabular data were compared using the Fisher’s exact test for 2 2 tables and chi-square for 2 n tables. Relative risks and confidence intervals were calculated using the contingency tables. Software used for the above analyses were GraphPad Prism 4 (GraphPad, La Jolla, CA) and SPSS 16 (IBM, Armonk, NY).
Results Patient characteristics Development of Abs to HLA, as well as Abs to self-Ags, ColIV and FN, were determined in KTx recipients with biopsyproven TG (n ¼ 26) and stable controls (n ¼ 10). Patient demographics are given in Table 1.
Detection of Abs to mismatched HLA/donor-specific antibodies All patients were screened once per 3 months for preformed Abs to HLA and after KTx for the development of donor-specific antibodies (DSA) using the LABScreen single antigen assay (One Lambda, Inc., Canoga Park, CA). A ‘‘positive’’ DSA was defined to be a ratio of sample to positive-control mean fluorescence intensity of 0.2 or higher (11). Donor kidneys were accepted with negative virtual crossmatch. All recipients had negative cytotoxicity crossmatch using serum obtained at surgery.
Detection of Abs to self-Ags Sera were tested for Abs to Col-IV and FN by enzyme-linked immunosorbent assay (ELISA) as detailed earlier for other self-Ags (12,13). Col-II was utilized as negative control. A sample was considered positive if values were greater than the mean 2 standard deviations (76 23 ng/mL for Col-IV, 80 40 ng/ mL for FN and 18 8 ng/mL for Col-II) from normal age-matched sera (n ¼ 33, age 47.8 12.4, male ¼ 19 and female ¼ 14). Abs concentration was calculated using a standard curve from known concentrations of Col-IV, FN or Col-II Abs (Santa Cruz Biotechnology, Santa Cruz, CA).
Cytokine measurement Serum levels of cytokines IL-10, interferon gamma (IFN-g) and IL-17 were detected by Luminex (Invitrogen, Carlsbad, CA) following the manufacturer’s protocols. The plates were analyzed by Bio-Plex Luminex 100TM (Invitrogen) and cytokine concentrations were calculated using the Bio-Plex Manager 3.0 software with a five parameter curve-fitting algorithm applied for standard curve calculations.
ELISPOT Frozen peripheral blood mononuclear cells (PBMCs) from patients were cultured overnight in RPMI-1640 and viability was determined by trypan blue exclusion. PBMCs with viability of >90% were used for ELISPOT assays. The plates were washed and developed to detect IFN-g, IL-10 and IL-17 using an ImmunoSpot analyzer (Cellular Technology, Shaker Heights, OH).
686
Table 1: Patient demographics
Recipient age at transplant Recipient sex (males %) Ethnicity Black Non-black Transplant type Living donor Deceased donor Cause of ESRD Diabetes PKD Reflux/obstruction FSGS Other/unknown HLA mismatch A B DR Time from transplant to biopsy Spot urine protein/creatinine >0.5 at biopsy Graft loss (%) Recipient death
TG patients (n ¼ 26)
Stable controls (n ¼ 10)
50.1 14.3 years 16 (62%)
45.8 9.8 5 (50%)
5 (19%) 21 (81%)
3 (30%) 7 (70%)
14 (54%) 12 (46%)
5 (50%) 5 (50%)
8 (31%) 2 (8%) 2 (8%) 0 14 (53%)
2 (20%) 3 (30%) 0 2 (20%) 3 (30%)
1.0 0.5 1.4 0.7 1.0 0.6 4.4 3.0 years 17 (65%)
1.2 0.7 1.4 0.7 1.4 0.7 1.0 1.9 1 (10%)
7 (27%) 1 (4%)
2 (20%) 0
ESRD, end-stage renal disease; FSGS, focal segmental glomerulosclerosis; PKD, polycystic kidney disease; TG, transplant glomerulopathy.
American Journal of Transplantation 2014; 14: 685–693
Autoimmunity in TG Table 2: Development of Abs to Col-IV and FN in KTx recipients with TG Patients
Pre/post-Tx
DSAþ
HLAþ
Col-IVþ
FNþ
Col-II
Biopsy proven TG
Pre-Tx Post-Tx
4/18 (22.2%) 22/26 (84.6%)
– –
Pre-Tx Post-Tx
– 4/26 (15.4%) I (2/4, 50%) II (2/4, 50%) – –
4/18 (22.2%) 22/26 (84.6%)
Biopsy proven stable
– 12/26 (46%) I (4/12, 33%) II (8/12, 67%) – 2/10 (20%)
1/10 (10%) 2/10 (20%)
1/10 (10%) 2/10 (20%)
– –
Abs, antibodies; Col-II/IV, Collagen-II/IV; DSA, donor-specific antibodies; FN, fibronectin; KTx, kidney transplantation; TG, transplant glomerulopathy.
De novo development of Abs to HLA in KTx recipients with biopsy proven TG To determine the correlation between alloimmune responses and TG, pre- and post-KTx sera were analyzed for Abs to HLA using Luminex single Ag beads. As shown in Table 2, 16 out of the 26 patients (61%) with TG developed Abs to HLA. Out of this, 12/16 (75%) were specific to donor HLA (DSA) and 4/16 (25%) to HLA not present on the donor, 2 had Abs to HLA class I and the other 2 to HLA class II. Out of the 12 TG who had DSA, 4/12 (33%) were to HLA class I and 8/12 (67%) to HLA class II antigens. In contrast, only 2/ 10 stable patients developed DSA (Table 2). Results presented in Table 3 demonstrate significant correlation for the posttransplant development of Abs to HLA and TG, odds ratio of 6.4, with a p-value of 0.026, thus suggesting that patients with TG often develop Abs to HLA and DSA. TG patients develop Abs to kidney-restricted self-Ags, Col-IV and FN which is not correlated with alloimmune responses To determine the correlation between Abs to kidneyrestricted self-Ags and TG, pre- and post-KTx sera were analyzed by ELISA for Abs to Col-IV and FN. Abs to Col-II was used as a control. A large proportion of the biopsy proven TG patients 22/26 (84%) had Abs to both Col-IV and FN and the majority developed posttransplant (Table 2). Not all of the patients with Abs to self-Ags had detectable Abs to HLA or DSA. Further, we noted Abs to Col-IV and FN in 4/18 (22%) in the pretransplant sera indicating that chronic renal diseases may contribute in the development of Abs to selfAgs. None of the patients tested had Abs to Col-II both preand posttransplant period. One of the 10 stable patients had Abs to self-Ags both in pre- and posttransplant sera. One additional patient developed Abs during the posttransplant period. Both of them developed Abs to donor HLA (DSA) following transplantation. None of the sera had reactivity to Col-II (Table 2 and Figure 1). Development of Abs to kidneyrestricted self-Ags during the posttransplant period also Table 3: Correlation between post-Tx Abs to HLA and TG
TG Stable Normal
Total
Post-Tx Abs (þ)
Odds ratio
p-Value
26 10 26
16 (61.54%) 2 (20%) n/a
6.4
0.026
Abs, antibodies; TG, transplant glomerulopathy; Tx, transplantation.
American Journal of Transplantation 2014; 14: 685–693
significantly increased the chance of developing TG with an odds ratio of 22 (p-value ¼ 0.001) (Table 4). Results presented in Figure 2 demonstrate the correlation between the development of Abs to self-Ags and development of either DSA or HLA. Abs to HLA is not a prerequisite for the development of Abs to self-Ags since 6 out of the 22 TG patients with Abs to self-Ag did not have Abs to HLA or DSA. However, there is a higher tendency for the development of Abs to self-Ags in TG patients having Abs to HLA or DSA suggesting that one may predispose development of others (16 out of 22 developed both). These results indicate that development of Abs to kidneyrestricted self-Ags correlated with development of TG. Correlation between increase in the concentration of Abs to self-Ags and development of TG following KTx Serial pre- and posttransplant sera from four TG and two stable patients were analyzed for Abs and their concentration at the time TG was diagnosed and proven by biopsy. Results presented in Figure 3 suggest that the Abs to self-Ags may play a role in TG since the concentration of Abs went significantly higher at the time of TG diagnosis. Two of the stable patients did not demonstrate presence and increases in Abs to self-Ags. As the number of patients in this study was too few, statistical analysis was not performed. Abs to self-Ags are both IgG and IgM isotypes DSA(þ)/anti-self-Ags(þ) (n ¼ 12), and anti-HLA(þ)/anti-selfAgs(þ) (n ¼ 4) and DSA()/anti-HLA()/anti-self-Ags(þ) were analyzed for their isotype using ELISA. Results presented in Table 5 demonstrate that both isotypes are present including in those patients who did not have any detectable Abs to HLA or DSA. The presence of IgG in 8/14 for anti-Col-IV and 6/10 for anti-FN suggest ongoing immune responses to these antigens. Pathologic characteristics are similar in TG patients with Abs to self-Ags independent of DSA status Biopsy slides from 23 of the 26 TG patients were scored according to Banff criteria (14). Results presented in Table 6 and Figure 4 show that severity of glomerulitis, peritubular capillaritis, C4d deposition and chronic glomerulopathy were equivalent in patients with Abs to self-Ags in presence or absence of DSA suggesting that Abs to self-Ags alone can be associated with histologic evidence of both acute and chronic Ab-mediated injury. 687
Angaswamy et al
Figure 1: Presence of post-Tx Abs to self-Ags in patients with TG. Serum concentration of Abs to Col-IV (A), FN (B) and Col-II (C) in patients with TG (n ¼ 26), stable (n ¼ 10) and normal (n ¼ 33) cohorts. Data represented as mean SD; statistical significance ( ) ascertained if p < 0.05; n/s if p > 0.05 (not significant). Abs to Col-II considered as a negative control in our studies and has demonstrated no significant difference between the three cohorts. Abs, antibodies; Col-II/IV, Collagen-II/IV; FN, fibronectin; self-Ags, self-antigens; TG, transplant glomerulopathy; Tx, transplantation.
Increased frequency of self-Ag specific IFN-g and IL17 with concomitant decrease in IL-10 in KTx recipients diagnosed with TG The CD4þ T cells isolated from PBMCs from 26 TG, 10 stable KTx recipients and normal (n ¼ 14) were analyzed for self-Ags-specific immune responses by ELISPOT. As shown in Figure 5, patients with TG demonstrated increased frequencies of IFN-g and IL-17 against Col-IV, Figure 5B,C (IFN-g, TG: 416 91 spm, stable: 178 69 spm, p ¼ 0.006; IL-17, TG: 278 62 spm, stable: 116 34 spm, p ¼ 0.009) and FN, Figure 5E,F (IFN-g, TG: 438 79 spm, stable: 193 73 spm, p ¼ 0.02; IL-17, TG: 342 92 spm, stable: 143 56 spm, p ¼ 0.003). In contrast, TG patients demonstrated decreased frequencies of IL-10 secreting CD4þ T cells against Col-IV (Figure 5A,D) (TG: 83 37 spm, stable: 378 132 spm, p ¼ 0.008); and FN (TG: 103 46 spm, stable: 285 81 spm, p ¼ 0.001). Frequency of IFN-g, Il-10 and IL-17 secreting T cells against Col-II (Figure 5G–I) demonstrated no significant differences (TG vs. stable, p ¼ ns; TG vs. normal, p ¼ ns). These results indicate a loss of peripheral tolerance to self-Ags along with a concomitant up-regulation of autoimmune cellular immune responses to self-Ags in TG. Increased serum concentrations of IFN-g and IL-17 in TG patients Serum concentrations of cytokines were analyzed using Luminex and data presented in Figure 6 support the findings Table 4: Correlation between post-Tx Abs to self-Ags and TG
TG Stable Normal
Total
Post-Tx Abs (þ)
Odds ratio
p-Value
26 10 26
22 (84.62%) 2 (20%) n/a
22
0.001
Abs, antibodies; self-Ags, self-antigens; TG, transplant glomerulopathy; Tx, transplantation.
688
of ELISPOT demonstrating increases in the frequency of selfAg specific IFN-g and IL-17 with reduction in IL-10. TG patients demonstrated higher concentrations of cytokines IFN-g (TG: 867 241 pg/mL, stable: 407 178 pg/mL, p ¼ 0.02) and IL-17 (TG: 918 197 pg/mL, stable: 327 142 pg/mL, p ¼ 0.009) with marked reduction in IL-10 (TG: 184 78 pg/mL, stable: 668 184 pg/mL, p ¼ 0.001).
Discussion De novo development of Abs to mismatched donor HLA (DSA) have been associated with CAN (15,16) and it has been proposed that Abs reactive to glomerular endothelial cells play an important role in the development of CAN. However, not all KTx recipients diagnosed with CAN have DSA raising the possibility that immune responses against tissue-restricted self-Ags might play a role in its pathogenesis. Joosten et al (5) using a rat KTx model of chronic rejection and TG patients identified Abs reactive to GBM antigen, heparan sulfate proteoglycan (HSPG), perlecan and Abs reactive to GBM-HSPG agrin, respectively, and postulated that immune responses to perlecan and agrin may play a role in the pathogenesis of TG. It has been reported that about 15–20% of KTx recipients diagnosed with CAN have TG and TG increases the risk for CAN (17,18). Therefore, we determined the immune responses to kidney-restricted self-Ags by analyzing sera from TG (with characteristic duplication of the GBM) for Abs to purified Col-IV and FN. As reported (8,9,19,20), about 61% of patients developed de novo Abs to HLA, of which 75% were DSA. Majority of the DSA (67%) were to HLA class II; however, 4/12 patients (33%) demonstrated HLA class I specificity alone indicating that immune responses to both HLA class I and II can be pathogenic for TG (Table 2). The development of Abs to Col-IV and FN were not always associated with Abs to HLA. Six of the 22 TG did not have any Abs to HLA or DSA. However, in 16 out of 26 patients, American Journal of Transplantation 2014; 14: 685–693
Autoimmunity in TG
Figure 2: Development of Abs to Col-IV and FN did not correlate with DSA. Serum concentration of Abs to Col-IV (A) and FN (B) in patients with DSA(þ) (n ¼ 12), DSA()/HLA() (n ¼ 6) and HLA(þ) (n ¼ 4) cohorts. Data represented as mean SD. As noted, patients with DSA(þ) and DSA()/HLA() cohorts did not reach statistical significance; n/s, not significant; p < 0.05; p < 0.01. Abs, antibodies; Col-IV, Collagen-IV; DSA, donor-specific antibodies; FN, fibronectin.
Abs to both HLA or DSA and Abs to self-Ags were detected (Figure 2). This result supports the concept that ongoing immune responses to self-Ags can be pathogenic for the development of TG. We propose that initial binding by Abs of IgG isotype will result in GBM duplication and this process exposes new antigenic determinants which then may lead to Abs of IgM isotype followed by IgG. This hypothesis needs to be tested. In our study, the temporal analysis of serum samples significantly varied between TG patients (4.4 3 years) and stable recipients (1.0 1.9 years), a future prospective monitoring of CoI-V and FN Abs and their isotypes are needed to define time frame of appearance in order to correlate it with TG.
To define the mechanisms for development of immune responses to kidney-restricted self-Ags, the frequency of T cells specific to Col-IV and FN and T cells secreting IFN-g, IL17 and IL-10 were determined using ELISPOT. There is significant activation of Col-IV and FN-specific T cells secreting both IFN-g and IL-17 in TG but not in stable patients (Figure 5). Significant drop in self-Ag specific IL-10 secreting cells in TG but not in stable KTx recipients was also noted. A parallel increase in circulating IFN-g, IL-17 with reduction in IL-10 was also seen in sera from TG (Figure 6). There is increasing evidence that IL-17 by itself can be profibrotic (21) and could potentially induce development of fibrosis in CAN.
Figure 3: Abs to Col-IV and FN increase following KTx in TG patients. Serial monitoring of Abs to Col-IV (A) and FN (B) in TG with pre-Tx Abs to self-Ags (n ¼ 4, solid line) and stable (n ¼ 2, dotted line) at three time points: pre-Tx, post-Tx and at time of clinical diagnosis of TG (or time-matched sample stable cohort). As noted, there is a serial increase in the Abs to Col-IV and FN in patients who had pre-Tx Abs to selfAgs and eventually developed TG. As noted in Table 1, due to widespread temporal distribution of the analysis of the serum samples (TG patients—4.4 3.0 years vs. stable—1.0 1.9 years) the x-axis is used only for depiction and not exact time sequence. Abs, antibodies; ColIV, Collagen-IV; FN, fibronectin; KTx, kidney transplantation; self-Ags, self-antigens; TG, transplant glomerulopathy.
American Journal of Transplantation 2014; 14: 685–693
689
Angaswamy et al Table 5: Abs to Col-IV and FN were both IgM and IgG isotypes
DSA (þ), n ¼ 12 HLA(þ), n ¼ 4 DSA()/HLA(), n ¼ 6
Anti-Col-IV
Anti-FN
IgG (7/12) IgM (12/12) Both (7/12) IgG (1/4) IgM (3/4) Both (1/4) IgG (4/6) IgM (4/6) Both (4/6)
IgG (10/12) IgM (12/12) Both (10/12) IgG (2/4) IgM (2/4) Both (2/4) IgG (3/6) IgM (4/6) Both (3/6)
Abs, antibodies; Col-IV, Collagen-IV; DSA, donor-specific antibodies; FN, fibronectin.
Since both cellular and humoral immune responses to both kidney-restricted self-Ags (Col-IV and FN) seem to go together, we propose that onset of immune response either to a kidney-restricted self-Ag or HLA initiates spreading of immune responses to various self-Ags as well as HLA molecules expressed on the kidney. These results suggest strongly that there is cross talk between immune responses to both allo-MHC molecules and tissuerestricted self-Ags and once either of them developed it can spread to other leading to the pathogenesis of TG. Further, selective expansion of IL-17 secreting T cells along with a decrease in the loss of peripheral tolerance induced by diminished IL-10 secreting T cells can result in immune Table 6: Banff scoring for glomerulitis, chronic glomerulopathy (CG), peritubular capillaritis (PTC) and C4d staining in TG patients with FN and Col-IV Abs stratified by donor-specific antibody (DSA) status DSAþ, N ¼ 14 Glomerulitis score, n (%) 0 1 2 3 CG score, n (%) 0 1 2 3 PTC score, n (%) 0 1 2 3 C4d, n (%) Positive Negative
DSA, N¼6
p-Value 1.0
3 4 5 2
(21.4) (28.6) (35.7) (14.3)
2 1 2 1
(33.3) (16.7) (33.3) (16.7)
5 4 2 3
(35.7) (28.6) (14.3) (21.4)
1 3 1 1
(16.7) (50.0) (16.7) (16.7)
0.9
0.6 4 (28.6) 0 8 (57.1) 2 (14.3)
3 (50.0) 0 3 (50.0) 0
4 (28.6) 10 (71.4)
3 (50.0) 3 (50.0)
0.6
Abs, antibodies; Col-IV, Collagen-IV; FN, fibronectin; TG, transplant glomerulopathy. Nineteen subjects had positive Col-IV and FN Abs, one subject was positive for Col-IV Abs only. Of the 14 subjects with positive DSAs, 6 were positive for class I and II, 3 for class I only and 5 for class II only. p-Values were calculated using Fisher’s exact test.
690
Figure 4: Histology of TG. (A) Representative image of a glomerulus from a patient that was Col-IV and FN Abþ and DSA. Features typical of TG are seen including splitting of the capillary loops (arrows) and glomerulitis ( ) (Periodic Acid Schiff stain, 400). (B) Representative image of a glomerulus from a patient that was Col-IV and FN Abþ and DSAþ. Focal capillary loop duplication (arrow) and glomerulitis ( ) are seen, similar to the findings depicted in A (Periodic Acid Schiff, 400). Ab, antibody; Col-IV, Collagen-IV; DSA, donor-specific antibody; FN, fibronectin; TG, transplant glomerulopathy.
responses to tissue-restricted self-Ags which we propose to play a vital role in the pathogenesis of TG. In conclusion, our results along with other published findings (16,22–24) support Ab-mediated mechanism for the development of TG following KTx. As this study is a retrospective analysis of patients with TG, it has its limitations, including small number, lack of serial samples for analysis and inability to perform multivariate analysis. However, it is clear that not only Abs to donor HLA but also Abs to tissue-restricted self-Ags, Col-IV, FN, agrin, AT1R, etc., are developed in patients with TG. We propose that serial monitoring for Ab development either to donor HLA or to kidney self-Ags during posttransplant may identify American Journal of Transplantation 2014; 14: 685–693
Autoimmunity in TG
Figure 5: Increased frequency of self-Ag specific Th1 and Th17 response in patients with TG. Frequency of CD4 þ T cells with specific reactivity to Col-IV (A–C), FN (D–F) and Col-II (G–I) in patients with TG (n ¼ 26), stable (n ¼ 10) and normal (n ¼ 33) cohorts as determined by ELISPOT. A and D represent CD4 þ T cells that induce IL-10 secretion upon specific reactivity to Col-IV and FN, respectively. B and E represent CD4 þ T cells that induce IFN-g secretion upon specific reactivity to Col-IV and FN, respectively. C and F represent CD4 þ T cells that induce IL-17 secretion upon specific reactivity to Col-IV and FN, respectively. Col-II (G–I) is used as negative control in this study. Data represented as mean SD; statistical significance ( ) ascertained if p < 0.05. n/s, not significant. Col-II/IV, Collagen-II/IV; FN, fibronectin; IFN-g, interferon gamma; self-Ags, self-antigens; TG, transplant glomerulopathy.
American Journal of Transplantation 2014; 14: 685–693
691
Angaswamy et al
Figure 6: Increased serum concentration of pro-inflammatory cytokines in patients with TG. Serum concentration of antiinflammatory cytokine, IL-10 (A) and pro-inflammatory cytokines, IFN-g (B) and IL-17 (C) in patients with TG (n ¼ 26), stable (n ¼ 10) and normal (n ¼ 33) cohorts as determined by Luminex. Data represented as mean SD; statistical significance ( ) ascertained if p < 0.05. IFN-g, interferon gamma; TG, transplant glomerulopathy.
patients at risk for developing TG. Since this report, as well as others, implicates Abs as an important initiator in the pathogenesis of TG and CAN, we propose that Ab directed therapy following early detection of de novo developed Abs during posttransplant may be beneficial in preventing TG. This approach has been successfully employed following human lung transplantation toward preventing the onset of chronic rejection following human lung transplantation (25,26). Since our results suggest an important role for IL-17 in the development of immune responses to selfAgs, it is likely that neutralization of IL-17 and IL-6 may represent another avenue to explore as possible targets to prevent TG following human KTx.
Acknowledgments This work was supported by Barnes-Jewish Foundation (TM). The authors would like to thank Billie Glasscock for her assistance in submitting this manuscript.
Disclosure The authors of this manuscript have no conflicts of interest to disclose as described by the American Journal of Transplantation.
References 1. Husain S, Sis B. Advances in the understanding of transplant glomerulopathy. Am J Kidney Dis 2013; 62: 352–363. 2. Tiriveedhi V, Sarma N, Mohanakumar T. An important role for autoimmunity in the immunopathogenesis of chronic allograft rejection. Int J Immunogenet 2012; 39: 373–380. 3. Dragun D, Muller DN, Brasen JH, et al. Angiotensin II type 1receptor activating antibodies in renal-allograft rejection. N Engl J Med 2005; 352: 558–569.
692
4. Giral M, Foucher Y, Dufay A, et al. Pretransplant sensitization against angiotensin II type 1 receptor is a risk factor for acute rejection and graft loss. Am J Transplant 2013; 13: 2567–2576. 5. Joosten SA, van Dixhoorn MG, Borrias MC, et al. Antibody response against perlecan and collagen types IV and VI in chronic renal allograft rejection in the rat. Am J Pathol 2002; 160: 1301– 1310. 6. Strom EH, Banfi G, Krapf R, et al. Glomerulopathy associated with predominant fibronectin deposits: A newly recognized hereditary disease. Kidney Int 1995; 48: 163–170. 7. Colvin RB. Antibody-mediated renal allograft rejection: Diagnosis and pathogenesis. J Am Soc Nephrol 2007; 18: 1046–1056. 8. Gloor JM, Sethi S, Stegall MD, et al. Transplant glomerulopathy: Subclinical incidence and association with alloantibody. Am J Transplant 2007; 7: 2124–2132. 9. Sis B, Campbell PM, Mueller T, et al. Transplant glomerulopathy, late antibody-mediated rejection and the ABCD tetrad in kidney allograft biopsies for cause. Am J Transplant 2007; 7: 1743–1752. 10. Ivanyi B. Transplant capillaropathy and transplant glomerulopathy: Ultrastructural markers of chronic renal allograft rejection. Nephrol Dial Transplant 2003; 18: 655–660. 11. Morris GP, Phelan DL, Jendrisak MD, Mohanakumar T. Virtual crossmatch by identification of donor-specific anti-human leukocyte antigen antibodies by solid-phase immunoassay: A 30-month analysis in living donor kidney transplantation. Hum Immunol 2010; 71: 268–273. 12. Borg BB, Seetharam A, Subramanian V, et al. Immune response to extracellular matrix collagen in chronic hepatitis C-induced liver fibrosis. Liver Transplant 2011; 17: 814–823. 13. Saini D, Weber J, Ramachandran S, et al. Alloimmunity-induced autoimmunity as a potential mechanism in the pathogenesis of chronic rejection of human lung allografts. J Heart Lung Transplant 2011; 30: 624–631. 14. Veronese FV, Manfro RC, Roman FR, et al. Reproducibility of the Banff classification in subclinical kidney transplant rejection. Clin Transplant 2005; 19: 518–521. 15. Li C, Yang CW. The pathogenesis and treatment of chronic allograft nephropathy. Nat Rev Nephrol 2009; 5: 513–519. 16. Gaston RS, Cecka JM, Kasiske BL, et al. Evidence for antibodymediated injury as a major determinant of late kidney allograft failure. Transplantation 2010; 90: 68–74.
American Journal of Transplantation 2014; 14: 685–693
Autoimmunity in TG 17. Shu KH, Lu YS, Chang CH, et al. Transplant glomerulopathy—A clinicopathological study. Transplant Proc 1996; 28: 1527–1528. 18. Kupin W, Nakhleh R, Lee M, et al. Separate risk factors for the development of transplant glomerulopathy vs chronic tubulointerstitial rejection. Transplant Proc 1997; 29: 245–246. 19. Mao Q, Terasaki PI, Cai J, et al. Extremely high association between appearance of HLA antibodies and failure of kidney grafts in a five-year longitudinal study. Am J Transplant 2007; 7: 864–871. 20. Campos EF, Tedesco-Silva H, Machado PG, Franco M, MedinaPestana JO, Gerbase-DeLima M. Post-transplant anti-HLA class II antibodies as risk factor for late kidney allograft failure. Am J Transplant 2006; 6: 2316–2320. 21. Al-Muhsen S, Letuve S, Vazquez-Tello A, et al. Th17 cytokines induce pro-fibrotic cytokines release from human eosinophils. Respir Res 2013; 14: 34–44.
American Journal of Transplantation 2014; 14: 685–693
22. Sellares J, de Freitas DG, Mengel M, et al. Understanding the causes of kidney transplant failure: The dominant role of antibodymediated rejection and nonadherence. Am J Transplant 2012; 12: 388–399. 23. Dinavahi R, George A, Tretin A, et al. Antibodies reactive to nonHLA antigens in transplant glomerulopathy. J Am Soc Nephrol 2011; 22: 1168–1178. 24. Cosio FG, Gloor JM, Sethi S, Stegall MD. Transplant glomerulopathy. Am J Transplant 2008; 8: 492–496. 25. Hachem RR, Yusen RD, Meyers BF, et al. Anti-human leukocyte antigen antibodies and preemptive antibody-directed therapy after lung transplantation. J Heart Lung Transplant 2010; 29: 973–980. 26. Hachem RR, Tiriveedhi V, Patterson GA, Aloush A, Trulock EP, Mohanakumar T. Antibodies to K-alpha 1 tubulin and Collagen V are associated with chronic rejection after lung transplantation. Am J Transplant 2012; 12: 2164–2171.
693
C
Copyright 2014 The American Society of Transplantation and the American Society of Transplant Surgeons doi: 10.1111/ajt.12592
Brief Communication
Immune Responses to Collagen-IV and Fibronectin in Renal Transplant Recipients With Transplant Glomerulopathy y
y
N. Angaswamy1, , C. Klein2, , V. Tiriveedhi1,z, J. Gaut3, S. Anwar2, A. Rossi2, D. Phelan4, J. R. Wellen1, S. Shenoy1, W. C. Chapman1 and T. Mohanakumar1,3,* 1
Department of Surgery, Washington University School of Medicine, St. Louis, MO 2 Department of Medicine, Washington University School of Medicine, St. Louis, MO 3 Department of Pathology & Immunology, Washington University School of Medicine, St. Louis, MO 4 HLA Laboratory—Barnes-Jewish Hospital, St. Louis, MO Corresponding author: Thalachallour Mohanakumar, [email protected] y Both authors contributed equally. z Present address: Department of Biology, Tennessee State University, Nashville, TN
Antibodies (Abs) to donor HLA (donor-specific antibodies [DSA]) have been associated with transplant glomerulopathy (TG) following kidney transplantation (KTx). Immune responses to tissue-restricted selfantigens (self-Ags) have been proposed to play a role in chronic rejection. We determined whether KTx with TG have immune responses to self-Ags, Collagen-IV (Col-IV) and fibronectin (FN). DSA were determined by solid phase assay, Abs against Col-IV and FN by enzyme-linked immunosorbent assay and CD4þ T cells secreting interferon gamma (IFN-g), IL-17 or IL-10 by ELISPOT. Development of Abs to self-Ags following KTx increased the risk for TG with an odds ratio of 22 (pvalue ¼ 0.001). Abs to self-Ags were IgG and IgM isotypes. Pretransplant Abs to self-Ags increased the risk of TG (22% vs. 10%, p < 0.05). Abs to self-Ags were identified frequently in KTx with DSA. TG patients demonstrated increased Col-IV and FN specific CD4þ T cells secreting IFN-g and IL-17 with reduction in IL-10. We conclude that development of Abs to self-Ags is a risk factor and having both DSA and Abs to self-Ags increases the risk for TG. The increased frequency of self-Ag-specific IFN-g and IL-17 cells with reduction in IL-10 demonstrate tolerance breakdown to self-Ags which we propose play a role in the pathogenesis of TG. Keywords: Alloimmunity, autoimmunity, Collagen-IV, fibronectin, kidney transplantation, transplant glomerulopathy
Abbreviations: Abs, antibodies; AMR, antibody-mediated; AT1R, angiotensin II type 1 receptor; CAN, chronic allograft nephropathy; Col-IV, Collagen-IVDSA, donorspecific antibodies; ELISA, enzyme-linked immunosorbent assay; FN, fibronectin; GBM, glomerular basement membrane; HSPG, heparan sulfate proteoglycan; IFN-g, interferon gamma; KAT-1, K-alpha-1 tubulin; KTx, kidney transplantation; MFI, mean fluorescence intensity; PBMC, peripheral blood mononuclear cell; selfAgs, self-antigens; spm, spots per million cells; TG, transplant glomerulopathy Received 27 June 2013, revised 08 November 2013 and accepted for publication 11 November 2013
Introduction Chronic rejection remains a major impediment to the longterm survival of renal allografts following human kidney transplantation (KTx). Transplant glomerulopathy (TG) is associated with chronic renal allograft rejection and is characterized by duplication of the glomerular basement membrane (GBM) and peritubular capillary suggesting endothelial injury (1). Although advances in the virtual crossmatch have significantly decreased the incidence of antibody mediated rejection, chronic rejection remains a major problem. A role for immune responses to donor HLA and tissue-restricted self-antigens (self-Ags) has been proposed in the pathogenesis of chronic rejection (2). Recently, immune responses to angiotensin II type 1 receptor (AT1R) have been shown to be detrimental for renal allograft function (3,4). To determine the correlation between the immune responses to kidney-restricted self-Ags (Collagen-IV [Col-IV] and fibronectin [FN]) in the development of chronic rejection following KTx, we analyzed a group of biopsy proven TG in KTx recipients at our institute. Biopsy proven stable transplant recipients were utilized as control. Immune responses to Col-IV have been documented in animal models of renal allograft rejection (5). Increased expression of FN has been reported in glomerulopathy (6). Studies have reported an association between TG and development of antibodies (Abs) to donor HLA, and/or C4d deposition suggesting an important role for Ab-mediated damage to the kidney parenchyma leading to TG (7–9). Studies have also shown that patients with TG often develop chronic allograft nephropathy (CAN) (10). Therefore, we postulated 685
Angaswamy et al
that TG patients will be an ideal candidate to determine the development of immune responses not only to HLA but also to kidney-restricted self-Ags. Col-IV and FN were chosen as renal tissue-restricted selfAgs. In this report, we confirm not only the published reports demonstrating a correlation between immune responses to HLA to development of TG, but also provide compelling evidence for immune responses to kidneyrestricted self-Ags in the pathogenesis of TG.
Materials and Methods Study design and patients Sera from KTx recipients (2000–2011) with biopsy proven TG (n ¼ 26) and with ‘‘for cause’’ biopsy showing no histopathologic abnormality (stable patients) (n ¼ 10) were analyzed. The mean age of TG and stable patients was 50 14.3 and 45.8 9.8 years (p ¼ 0.19) respectively. The two cohorts contained 16, five male and 10, five female, respectively. The major indications for KTx in both the groups included focal segmental glomerulosclerosis, hereditary nephritis, diabetes mellitus, hypertension, obstruction, polycystic kidney diseases, and hemolytic uremic syndrome. Immunosuppression treatment was similar for TG and stable groups. The statistical analyses were performed by comparison to the normal cohort of 33 subjects with no kidney diseases (17 male, 16 female; mean age: 49.3 13.6). All patients were included in the study following approval by the Washington Universal human studies committee.
CD4þ T cells plus autologous antigen presenting cells was negative control while CD4þ T cells plus autologous antigen presenting cells with phytohemagglutinin (5 mg/mL) was positive control. Number of spots in negative control subtracted from spots in experimental wells was reported as results in spots per million cells (spm).
Histopathologic scoring Original biopsy slides were available for detailed Banff scoring (14) in 23 of 26 TG patients. Scoring was performed by a transplant pathologist blinded to the DSA and clinical status of the patient.
Statistical analysis Continuous data were checked for normality using the Shapiro–Wilk test. Nonnormal data were transformed with a log transformation. Type 1 error was controlled when performing multiple t-tests using the Dunn–Sidak correction. Tabular data were compared using the Fisher’s exact test for 2 2 tables and chi-square for 2 n tables. Relative risks and confidence intervals were calculated using the contingency tables. Software used for the above analyses were GraphPad Prism 4 (GraphPad, La Jolla, CA) and SPSS 16 (IBM, Armonk, NY).
Results Patient characteristics Development of Abs to HLA, as well as Abs to self-Ags, ColIV and FN, were determined in KTx recipients with biopsyproven TG (n ¼ 26) and stable controls (n ¼ 10). Patient demographics are given in Table 1.
Detection of Abs to mismatched HLA/donor-specific antibodies All patients were screened once per 3 months for preformed Abs to HLA and after KTx for the development of donor-specific antibodies (DSA) using the LABScreen single antigen assay (One Lambda, Inc., Canoga Park, CA). A ‘‘positive’’ DSA was defined to be a ratio of sample to positive-control mean fluorescence intensity of 0.2 or higher (11). Donor kidneys were accepted with negative virtual crossmatch. All recipients had negative cytotoxicity crossmatch using serum obtained at surgery.
Detection of Abs to self-Ags Sera were tested for Abs to Col-IV and FN by enzyme-linked immunosorbent assay (ELISA) as detailed earlier for other self-Ags (12,13). Col-II was utilized as negative control. A sample was considered positive if values were greater than the mean 2 standard deviations (76 23 ng/mL for Col-IV, 80 40 ng/ mL for FN and 18 8 ng/mL for Col-II) from normal age-matched sera (n ¼ 33, age 47.8 12.4, male ¼ 19 and female ¼ 14). Abs concentration was calculated using a standard curve from known concentrations of Col-IV, FN or Col-II Abs (Santa Cruz Biotechnology, Santa Cruz, CA).
Cytokine measurement Serum levels of cytokines IL-10, interferon gamma (IFN-g) and IL-17 were detected by Luminex (Invitrogen, Carlsbad, CA) following the manufacturer’s protocols. The plates were analyzed by Bio-Plex Luminex 100TM (Invitrogen) and cytokine concentrations were calculated using the Bio-Plex Manager 3.0 software with a five parameter curve-fitting algorithm applied for standard curve calculations.
ELISPOT Frozen peripheral blood mononuclear cells (PBMCs) from patients were cultured overnight in RPMI-1640 and viability was determined by trypan blue exclusion. PBMCs with viability of >90% were used for ELISPOT assays. The plates were washed and developed to detect IFN-g, IL-10 and IL-17 using an ImmunoSpot analyzer (Cellular Technology, Shaker Heights, OH).
686
Table 1: Patient demographics
Recipient age at transplant Recipient sex (males %) Ethnicity Black Non-black Transplant type Living donor Deceased donor Cause of ESRD Diabetes PKD Reflux/obstruction FSGS Other/unknown HLA mismatch A B DR Time from transplant to biopsy Spot urine protein/creatinine >0.5 at biopsy Graft loss (%) Recipient death
TG patients (n ¼ 26)
Stable controls (n ¼ 10)
50.1 14.3 years 16 (62%)
45.8 9.8 5 (50%)
5 (19%) 21 (81%)
3 (30%) 7 (70%)
14 (54%) 12 (46%)
5 (50%) 5 (50%)
8 (31%) 2 (8%) 2 (8%) 0 14 (53%)
2 (20%) 3 (30%) 0 2 (20%) 3 (30%)
1.0 0.5 1.4 0.7 1.0 0.6 4.4 3.0 years 17 (65%)
1.2 0.7 1.4 0.7 1.4 0.7 1.0 1.9 1 (10%)
7 (27%) 1 (4%)
2 (20%) 0
ESRD, end-stage renal disease; FSGS, focal segmental glomerulosclerosis; PKD, polycystic kidney disease; TG, transplant glomerulopathy.
American Journal of Transplantation 2014; 14: 685–693
Autoimmunity in TG Table 2: Development of Abs to Col-IV and FN in KTx recipients with TG Patients
Pre/post-Tx
DSAþ
HLAþ
Col-IVþ
FNþ
Col-II
Biopsy proven TG
Pre-Tx Post-Tx
4/18 (22.2%) 22/26 (84.6%)
– –
Pre-Tx Post-Tx
– 4/26 (15.4%) I (2/4, 50%) II (2/4, 50%) – –
4/18 (22.2%) 22/26 (84.6%)
Biopsy proven stable
– 12/26 (46%) I (4/12, 33%) II (8/12, 67%) – 2/10 (20%)
1/10 (10%) 2/10 (20%)
1/10 (10%) 2/10 (20%)
– –
Abs, antibodies; Col-II/IV, Collagen-II/IV; DSA, donor-specific antibodies; FN, fibronectin; KTx, kidney transplantation; TG, transplant glomerulopathy.
De novo development of Abs to HLA in KTx recipients with biopsy proven TG To determine the correlation between alloimmune responses and TG, pre- and post-KTx sera were analyzed for Abs to HLA using Luminex single Ag beads. As shown in Table 2, 16 out of the 26 patients (61%) with TG developed Abs to HLA. Out of this, 12/16 (75%) were specific to donor HLA (DSA) and 4/16 (25%) to HLA not present on the donor, 2 had Abs to HLA class I and the other 2 to HLA class II. Out of the 12 TG who had DSA, 4/12 (33%) were to HLA class I and 8/12 (67%) to HLA class II antigens. In contrast, only 2/ 10 stable patients developed DSA (Table 2). Results presented in Table 3 demonstrate significant correlation for the posttransplant development of Abs to HLA and TG, odds ratio of 6.4, with a p-value of 0.026, thus suggesting that patients with TG often develop Abs to HLA and DSA. TG patients develop Abs to kidney-restricted self-Ags, Col-IV and FN which is not correlated with alloimmune responses To determine the correlation between Abs to kidneyrestricted self-Ags and TG, pre- and post-KTx sera were analyzed by ELISA for Abs to Col-IV and FN. Abs to Col-II was used as a control. A large proportion of the biopsy proven TG patients 22/26 (84%) had Abs to both Col-IV and FN and the majority developed posttransplant (Table 2). Not all of the patients with Abs to self-Ags had detectable Abs to HLA or DSA. Further, we noted Abs to Col-IV and FN in 4/18 (22%) in the pretransplant sera indicating that chronic renal diseases may contribute in the development of Abs to selfAgs. None of the patients tested had Abs to Col-II both preand posttransplant period. One of the 10 stable patients had Abs to self-Ags both in pre- and posttransplant sera. One additional patient developed Abs during the posttransplant period. Both of them developed Abs to donor HLA (DSA) following transplantation. None of the sera had reactivity to Col-II (Table 2 and Figure 1). Development of Abs to kidneyrestricted self-Ags during the posttransplant period also Table 3: Correlation between post-Tx Abs to HLA and TG
TG Stable Normal
Total
Post-Tx Abs (þ)
Odds ratio
p-Value
26 10 26
16 (61.54%) 2 (20%) n/a
6.4
0.026
Abs, antibodies; TG, transplant glomerulopathy; Tx, transplantation.
American Journal of Transplantation 2014; 14: 685–693
significantly increased the chance of developing TG with an odds ratio of 22 (p-value ¼ 0.001) (Table 4). Results presented in Figure 2 demonstrate the correlation between the development of Abs to self-Ags and development of either DSA or HLA. Abs to HLA is not a prerequisite for the development of Abs to self-Ags since 6 out of the 22 TG patients with Abs to self-Ag did not have Abs to HLA or DSA. However, there is a higher tendency for the development of Abs to self-Ags in TG patients having Abs to HLA or DSA suggesting that one may predispose development of others (16 out of 22 developed both). These results indicate that development of Abs to kidneyrestricted self-Ags correlated with development of TG. Correlation between increase in the concentration of Abs to self-Ags and development of TG following KTx Serial pre- and posttransplant sera from four TG and two stable patients were analyzed for Abs and their concentration at the time TG was diagnosed and proven by biopsy. Results presented in Figure 3 suggest that the Abs to self-Ags may play a role in TG since the concentration of Abs went significantly higher at the time of TG diagnosis. Two of the stable patients did not demonstrate presence and increases in Abs to self-Ags. As the number of patients in this study was too few, statistical analysis was not performed. Abs to self-Ags are both IgG and IgM isotypes DSA(þ)/anti-self-Ags(þ) (n ¼ 12), and anti-HLA(þ)/anti-selfAgs(þ) (n ¼ 4) and DSA()/anti-HLA()/anti-self-Ags(þ) were analyzed for their isotype using ELISA. Results presented in Table 5 demonstrate that both isotypes are present including in those patients who did not have any detectable Abs to HLA or DSA. The presence of IgG in 8/14 for anti-Col-IV and 6/10 for anti-FN suggest ongoing immune responses to these antigens. Pathologic characteristics are similar in TG patients with Abs to self-Ags independent of DSA status Biopsy slides from 23 of the 26 TG patients were scored according to Banff criteria (14). Results presented in Table 6 and Figure 4 show that severity of glomerulitis, peritubular capillaritis, C4d deposition and chronic glomerulopathy were equivalent in patients with Abs to self-Ags in presence or absence of DSA suggesting that Abs to self-Ags alone can be associated with histologic evidence of both acute and chronic Ab-mediated injury. 687
Angaswamy et al
Figure 1: Presence of post-Tx Abs to self-Ags in patients with TG. Serum concentration of Abs to Col-IV (A), FN (B) and Col-II (C) in patients with TG (n ¼ 26), stable (n ¼ 10) and normal (n ¼ 33) cohorts. Data represented as mean SD; statistical significance ( ) ascertained if p < 0.05; n/s if p > 0.05 (not significant). Abs to Col-II considered as a negative control in our studies and has demonstrated no significant difference between the three cohorts. Abs, antibodies; Col-II/IV, Collagen-II/IV; FN, fibronectin; self-Ags, self-antigens; TG, transplant glomerulopathy; Tx, transplantation.
Increased frequency of self-Ag specific IFN-g and IL17 with concomitant decrease in IL-10 in KTx recipients diagnosed with TG The CD4þ T cells isolated from PBMCs from 26 TG, 10 stable KTx recipients and normal (n ¼ 14) were analyzed for self-Ags-specific immune responses by ELISPOT. As shown in Figure 5, patients with TG demonstrated increased frequencies of IFN-g and IL-17 against Col-IV, Figure 5B,C (IFN-g, TG: 416 91 spm, stable: 178 69 spm, p ¼ 0.006; IL-17, TG: 278 62 spm, stable: 116 34 spm, p ¼ 0.009) and FN, Figure 5E,F (IFN-g, TG: 438 79 spm, stable: 193 73 spm, p ¼ 0.02; IL-17, TG: 342 92 spm, stable: 143 56 spm, p ¼ 0.003). In contrast, TG patients demonstrated decreased frequencies of IL-10 secreting CD4þ T cells against Col-IV (Figure 5A,D) (TG: 83 37 spm, stable: 378 132 spm, p ¼ 0.008); and FN (TG: 103 46 spm, stable: 285 81 spm, p ¼ 0.001). Frequency of IFN-g, Il-10 and IL-17 secreting T cells against Col-II (Figure 5G–I) demonstrated no significant differences (TG vs. stable, p ¼ ns; TG vs. normal, p ¼ ns). These results indicate a loss of peripheral tolerance to self-Ags along with a concomitant up-regulation of autoimmune cellular immune responses to self-Ags in TG. Increased serum concentrations of IFN-g and IL-17 in TG patients Serum concentrations of cytokines were analyzed using Luminex and data presented in Figure 6 support the findings Table 4: Correlation between post-Tx Abs to self-Ags and TG
TG Stable Normal
Total
Post-Tx Abs (þ)
Odds ratio
p-Value
26 10 26
22 (84.62%) 2 (20%) n/a
22
0.001
Abs, antibodies; self-Ags, self-antigens; TG, transplant glomerulopathy; Tx, transplantation.
688
of ELISPOT demonstrating increases in the frequency of selfAg specific IFN-g and IL-17 with reduction in IL-10. TG patients demonstrated higher concentrations of cytokines IFN-g (TG: 867 241 pg/mL, stable: 407 178 pg/mL, p ¼ 0.02) and IL-17 (TG: 918 197 pg/mL, stable: 327 142 pg/mL, p ¼ 0.009) with marked reduction in IL-10 (TG: 184 78 pg/mL, stable: 668 184 pg/mL, p ¼ 0.001).
Discussion De novo development of Abs to mismatched donor HLA (DSA) have been associated with CAN (15,16) and it has been proposed that Abs reactive to glomerular endothelial cells play an important role in the development of CAN. However, not all KTx recipients diagnosed with CAN have DSA raising the possibility that immune responses against tissue-restricted self-Ags might play a role in its pathogenesis. Joosten et al (5) using a rat KTx model of chronic rejection and TG patients identified Abs reactive to GBM antigen, heparan sulfate proteoglycan (HSPG), perlecan and Abs reactive to GBM-HSPG agrin, respectively, and postulated that immune responses to perlecan and agrin may play a role in the pathogenesis of TG. It has been reported that about 15–20% of KTx recipients diagnosed with CAN have TG and TG increases the risk for CAN (17,18). Therefore, we determined the immune responses to kidney-restricted self-Ags by analyzing sera from TG (with characteristic duplication of the GBM) for Abs to purified Col-IV and FN. As reported (8,9,19,20), about 61% of patients developed de novo Abs to HLA, of which 75% were DSA. Majority of the DSA (67%) were to HLA class II; however, 4/12 patients (33%) demonstrated HLA class I specificity alone indicating that immune responses to both HLA class I and II can be pathogenic for TG (Table 2). The development of Abs to Col-IV and FN were not always associated with Abs to HLA. Six of the 22 TG did not have any Abs to HLA or DSA. However, in 16 out of 26 patients, American Journal of Transplantation 2014; 14: 685–693
Autoimmunity in TG
Figure 2: Development of Abs to Col-IV and FN did not correlate with DSA. Serum concentration of Abs to Col-IV (A) and FN (B) in patients with DSA(þ) (n ¼ 12), DSA()/HLA() (n ¼ 6) and HLA(þ) (n ¼ 4) cohorts. Data represented as mean SD. As noted, patients with DSA(þ) and DSA()/HLA() cohorts did not reach statistical significance; n/s, not significant; p < 0.05; p < 0.01. Abs, antibodies; Col-IV, Collagen-IV; DSA, donor-specific antibodies; FN, fibronectin.
Abs to both HLA or DSA and Abs to self-Ags were detected (Figure 2). This result supports the concept that ongoing immune responses to self-Ags can be pathogenic for the development of TG. We propose that initial binding by Abs of IgG isotype will result in GBM duplication and this process exposes new antigenic determinants which then may lead to Abs of IgM isotype followed by IgG. This hypothesis needs to be tested. In our study, the temporal analysis of serum samples significantly varied between TG patients (4.4 3 years) and stable recipients (1.0 1.9 years), a future prospective monitoring of CoI-V and FN Abs and their isotypes are needed to define time frame of appearance in order to correlate it with TG.
To define the mechanisms for development of immune responses to kidney-restricted self-Ags, the frequency of T cells specific to Col-IV and FN and T cells secreting IFN-g, IL17 and IL-10 were determined using ELISPOT. There is significant activation of Col-IV and FN-specific T cells secreting both IFN-g and IL-17 in TG but not in stable patients (Figure 5). Significant drop in self-Ag specific IL-10 secreting cells in TG but not in stable KTx recipients was also noted. A parallel increase in circulating IFN-g, IL-17 with reduction in IL-10 was also seen in sera from TG (Figure 6). There is increasing evidence that IL-17 by itself can be profibrotic (21) and could potentially induce development of fibrosis in CAN.
Figure 3: Abs to Col-IV and FN increase following KTx in TG patients. Serial monitoring of Abs to Col-IV (A) and FN (B) in TG with pre-Tx Abs to self-Ags (n ¼ 4, solid line) and stable (n ¼ 2, dotted line) at three time points: pre-Tx, post-Tx and at time of clinical diagnosis of TG (or time-matched sample stable cohort). As noted, there is a serial increase in the Abs to Col-IV and FN in patients who had pre-Tx Abs to selfAgs and eventually developed TG. As noted in Table 1, due to widespread temporal distribution of the analysis of the serum samples (TG patients—4.4 3.0 years vs. stable—1.0 1.9 years) the x-axis is used only for depiction and not exact time sequence. Abs, antibodies; ColIV, Collagen-IV; FN, fibronectin; KTx, kidney transplantation; self-Ags, self-antigens; TG, transplant glomerulopathy.
American Journal of Transplantation 2014; 14: 685–693
689
Angaswamy et al Table 5: Abs to Col-IV and FN were both IgM and IgG isotypes
DSA (þ), n ¼ 12 HLA(þ), n ¼ 4 DSA()/HLA(), n ¼ 6
Anti-Col-IV
Anti-FN
IgG (7/12) IgM (12/12) Both (7/12) IgG (1/4) IgM (3/4) Both (1/4) IgG (4/6) IgM (4/6) Both (4/6)
IgG (10/12) IgM (12/12) Both (10/12) IgG (2/4) IgM (2/4) Both (2/4) IgG (3/6) IgM (4/6) Both (3/6)
Abs, antibodies; Col-IV, Collagen-IV; DSA, donor-specific antibodies; FN, fibronectin.
Since both cellular and humoral immune responses to both kidney-restricted self-Ags (Col-IV and FN) seem to go together, we propose that onset of immune response either to a kidney-restricted self-Ag or HLA initiates spreading of immune responses to various self-Ags as well as HLA molecules expressed on the kidney. These results suggest strongly that there is cross talk between immune responses to both allo-MHC molecules and tissuerestricted self-Ags and once either of them developed it can spread to other leading to the pathogenesis of TG. Further, selective expansion of IL-17 secreting T cells along with a decrease in the loss of peripheral tolerance induced by diminished IL-10 secreting T cells can result in immune Table 6: Banff scoring for glomerulitis, chronic glomerulopathy (CG), peritubular capillaritis (PTC) and C4d staining in TG patients with FN and Col-IV Abs stratified by donor-specific antibody (DSA) status DSAþ, N ¼ 14 Glomerulitis score, n (%) 0 1 2 3 CG score, n (%) 0 1 2 3 PTC score, n (%) 0 1 2 3 C4d, n (%) Positive Negative
DSA, N¼6
p-Value 1.0
3 4 5 2
(21.4) (28.6) (35.7) (14.3)
2 1 2 1
(33.3) (16.7) (33.3) (16.7)
5 4 2 3
(35.7) (28.6) (14.3) (21.4)
1 3 1 1
(16.7) (50.0) (16.7) (16.7)
0.9
0.6 4 (28.6) 0 8 (57.1) 2 (14.3)
3 (50.0) 0 3 (50.0) 0
4 (28.6) 10 (71.4)
3 (50.0) 3 (50.0)
0.6
Abs, antibodies; Col-IV, Collagen-IV; FN, fibronectin; TG, transplant glomerulopathy. Nineteen subjects had positive Col-IV and FN Abs, one subject was positive for Col-IV Abs only. Of the 14 subjects with positive DSAs, 6 were positive for class I and II, 3 for class I only and 5 for class II only. p-Values were calculated using Fisher’s exact test.
690
Figure 4: Histology of TG. (A) Representative image of a glomerulus from a patient that was Col-IV and FN Abþ and DSA. Features typical of TG are seen including splitting of the capillary loops (arrows) and glomerulitis ( ) (Periodic Acid Schiff stain, 400). (B) Representative image of a glomerulus from a patient that was Col-IV and FN Abþ and DSAþ. Focal capillary loop duplication (arrow) and glomerulitis ( ) are seen, similar to the findings depicted in A (Periodic Acid Schiff, 400). Ab, antibody; Col-IV, Collagen-IV; DSA, donor-specific antibody; FN, fibronectin; TG, transplant glomerulopathy.
responses to tissue-restricted self-Ags which we propose to play a vital role in the pathogenesis of TG. In conclusion, our results along with other published findings (16,22–24) support Ab-mediated mechanism for the development of TG following KTx. As this study is a retrospective analysis of patients with TG, it has its limitations, including small number, lack of serial samples for analysis and inability to perform multivariate analysis. However, it is clear that not only Abs to donor HLA but also Abs to tissue-restricted self-Ags, Col-IV, FN, agrin, AT1R, etc., are developed in patients with TG. We propose that serial monitoring for Ab development either to donor HLA or to kidney self-Ags during posttransplant may identify American Journal of Transplantation 2014; 14: 685–693
Autoimmunity in TG
Figure 5: Increased frequency of self-Ag specific Th1 and Th17 response in patients with TG. Frequency of CD4 þ T cells with specific reactivity to Col-IV (A–C), FN (D–F) and Col-II (G–I) in patients with TG (n ¼ 26), stable (n ¼ 10) and normal (n ¼ 33) cohorts as determined by ELISPOT. A and D represent CD4 þ T cells that induce IL-10 secretion upon specific reactivity to Col-IV and FN, respectively. B and E represent CD4 þ T cells that induce IFN-g secretion upon specific reactivity to Col-IV and FN, respectively. C and F represent CD4 þ T cells that induce IL-17 secretion upon specific reactivity to Col-IV and FN, respectively. Col-II (G–I) is used as negative control in this study. Data represented as mean SD; statistical significance ( ) ascertained if p < 0.05. n/s, not significant. Col-II/IV, Collagen-II/IV; FN, fibronectin; IFN-g, interferon gamma; self-Ags, self-antigens; TG, transplant glomerulopathy.
American Journal of Transplantation 2014; 14: 685–693
691
Angaswamy et al
Figure 6: Increased serum concentration of pro-inflammatory cytokines in patients with TG. Serum concentration of antiinflammatory cytokine, IL-10 (A) and pro-inflammatory cytokines, IFN-g (B) and IL-17 (C) in patients with TG (n ¼ 26), stable (n ¼ 10) and normal (n ¼ 33) cohorts as determined by Luminex. Data represented as mean SD; statistical significance ( ) ascertained if p < 0.05. IFN-g, interferon gamma; TG, transplant glomerulopathy.
patients at risk for developing TG. Since this report, as well as others, implicates Abs as an important initiator in the pathogenesis of TG and CAN, we propose that Ab directed therapy following early detection of de novo developed Abs during posttransplant may be beneficial in preventing TG. This approach has been successfully employed following human lung transplantation toward preventing the onset of chronic rejection following human lung transplantation (25,26). Since our results suggest an important role for IL-17 in the development of immune responses to selfAgs, it is likely that neutralization of IL-17 and IL-6 may represent another avenue to explore as possible targets to prevent TG following human KTx.
Acknowledgments This work was supported by Barnes-Jewish Foundation (TM). The authors would like to thank Billie Glasscock for her assistance in submitting this manuscript.
Disclosure The authors of this manuscript have no conflicts of interest to disclose as described by the American Journal of Transplantation.
References 1. Husain S, Sis B. Advances in the understanding of transplant glomerulopathy. Am J Kidney Dis 2013; 62: 352–363. 2. Tiriveedhi V, Sarma N, Mohanakumar T. An important role for autoimmunity in the immunopathogenesis of chronic allograft rejection. Int J Immunogenet 2012; 39: 373–380. 3. Dragun D, Muller DN, Brasen JH, et al. Angiotensin II type 1receptor activating antibodies in renal-allograft rejection. N Engl J Med 2005; 352: 558–569.
692
4. Giral M, Foucher Y, Dufay A, et al. Pretransplant sensitization against angiotensin II type 1 receptor is a risk factor for acute rejection and graft loss. Am J Transplant 2013; 13: 2567–2576. 5. Joosten SA, van Dixhoorn MG, Borrias MC, et al. Antibody response against perlecan and collagen types IV and VI in chronic renal allograft rejection in the rat. Am J Pathol 2002; 160: 1301– 1310. 6. Strom EH, Banfi G, Krapf R, et al. Glomerulopathy associated with predominant fibronectin deposits: A newly recognized hereditary disease. Kidney Int 1995; 48: 163–170. 7. Colvin RB. Antibody-mediated renal allograft rejection: Diagnosis and pathogenesis. J Am Soc Nephrol 2007; 18: 1046–1056. 8. Gloor JM, Sethi S, Stegall MD, et al. Transplant glomerulopathy: Subclinical incidence and association with alloantibody. Am J Transplant 2007; 7: 2124–2132. 9. Sis B, Campbell PM, Mueller T, et al. Transplant glomerulopathy, late antibody-mediated rejection and the ABCD tetrad in kidney allograft biopsies for cause. Am J Transplant 2007; 7: 1743–1752. 10. Ivanyi B. Transplant capillaropathy and transplant glomerulopathy: Ultrastructural markers of chronic renal allograft rejection. Nephrol Dial Transplant 2003; 18: 655–660. 11. Morris GP, Phelan DL, Jendrisak MD, Mohanakumar T. Virtual crossmatch by identification of donor-specific anti-human leukocyte antigen antibodies by solid-phase immunoassay: A 30-month analysis in living donor kidney transplantation. Hum Immunol 2010; 71: 268–273. 12. Borg BB, Seetharam A, Subramanian V, et al. Immune response to extracellular matrix collagen in chronic hepatitis C-induced liver fibrosis. Liver Transplant 2011; 17: 814–823. 13. Saini D, Weber J, Ramachandran S, et al. Alloimmunity-induced autoimmunity as a potential mechanism in the pathogenesis of chronic rejection of human lung allografts. J Heart Lung Transplant 2011; 30: 624–631. 14. Veronese FV, Manfro RC, Roman FR, et al. Reproducibility of the Banff classification in subclinical kidney transplant rejection. Clin Transplant 2005; 19: 518–521. 15. Li C, Yang CW. The pathogenesis and treatment of chronic allograft nephropathy. Nat Rev Nephrol 2009; 5: 513–519. 16. Gaston RS, Cecka JM, Kasiske BL, et al. Evidence for antibodymediated injury as a major determinant of late kidney allograft failure. Transplantation 2010; 90: 68–74.
American Journal of Transplantation 2014; 14: 685–693
Autoimmunity in TG 17. Shu KH, Lu YS, Chang CH, et al. Transplant glomerulopathy—A clinicopathological study. Transplant Proc 1996; 28: 1527–1528. 18. Kupin W, Nakhleh R, Lee M, et al. Separate risk factors for the development of transplant glomerulopathy vs chronic tubulointerstitial rejection. Transplant Proc 1997; 29: 245–246. 19. Mao Q, Terasaki PI, Cai J, et al. Extremely high association between appearance of HLA antibodies and failure of kidney grafts in a five-year longitudinal study. Am J Transplant 2007; 7: 864–871. 20. Campos EF, Tedesco-Silva H, Machado PG, Franco M, MedinaPestana JO, Gerbase-DeLima M. Post-transplant anti-HLA class II antibodies as risk factor for late kidney allograft failure. Am J Transplant 2006; 6: 2316–2320. 21. Al-Muhsen S, Letuve S, Vazquez-Tello A, et al. Th17 cytokines induce pro-fibrotic cytokines release from human eosinophils. Respir Res 2013; 14: 34–44.
American Journal of Transplantation 2014; 14: 685–693
22. Sellares J, de Freitas DG, Mengel M, et al. Understanding the causes of kidney transplant failure: The dominant role of antibodymediated rejection and nonadherence. Am J Transplant 2012; 12: 388–399. 23. Dinavahi R, George A, Tretin A, et al. Antibodies reactive to nonHLA antigens in transplant glomerulopathy. J Am Soc Nephrol 2011; 22: 1168–1178. 24. Cosio FG, Gloor JM, Sethi S, Stegall MD. Transplant glomerulopathy. Am J Transplant 2008; 8: 492–496. 25. Hachem RR, Yusen RD, Meyers BF, et al. Anti-human leukocyte antigen antibodies and preemptive antibody-directed therapy after lung transplantation. J Heart Lung Transplant 2010; 29: 973–980. 26. Hachem RR, Tiriveedhi V, Patterson GA, Aloush A, Trulock EP, Mohanakumar T. Antibodies to K-alpha 1 tubulin and Collagen V are associated with chronic rejection after lung transplantation. Am J Transplant 2012; 12: 2164–2171.
693
