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Keywords:

  • CTL;
  • kidney rejection;
  • transplantation;
  • tubulitis

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

The usual phenotype of clinical kidney allograft rejection is infiltration by lymphocytes and macrophages and evolution of histologic Banff lesions, particularly tubulitis, which indicate parenchymal injury. Using Affymetrix microarrays, we evaluated the relationship between the evolution of pathologic lesions and the transcriptome. We studied CBA/J into C57Bl/6 mouse kidney allografts in which one host kidney is left in place to permit observation of lesion development. Histology was dominated by early infiltration by mononuclear cells from day 3 and slower evolution of tubulitis after day 7. We defined a set of cytotoxic T lymphocyte-associated transcripts (CATs) on the basis of expression in purified cytotoxic T lymphocytes (CTL) and in a mixed lymphocyte culture, and absence in normal kidney. CATs were detectable by day 3 and highly expressed by day 5 in rejecting kidneys, with a median signal 14% of that in CTL, compared to 4% in isografts and normal kidneys, and persisted through day 42. Lack of mature B cells had little effect on CAT expression, confirming that CATs reflect T-cell-mediated rejection. Expression of CATs was established before diagnostic lesions and remained remarkably consistent through day 42 despite massive alterations in the pathology, and probably reflects T cells recruited to the graft.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Alloantigens elicit an immune response that generates effector T lymphocytes (cytotoxic T lymphocytes, CTL) and alloantibody production, probably in the secondary lymphoid organs (1). Alloantibody responses contribute to the pathology of rejecting kidney transplants under some circumstances, typically damaging the microcirculation (2). However, human kidney rejection is usually T-cell mediated, characterized by interstitial infiltration by T cells (predominantly CD8+) and macrophages (3), resulting in injury to the tubular epithelium, as manifested by the principal diagnostic lesion, tubulitis (4). To explore these lesions, we adapted a mouse kidney transplant model by leaving one host kidney in place to prevent uremic death, permitting us to study the evolution of the histology. This model develops the same lesions as human allografts and simulates human clinical transplant practice, where rejection is defined by histologic lesions and uremia is prevented by dialysis. The tubulitis and interstitial infiltrate are T-cell-dependent (5,6) and associated with expression of perforin (Prf1) and granzymes A and B (GzmA, GzmB) (7,8). However, these cytotoxic molecules are not necessary: rejection lesions develop in mice lacking Prf1 or GzmA and GzmB (6), and may be mediated by CTL recruiting and activating macrophages, as in a delayed-type hypersensitivity (9–11). B cells and plasma cells appear late in smaller numbers (12). B-cell infiltration does not imply alloantibody-mediated rejection (2): human kidney allografts undergoing antibody-mediated rejection lack B cell/plasma cell infiltrates (13), presumably because the alloantibody is produced in secondary lymphoid organs.

An emerging tool for exploring the relationship between molecular events and pathologic lesions is assessment of the transcriptome using high-density microarrays (12,14). Biopsies from rejecting human kidney transplants show complex patterns of transcript expression (12), with potential to distinguish rejection from other processes (15). Rejecting heart and kidney allografts (14,16) show striking induction of IFN-γ-inducible transcripts and features of ischemia/hypoxia (17,18), innate immunity (19,20), cold injury (21) and fibrosis (22). The present experiments examined the relationship between the transcriptome changes and the diagnostic Banff lesions that define clinical rejection (4). We concentrated, in particular, on the contribution of cytotoxic T lymphocyte-associated transcripts (CATs) to the transcriptome of rejecting kidney in relationship to the Banff lesions of interstitial infiltration and tubulitis.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Mice

Male CBA/J (CBA), C57Bl/6 (B6), B6.129P2-Igh-Jtm1Cgn (Igh-j) and B6.129S2-Igh-6tm1,Cgn (Igh-6) mice were obtained from Jackson Laboratory (Bar Harbor, ME) and were maintained in the Health Sciences Laboratory Animal Services at the University of Alberta. All maintenance and experiments conformed to approved animal care protocols. CBA (H-2K, I-Ak) into C57Bl/6 (B6; H-2KbDb, I-Ab) mice strain combinations were studied across full major histocompatibility complex (MHC) and non-MHC disparities. To ensure robust findings, we used two types of IghKO hosts (Igh-j and Igh-6), which have similar phenotypes (5).

Renal transplantation

Renal transplantation was performed as a non-life-supporting transplant model as previously described (23). Recovered mice were killed at days 3, 4, 5, 7, 14, 21 or 42 post-transplant. Kidneys were removed, snap frozen in liquid nitrogen and stored at −70°C. No mice received immunosuppressive therapy. Kidneys with technical complications or infection at the time of harvesting were removed from the study.

Mixed leukocyte reaction (MLR)

CTL effectors were generated by coculturing C57BL/6l responder splenocytes with mitomycin C-treated (5 μg/mL, Sigma Chemicals, St. Louis, MO) CBA splenocytes in complete RPMI 1640 medium (10% FCS, 1% antibiotic-antimycotic (Life Technologies, Grand Island, NY), 1% non-essential amino acids, 1% sodium pyruvate (Flow Laboratories, McLean, VA) and 50 μM β-ME) at 3×106 cells/mL. Cultures were kept at 37°C, 5% CO2 in 25 cm2 cell culture flasks standing upright for 4 days. Cytolytic activity was confirmed by 51Cr release assay.

CTL culture

Purified CTL were propagated from a frozen stock of a long-term culture of C57/B6 anti C3H. C57Bl/6 splenocytes were cocultured for 3 days at a ratio of 1:1 with irradiated (2500 rads) C3H splenocytes (1×106 cells/mL) in RPMI 1640 medium (the same composition as for the d4MLR plus 80 μg/mL human IL2). CTL were purified by Ficoll gradient and cultured for 4 days at 5×105 cells/mL. C57/B6 splenocytes were restimulated at a ratio of 1:14 with irradiated C3H splenocytes for 3 days. After Ficoll purification and cultivation for 4 days, RNA was extracted. Cytolytic activity was confirmed by 51Cr release assay.

RNA preparation

Total RNA was extracted from individual kidneys by the guanidinium-caesium chloride method (transplants) or by Trizol extraction (d4MLR and CTL cultures). RNA yields were measured by UV absorbance. Quality was assessed by absorbance ratio, agarose gel electrophoresis, and in select samples by Affymetrix T3 Test arrays (Affymetrix, Santa Clara, CA). Equal amounts of RNA from 3 mice (20–25 μg each) were pooled and purified using the RNeasy Mini Kit (Quiagen, Ont. Canada). dsDNA and cRNA synthesis, hybridization to MOE430A or MOE430 2.0 oligonucleotide arrays (Affymetrix), washing and staining were carried out according to the Affymetrix Technical Manual (http://www.affymetrix.com).

Real-time RT-PCR

Expression of selected transcripts was confirmed by TaqMan real-time RT-PCR. Probe/primer combinations were designed using Primer Express software version 1.5 or purchased as assay on demand (PE Applied Biosystems, Foster City, CA). RNA was transcribed using M-MLV reverse transcriptase and random primers. cDNA was amplified in a multiplex system using murine hypoxanthine phosphoribosyltransferase cDNA as the control. Quantification was performed utilizing the ABI prism 7700 Sequence Detection System (PE Applied Biosystems) (24). Fold changes were determined using the ΔCt or ΔΔCt methods as described by the manufacturer.

Sample designation and analysis

Normal control kidneys were from CBA mice (NCBA). CBA allografts rejecting in wild-type hosts (B6) at days 3, 4, 5, 7, 14, 21 and 42 post-transplant were designated WTD3, WTD4, WTD5, WTD7, WTD14, WTD21 and WTD42, respectively. CBA isografts in CBA hosts were designated IsoD5, IsoD7 and IsoD21. CBA allografts rejecting in mature B-cell-deficient B6 hosts at days 7 (Igh-6) and 21 (Igh-6 or Igh-j) were designated IghKOD7 and IghKOD21, respectively. Mixed leukocyte reaction, day 4, and purified CTL were designated d4MLR and CTL, respectively. Two biological replicates (each consisting of RNA pooled from 3 mice) were tested for WTD3, WTD4, WTD7, WTD14, WTD21, WTD42, IsoD7, IghKOD7 and host kidneys at day 5. Biological triplicates were analyzed in NCBA, WTD5, IghKOD21 (2 arrays for Igh-6 hosts, 1 array for Igh-j hosts). A single analysis was done in IsoD5, IsoD21, d4MLR and CTL. Samples were hybridized to Affymetrix MOE430A arrays; WTD3, WTD4 and host kidneys at day 5 were analyzed on MOE430 2.0 arrays. Microarray data are available in supplementary Table S1 at http://transplants.med.uaberta.ca/.

Microarray Suite Expression Analysis 5.0 software (Affymetrix) was used to calculate absolute signal strength and to flag transcripts as present or absent, based on software default conditions. Total fluorescence for each array was globally scaled to a target value of 500. Using GeneSpring™ software (Version 6.1, Silicon Genetics, CA), we adjusted intensity values below 20 to a value of 20 and performed a per chip normalization to the 50th percentile and a per gene normalization using NCBA or CTL as control samples. Replicate samples were expressed as mean normalized value. For unsupervised hierarchical cluster analysis, similarity measurements were based on distance and visualized by a tree diagram (25).

CTL-associated transcripts (CATs) were defined as transcripts with high expression in d4MLR and CTL (≥5-fold vs. NCBA, significant by ANOVA), and absent in NCBA. CATs were analyzed using a K-means cluster algorithm based on expression data normalized to CTL.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Pathological lesions in rejecting kidneys

As previously described (5,6), isografts at days 5 (Figure 1, panel A), 7 and 21 post-transplant appeared normal with little inflammation or tubular necrosis. Allografts showed focal periarterial mononuclear infiltrates at days 3 and 4 (Panel B) and interstitial mononuclear infiltration by day 5, which increased at day 7 and persisted through day 42 (panels C, D and E respectively). Tubulitis was absent at days 3, 4 and 5, mild at day 7, and severe at days 14, 21 and 42. The late grafts at days 14, 21 and 42 showed severe tubular damage with patchy cortical necrosis of 30% of the cortex by day 42 (Panel F). Allografts from days 14 through 42 showed severe loss of tubule diameter, and of E-cadherin and Ksp-cadherin, reflecting parenchymal injury (Fairhead T, Einecke G, Turner P, Zhu L-F, Hadley GA, Famulski KS, Halloran PF: Tubu lesions of kidney transplant rejection are associated with loss of cadherins and do not require CD103. Manuscript in preparation.). The day 42 kidneys resembled some end-stage rejecting human kidneys, which may remain largely viable despite severe tubulitis, arteritis and patchy necrosis. By immunostaining, the infiltrate in kidney allografts at days 5, 7 and 21 contained 40–60% CD3+ T cells. At day 21, T cells were present in the interstitium and tubules, with CD3/CD8+ cells exceeding CD3/CD4+ cells by 8 to 1 (34 ± 4 vs. 4 ± 2 cells per 10 high power field, n = 9). The infiltrate was 35–50% CD68+ (macrophages), with late appearance of 5% CD19+ B cells at day 21. Hosts deficient in mature B cells (Igh6KO or IghJKO) showed similar infiltrate and tubulitis but less necrosis and hemorrhage at day 21 (5), and 19% lower kidney weight (260 ± 58 mg, n = 8 vs. 319 ± 70 mg, n = 6 in wild-type hosts). Detailed histology is shown in supplementary table S1 at http://transplants.med.ualberta.ca/.

image

Figure 1. Histopathology of rejecting mouse allografts. All panels show PAS staining (magnification 40×). Panel A: isograft (CBA into CBA) at day 5 with normal histology. Panel B: rejecting kidney allograft (CBA into B6) at day 3 showing focal periarterial mononuclear infiltrate. Panel C: rejecting kidney allograft (CBA into B6) at day 5 showing periarterial and beginning interstitial mononuclear infiltration. Panel D: rejecting kidney allograft at D7 (CBA into B6) with mononuclear interstitial infiltration and mild tubulitis. Panel E: kidney transplant (CBA into B6) at day 21 showing heavy tubulitis. Panel F: Allografts show severe tubular damage, with patchy cortical necrosis at day 42.

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These kidneys manifested functional impairment. Removing the host kidney in selected mice resulted in elevated serum creatinine at day 7 (data not shown). Moreover, tubulitis per se correlates with loss of function (26), and severe deterioration of the renal epithelium in the tubulitis lesions at days 14, 21 and 42 in our mouse model indicates severe loss of function.

Affymetrix microarray analysis and validation

For each experimental group, we performed biological replicate microarrays, each representing a different pool of three kidneys. Global transcript expression correlated well in biological replicates of each experimental group (NCBA: r= 0.96; Iso D7: r= 0.96; WT D5: r= 0.92; WT D7: r= 0.96; WT D14: r= 0.98; WT D21: r= 0.86; WT D42: r= 0.90). The results for WT D5 transplants are shown in Figure 2. The correlation between the d4MLR and purified CTL was r= 0.82. Microarray results were compared with real-time RT-PCR for a set of 11 transcripts that represent cytotoxic T-cell and B-cell markers (Figure 3). The patterns of transcript expression were similar for microarray and RT-PCR (r= 0.87), but fold increase in RT-PCR results was approximately 10 to 30 times higher compared to microarrays, presumably reflecting the lower background and greater sensitivity of RT-PCR.

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Figure 2. Reproducibility of transcript expression analysis. Transcript expression values (n = 22 690) from two biological replicates of pools of three kidneys rejecting in wild type hosts at day 5 (WTD5) are shown (r= 0.92).

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image

Figure 3. Correlation of transcript expression analysis between microarrays and real-time RT-PCR. The plots show the time course of transcript expression in kidneys rejecting at days 5, 7 and 21 post-transplant for 11 transcripts: left panel: - RT-PCR data; right panel - microarray data.

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Hierarchical clustering of the global transcript expression in rejecting kidneys, isografts, CTL and d4 MLR

We used unsupervised hierarchical cluster analysis to compare transcript expression between control kidneys, isografts, allografts rejecting in WT and IghKO hosts, d4MLR and CTL. The transcriptomes clustered into three groups (Figure 4): normal kidneys and isografts, with Iso D21 being more similar to NCBA than Iso D5 or Iso D7; allografts, with WT D5, WT D7, IghKO D7 and IghKO D21 in one subcluster and WT D14, WT D21 and WT D42 in a second sub-cluster; and d4MLR and CTL formed a third cluster.

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Figure 4. Unsupervised hierarchical clustering of experimental groups. Unsupervised clustering of all transcripts, based on distance, was performed on isografts (ISO), allografts rejecting in wild type hosts (WT) and B-cell deficient hosts (IghKO) and lymphocyte cultures (MLR = mixed lymphocyte culture; CTL = purified CTL from a long-term culture).

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CD antigen transcript expression

We analyzed expression of CD transcripts as a reflection of cellular infiltration (Table 1). The 18 CD transcripts in normal kidney may reflect immature dendritic cells in the interstitium (27). T-cell-specific transcripts were absent, making it unlikely that the transcripts detected reflect peripheral blood. Expression of CD transcripts was similar between CTL and d4MLR, with both reflecting effector CD8 cells, with high expression of CD44 (43 and 25-fold vs. NCBA in CTL and MLR, respectively) and no L-selectin. D4MLR contained B-cell-specific transcripts CD79a and CD79b.

Table 1.  CD antigen transcripts in isografts and in allografts transplanted into wildtype hosts and B cell deficient hosts. Transcripts had to be at least two fold increased compared to NCBA and flagged as present in at least one of the samples to be considered. The table shows the signal strength for controls and fold changes for the transplants. (−) indicates that a given transcript was not increased; bolded signal values indicate that a transcript was classified as present. In case of multiple probe sets querying the same gene, data obtained from probe sets with suffixes _s_at and _x_at were not considered, and a probe set displaying the most robust signal was selected.
SymbolNCBA Signal NCBAIsografts Fold ChangeWT Allografts Fold ChangeIghKO Allografts Fold ChangeLymphocytes Fold Change
D5D7D21D5D7D14D21D42D7D21CTLMLR
Cd1d148-----5.23.14.12.62.79.45.3
Cd215---10.413.615.812.79.310.29.9269.5166.0
Cd2f10112---4.25.510.87.97.43.94.8--
Cd3d8---42.159.060.848.931.866.938.2812.4910.7
Cd3e60---9.316.311.216.16.319.913.864.881.1
Cd3g43---22.935.941.442.423.437.628.0252.4174.9
Cd3z39---6.99.18.18.64.39.56.654.864.5
Cd5112---2.94.12.43.2-3.82.714.617.1
Cd654---6.86.6-7.2-7.26.918.416.2
Cd724---------9.6--
Cd8a97---9.318.817.611.68.827.910.939.732.8
Cd8b22---26.939.650.340.024.847.129.1251.6111.8
Cd14424-3.6-7.32.85.44.24.65.53.1--
Cd22153-----2.43.13.2-2.6-14.4
Cd2841---8.68.110.75.26.17.95.176.245.8
Cd38343-----3.12.32.93.12.0-3.2
Cd44652.13.7-9.814.329.628.925.015.616.843.025.6
CD45187-2.5-20.121.328.024.316.723.818.988.272.9
Cd47990---3.22.94.03.72.93.83.113.39.5
Cd4820-4.0-23.629.645.831.233.532.122.3269.663.1
Cd52287-2.1-15.119.130.619.819.721.615.371.058.8
Cd53134-2.8-11.417.322.618.219.618.013.073.971.7
Cd68161---4.86.713.410.618.39.88.92.52.7
Cd7241---7.813.427.714.920.29.414.413.630.9
Cd79a85-----2.02.92.7---35.6
Cd79b67-----3.03.83.9---35.6
Cd8054----2.03.02.32.4--6.33.1
Cd8381---3.84.79.312.212.74.19.2-35.6
Cd8471---2.93.811.610.512.76.98.920.812.3
Cd8682---2.93.48.45.86.83.64.22.75.9
CD90132---9.210.67.611.25.712.011.671.991.6
Cd97272---2.12.82.83.63.12.82.914.28.9
CD138247-----3.33.63.83.12.8--

Thirty-three CD transcripts were increased at least 2-fold in allografts compared to NCBA kidney. Twenty-one had high expression in d4MLR and CTL (> 5-fold compared to NCBA). CD2f10, CD14 and CD68 macrophage-associated transcripts (28) were increased in rejecting allografts but absent or low in d4MLR or CTL, suggesting that they represent infiltrating activated macrophages, which are not represented in d4MLR or CTL. The B-cell-specific transcripts CD79a and CD79b appeared at days 14, 21, and 42 in WT but not in IghKO hosts, consistent with antibody-producing cells. The analysis of CD transcripts is consistent with an early and sustained CTL/macrophage infiltrate in WT and IghKO hosts, and with late B-cell infiltration in WT hosts. CD4 transcripts were flagged absent in the allografts, indicating a minor presence of CD4 cells in rejecting kidneys, as opposed to d4MLR, which contains both naïve and effector CD4 cells (60-fold increase vs. NCBA).

CATs in rejecting mouse kidney allografts

CATs (287 transcripts) were defined by high expression in both CTL and in d4MLR but absent in normal kidney. CAT expression was lower in d4MLR than in CTL (median 87%, expressed as a percentage of CTL signal). In NCBA, expression of CATs was called ‘absent’ by Affymetrix definition. CAT signal was unchanged in isografts (Iso D5, 1.0; Iso D7, 1.1; Iso D21, 1.0-fold vs. NCBA). In host kidneys of transplanted animals at day 5, CAT signals were not detectable (median 1.1 fold vs. NCBA), showing that circulating blood does not contribute to CAT expression in rejecting transplants.

In contrast to NCBA and isografts, CATs were strongly expressed in allografts. CATs were detectable by days 3 and 4, with a median 2.2-fold change in WT D3 and 3.3-fold change in WT D4 compared to NCBA. Expression of CATs was well established by day 5 with a median 3.8-fold increase compared to NCBA (Figure 5a). After day 5, expression of CATs was stable and persisted throughout the rejection process (D7, 4.3-fold; D14, 4.8-fold; D21, 4.6-fold; D42, 3.3-fold compared to the signal in NCBA) (Figure 5b).

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Figure 5. Expression of CTL associated transcripts (CATs) in isografts and WT allografts. CATs were defined as absent in normal kidney, but highly expressed in lymphocyte cultures. (A) CATs were readily detected by day 3 although at a lower level than at day 5.

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Figure 5. (B) Expression of CATs was low in isografts but high in rejecting kidneys at day 5 and persisted throughout the rejection process.

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The level of CAT expression gives us an indication of the T-cell burden in the graft. However, the quantification of T-cell infiltration based on fold changes over a low background signal of CATs in NCBA may not be accurate. We therefore took a different approach: as CAT expression by definition was very high in CTL, the signal in this sample is much more reliable than the low background in NCBA. Given that the RNA from purified CTL is not diluted by RNA from other cell populations, the signal in this sample should exclusively represent transcript expression in CTL. Therefore, we chose to set the signal in CTL as 100% and quantified CAT expression in the rejecting transplants relative to the expression in CTL as an estimate for the relative amount of lymphocyte RNA in rejecting transplants.

The background signal of CATs in NCBA was median 4% of that in CTL and unchanged in isografts (D5, 4%, D7, 5%, D21, 4% of expression in CTL). At day 5 post-transplant, the signal for CATs was median 14% of that in CTL. After day 5, CAT expression remained very stable (D7, 16%; D14, 16%; D21, 16%; D42, 12% expressed as median% of expression in CTL). When we diluted RNA from d4MLR with kidney RNA in a ratio 1:4, the resulting signal was similar to that in all rejecting kidneys (median 15% of expression in CTL) and adequately reflected the dilution (median 20% of the d4MLR). This suggests that the RNA from lymphocytes infiltrating the kidney is diluted about 7-fold compared to CTL.

We analyzed the consistency of expression of individual CATs in all conditions by a nonparametric regression test. The CAT signal correlated well in all transplants, MLR and CTL, indicating robust maintenance of CAT expression in vivo and in vitro. The d4MLR correlated with diluted MLR (r= 0.91), despite 80% decrease in signal, and with the CTL (r= 0.81; p < 0.001). In rejecting transplants in WT hosts, the CAT signals correlated among all days (day 5 throughout day 42; r= 0.90–0.96; p < 0.001), indicating consistency of CAT expression over many days in all allografts. The CATs in the d4MLR correlated with the transplants at all days (r= 0.70–0.78; p < 0.001), as did the expression in CTL (r= 0.66–0.74).

K-means cluster analysis of CATs, based on their expression level in WT allografts relative to purified CTL, resulted in five clusters (Figure 6). Expression values for each cluster are given in supplementary tables S2–6. Cluster 1 included 140 transcripts (e.g. CD2, CD3g, GzmB, Tcrb, mes), and displayed low expression in allografts relative to CTL but was stable throughout the time course, with median 3.6-fold increase versus NCBA at day 5. Cluster 2 included 23 transcripts, including Ccl3, CD44, Il16, Il10ra, Il21r and Runx3. Expression of cluster 2 CATs was higher in d4MLR and in rejecting allografts relative to CTL than in cluster 1, with median 5-fold increase in rejecting kidneys at day 5, a further 2-fold increase from day 5 to day 14, and stable thereafter. Cluster 3 (n = 74) CAT expression was relatively high in d4MLR versus CTL but low in rejecting kidney. Cluster 4 (n = 46) CATs were less expressed in d4MLR than CTL, increased 1.9 fold between day 5 and day 14 and decreased thereafter by 1.3-fold. Cluster 5 consisted of Pdcd1, Socs1 and Stat1, which were as highly expressed in rejecting grafts as in CTL or d4MLR.

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Figure 6. K-means clustering of CATs in WT allografts. Expression in WT allografts is shown as % of expression in CTL. CATs (n = 287) cluster in five groups according to their relative expression in the allografts versus CTL. The boxplots represent the median and quartiles of expression of CATs for each timepoint.

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CATs in allografts rejecting in B-cell-deficient hosts

We analyzed whether the absence of B cells affects T-cell-mediated rejection by comparing CAT expression in kidneys rejecting in WT hosts to those rejecting in IghKO hosts at days 7 and 21. The expression of CATs in grafts rejecting in IghKO hosts was highly correlated with that in WT hosts (D7: r= 0.98; D21: r= 0.98). The mean expression of the five clusters of CAT transcripts was also similar in IghKO versus WT hosts (Figure 7), but was slightly higher in IghKO at day 7 (mean 23.2% in WT vs. 25.3% in IghKO, p < 0.01) and lower in IghKO at day 21 (mean 26.2% in WT compared to 21.1% in IghKO, p < 0.01).

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Figure 7. Comparison of CATs expression in allografts grafted into wild type hosts and B-cell deficient hosts at days 7 and 21. CATs were clustered based on expression in WT allografts (Figure 6). CAT expression in k-means clusters (Figure 6) in transplants rejecting in WT compared to IghKO hosts at days 7 and 21. Expression in allografts is shown as % of expression in CTL. The boxplots represent the median and quartiles of expression of CATs for each timepoint. Expression of CATs was slightly higher in IghKO compared to WT at D7 but showed some attenuation in IghKO compared to their wild-type counterparts at D21.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

We explored the relationship between the pathologic Banff lesions of kidney rejection and the changes in the transcriptome, particularly the appearance of the CATs. Mononuclear infiltrate was detectable at day 3 and established by day 5, whereas tubulitis and arteritis evolved slowly and progressively, being absent at day 5 and fully developed only after 14 days. We defined CATs by their high expression in d4MLR and CTL and absence in normal kidney. CAT expression was detectable in rejecting transplants at day 3 and reached a median signal intensity 14% of that in CTL after day 5. CAT expression was essentially constant from day 5 through 42, despite massive changes in the histopathology, and was minimally affected by B-cell deficiency. Thus CATs appeared early before the diagnostic Banff lesion of rejection and then persisted for at least 6 weeks. While the relative contribution of antigen-specific versus non-antigen-specific lymphocytes in the infiltrate is not known, we propose that CAT expression reflects the lymphoid infiltrate in the graft, and provides a measure of this component of the rejection process.

The CD transcripts provide an overview of leukocyte population changes and support the concept of a CTL and macrophage infiltrate with late B-cell infiltration indicated by the histologic analysis (5). There is no real ‘gold standard’ that provides an unbiased assessment of the composition of the infiltrate: both immunostaining of sections and cell isolation have potential for errors. Nevertheless the array estimates are compatible with the conventional methods. CD transcripts with high expression in CTL and d4MLR increased early during rejection and persisted throughout the time course, consistent with lymphocyte infiltration. There were few CD4+ cells in the infiltrate by immunostaining, and CD4 transcript expression in the microarrays was low. The macrophage markers, CD14 and CD68, were present in rejecting kidneys, with low expression in CTL and d4MLR, consistent with macrophage infiltration. B-cell markers, CD79A and CD79B, were present in d4MLR but not CTL, and appeared late in rejection, reflecting late B-cell infiltration.

Although non-antigen-stimulated lymphocytes may home to the graft because of the inflammation induced by the antigen-specific T cells, many transcripts expected in the antigen-experienced effector T cells that home to inflamed sites are prominent among the CATs, suggesting that antigen-stimulated effector T cells make a major contribution to CAT expression. Examples of such transcripts are Prf1, GzmA and GzmB, IFN-γ, P selectin ligand, CXCR3, CD44 and lymphotoxin B. Moreover, the stability of CAT expression over weeks despite massive parenchymal changes suggests that in T-cell-mediated rejection the CTL generated in lymphoid organs establish and maintain a condition in the rejecting organ that triggers parenchymal changes, yielding the pathologic lesions. Perhaps the infiltrating cells in the graft occupy a finite ‘space’, similar to the emerging concepts of space in the secondary lymphoid organs (29). The relative absence of CD4 and CCR7 transcripts and of B-cell transcripts in the early specimens (days 5 and 7) indicates that the infiltrate is structured, not ‘nonspecific’.

Although B-cell transcripts appeared in the late rejecting kidneys, absence of B cells and alloantibody in IghKO hosts had little effect on CAT expression. Taken with our previous observations that the infiltrate and tubulitis evolve normally in kidneys rejecting in B-cell-deficient hosts (5), this observation supports our contention that CAT expression reflects T-cell-mediated rejection. As we noted in the introduction, B-cell infiltrates are not associated with human antibody-mediated rejection, presumably because the alloantibody is produced in lymphoid organs. The small decrease in CAT expression in kidney rejecting in B-cell-deficient hosts suggests that the antigen-presenting function of B cells helps sustain the T-cell infiltrate, but further studies will be needed to explore this issue.

The sustained expression of transcripts associated with cytotoxicity (e.g. perforin, granzymes A and B) in rejecting grafts raises the question of the role of cytotoxic mechanisms, given that typical lesions develop in mice lacking Prf1 or GzmA plus GzmB (6). Fas ligand (Tnfsf6) is expressed in CTL and rejecting grafts, but is not necessary for organ rejection across MHC disparities (30). The epithelial deterioration and tubulitis could reflect CTL and macrophage products acting either directly on the parenchyma or indirectly, e.g. on extracellular matrix, triggering secondary changes in the epithelium. One possible role of Prf1, GzmA, GzmB and Fas ligand could be regulatory, e.g. fratricide of T cells (31) or regulatory through effects on dendritic cells (32).

CAT expression may prove useful for estimating the burden of infiltrating T cells in rejecting grafts, as one component of assessment of transplanted organs. We used CD8+ CTL to define CATs, but this definition probably includes most transcripts in the CD4+ effector T cells that home to inflamed sites, because they share many properties with CD8+ effectors (33). Less is known about effector CD4+ T cells in rejection across full MHC plus non-MHC disparities, perhaps because CD8+ effectors develop more rapidly (34). However, given the major role that class II mismatches play in human kidney graft rejection, it will be of interest to determine whether CD4+ T cells play a bigger role in human rejection than in this mouse model, and what transcripts may distinguish such cells in the rejecting graft.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Research support was received from Genome Canada, Roche Organ Transplant Research Foundation, the Canadian Institutes of Health Research, Kidney Foundation of Canada, Roche Canada, Roche Germany, Fujisawa Canada, and The Muttart Chair in Clinical Immunology. Dr Halloran holds a Canada Research Chair in Life Sciences Related to Human Health and Disease. Dr. Bleackley holds a Canada Research Chair in Life Sciences and is a Howard Hughes International Research Scholar. His laboratory is supported by grants from CIHR and NCIC.

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  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
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