Lower Circulating Cytotoxic T-Cell Frequency and Higher Intragraft Granzyme-B Expression Are Associated with Inflammatory Interstitial Fibrosis and Tubular Atrophy in Renal Allograft Recipients
Abstract
:1. Introduction
2. Materials and Methods
2.1. Patient Recruitment
2.2. Differential Diagnosis of Allograft Dysfunction
2.3. Definition of Differential Diagnosis
2.4. Ethical Clearance
2.5. Sample Collection
2.6. Cytotoxic T-Cell Staining and Frequency Analysis
2.6.1. Cell Stimulation
2.6.2. Cytotoxic T-Cell Staining
2.6.3. Cell Analysis and Gating Strategy
2.7. Peripheral Blood Mononuclear Cell Intact Granzyme-B Level Measurement
2.7.1. Serum Granzyme-B Level Analysis
2.7.2. Intragraft Granzyme-B Gene mRNA Transcript Expression Analysis
2.7.3. Granzyme-B Gene mRNA Transcript Expression Analysis
2.8. Statistical Data Analysis
3. Results
3.1. Demographic and Clinical Profiles of Patients
3.2. Circulating Cytotoxic T-Cell Frequency Was Lower in i-IFTA Patients
3.3. Serum Soluble and Cell Intact Granzyme-B Level
3.4. Intragraft Granzyme-B Gene mRNA Transcript Expression Analysis
3.5. Cytotoxic T-Cell Profiles Correlation with Kidney Function Markers
4. Discussion
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
References
- Loupy, A.; Haas, M.; Solez, K.; Racusen, L.; Glotz, D.; Seron, D.; Nankivell, B.J.; Colvin, R.B.; Afrouzian, M.; Akalin, E.; et al. The Banff 2015 Kidney Meeting Report: Current Challenges in Rejection Classification and Prospects for Adopting Molecular Pathology. Am. J. Transplant. 2017, 17, 28–41. [Google Scholar] [CrossRef]
- Prasad, N.; Yadav, B.; Agrawal, V.; Jain, M.; Agarwal, V. Role of pathogenic T-helper cells-17 in chronic antibody-mediated rejection in renal allograft recipients. Indian J. Transplant. 2022, 16, 88. [Google Scholar] [CrossRef]
- Mannon, R.B.; Matas, A.J.; Grande, J.; Leduc, R.; Connett, J.; Kasiske, B.; Cecka, J.M.; Gaston, R.S.; Cosio, F.; Gourishankar, S.; et al. Inflammation in areas of tubular atrophy in kidney allograft biopsies: A potent predictor of allograft failure. Am. J. Transplant. 2010, 10, 2066–2073. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Nankivell, B.J.; Borrows, R.J.; Fung, C.L.S.; O’Connell, P.J.; Chapman, J.R.; Allen, R.D.M. Delta Analysis of Posttransplantation Tubulointerstitial Damage. Transplantation 2004, 78, 434–441. [Google Scholar] [CrossRef] [PubMed]
- Mengel, M.; Reeve, J.; Bunnag, S.; Einecke, G.; Jhangri, G.S.; Sis, B.; Famulski, K.; Guembes-Hidalgo, L.; Halloran, P.F. Scoring Total Inflammation Is Superior to the Current Banff Inflammation Score in Predicting Outcome and the Degree of Molecular Disturbance in Renal Allografts. Am. J. Transplant. 2009, 9, 1859–1867. [Google Scholar] [CrossRef]
- Yadav, B.; Prasad, N.; Agrawal, V.; Jain, M.; Agarwal, V.; Jaiswal, A.; Bhadauria, D.; Sharma, R.K.; Gupta, A. T-bet-positive mononuclear cell infiltration is associated with transplant glomerulopathy and interstitial fibrosis and tubular atrophy in renal allograft recipients. Exp. Clin. Transplant. 2015, 13, 145–151. [Google Scholar] [PubMed]
- Ashton-Chess, J.; Dugast, E.; Colvin, R.B.; Giral, M.; Foucher, Y.; Moreau, A.; Renaudin, K.; Braud, C.; Devys, A.; Brouard, S.; et al. Regulatory, Effector, and Cytotoxic T Cell Profiles in Long-Term Kidney Transplant Patients. J. Am. Soc. Nephrol. 2009, 20, 1113–1122. [Google Scholar] [CrossRef] [Green Version]
- Vardhan, H.; Prasad, N.; Jaiswal, A.; Yadav, B.; Kumar, S.; Bhadauria, D.; Kaul, A.; Gupta, A.; Srivartava, A.; Sharma, R.K. Outcomes of living donor renal transplant recipients with and without basiliximab induction: A long-term follow-up study. Indian J. Transplant. 2014, 8, 44–50. [Google Scholar] [CrossRef]
- Williams, M.A.; Tyznik, A.J.; Bevan, M.J. Interleukin-2 signals during priming are required for secondary expansion of CD8+ memory T cells. Nature 2006, 441, 890–893. [Google Scholar] [CrossRef] [Green Version]
- Yadav, B.; Prasad, N.; Agarwal, V.; Agrawal, V.; Jain, M. Immunophenotyping of Granzyme-B Expressing Lymphocyte Subset in Renal Allograft Recipients with Chronic Allograft Dysfunction. Int. J. Org. Transplant. Med. 2023, 13, 20–27. [Google Scholar]
- Afonina, I.S.; Cullen, S.P.; Martin, S.J. Cytotoxic and non-cytotoxic roles of the CTL/NK protease granzyme B. Immunol. Rev. 2010, 235, 105–116. [Google Scholar] [CrossRef] [PubMed]
- Boivin, W.A.; Shackleford, M.; Vanden Hoek, A.; Zhao, H.; Hackett, T.L.; Knight, D.A.; Granville, D.J. Granzyme B cleaves decorin, biglycan and soluble betaglycan, releasing active transforming growth factor-β1. PLoS ONE 2012, 7, e33163. [Google Scholar] [CrossRef]
- Sharma, V.K.; Bologa, R.M.; Li, B.; Xu, G.P.; Lagman, M.; Hiscock, W.; Mouradian, J.; Wang, J.; Serur, D.; Rao, V.K.; et al. Molecular executors of cell death--differential intrarenal expression of Fas ligand, Fas, granzyme B, and perforin during acute and/or chronic rejection of human renal allografts. Transplantation 1996, 62, 1860–1866. [Google Scholar] [CrossRef] [PubMed]
- Strehlau, J.; Pavlakis, M.; Lipman, M.; Shapiro, M.; Vasconcellos, L.; Harmon, W.; Strom, T.B. Quantitative detection of immune activation transcripts as a diagnostic tool in kidney transplantation. Proc. Natl. Acad. Sci. USA 1997, 94, 695–700. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Yadav, B.; Prasad, N.; Agarwal, V.; Agarwal, V.; Jain, M. Hidden Granzyme B-Mediated Injury in Chronic Active Antibody-Mediated Rejection. Exp. Clin. Transplant. 2020, 18, 778–784. [Google Scholar] [CrossRef]
- Livak, K.J.; Schmittgen, T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2−∆∆CT Method. Methods 2001, 25, 402–408. [Google Scholar] [CrossRef]
- Li, B.; Hartono, C.; Ding, R.; Sharma, V.K.; Ramaswamy, R.; Qian, B.; Serur, D.; Mouradian, J.; Schwartz, J.E.; Suthanthiran, M. Noninvasive diagnosis of renal-allograft rejection by measurement of messenger RNA for perforin and granzyme B in urine. N. Engl. J. Med. 2001, 344, 947–954. [Google Scholar] [CrossRef]
- Wagner, C.; Iking-Konert, C.; Denefleh, B.; Stegmaier, S.; Hug, F.; Hänsch, G.M. Granzyme B and perforin: Constitutive expression in human polymorphonuclear neutrophils. Blood 2004, 103, 1099–1104. [Google Scholar] [CrossRef] [Green Version]
- Kim, W.-J.; Kim, H.; Suk, K.; Lee, W.-H. Macrophages express granzyme B in the lesion areas of atherosclerosis and rheumatoid arthritis. Immunol. Lett. 2007, 111, 57–65. [Google Scholar] [CrossRef]
- Hagn, M.; Jahrsdörfer, B. Why do human B cells secrete granzyme B? Insights into a novel B-cell differentiation pathway. Oncoimmunology 2012, 1, 1368–1375. [Google Scholar] [CrossRef] [Green Version]
- Ashraf, M.I.; Sarwar, A.; Kühl, A.A.; Hunger, E.; Sattler, A.; Aigner, F.; Regele, H.; Sauter, M.; Klingel, K.; Schneeberger, S.; et al. Natural Killer Cells Promote Kidney Graft Rejection Independently of Cyclosporine A Therapy. Front. Immunol. 2019, 10, 2279. [Google Scholar] [CrossRef] [PubMed]
- Boivin, W.A.; Cooper, D.M.; Hiebert, P.R.; Granville, D.J. Intracellular versus extracellular granzyme B in immunity and disease: Challenging the dogma. Lab. Investig. 2009, 89, 1195–1220. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Shen, Y.; Cheng, F.; Sharma, M.; Merkulova, Y.; Raithatha, S.A.; Parkinson, L.G.; Zhao, H.; Westendorf, K.; Bohunek, L.; Bozin, T.; et al. Granzyme B Deficiency Protects against Angiotensin II-Induced Cardiac Fibrosis. Am. J. Pathol. 2016, 186, 87–100. [Google Scholar] [CrossRef]
- Tang, P.M.-K.; Zhang, Y.-Y.; Mak, T.S.-K.; Tang, P.C.-T.; Huang, X.-R.; Lan, H.-Y. Transforming growth factor-β signalling in renal fibrosis: From Smads to non-coding RNAs. J. Physiol. 2018, 596, 3493–3503. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Weng, C.-H.; Li, Y.-J.; Wu, H.-H.; Liu, S.-H.; Hsu, H.-H.; Chen, Y.-C.; Yang, C.-W.; Chu, P.-H.; Tian, Y.-C. Interleukin-17A induces renal fibrosis through the ERK and Smad signaling pathways. Biomed. Pharmacother. 2020, 123, 109741. [Google Scholar] [CrossRef]
- AkimoAkimova, T.; Kamath, B.M.; Goebel, J.W.; Meyers, K.E.C.; Rand, E.B.; Hawkins, A.; Levine, M.H.; Bucuvalas, J.C.; Hancock, W.W. Differing Effects of Rapamycin or Calcineurin Inhibitor on T-Regulatory Cells in Pediatric Liver and Kidney Transplant Recipients. Am. J. Transplant. 2012, 12, 3449–3461. [Google Scholar] [CrossRef] [Green Version]
- Meng, X.M.; Tang, P.M.K.; Li, J.; Lan, H.Y. TGF-β/Smad signaling in renal fibrosis. Front. Physiol. 2015, 6, 82. [Google Scholar] [CrossRef] [Green Version]
- Afonina, I.S.; Tynan, G.A.; Logue, S.E.; Cullen, S.P.; Bots, M.; Lüthi, A.U.; Reeves, E.P.; McElvaney, N.G.; Medema, J.P.; Lavelle, E.C.; et al. Granzyme B-Dependent Proteolysis Acts as a Switch to Enhance the Proinflammatory Activity of IL-1α. Mol. Cell 2011, 44, 265–278. [Google Scholar] [CrossRef] [Green Version]
- Borthwick, L.A. The IL-1 cytokine family and its role in inflammation and fibrosis in the lung. Semin. Immunopathol. 2016, 38, 517–534. [Google Scholar] [CrossRef] [Green Version]
- Lonnemann, G.; Shapiro, L.; Engler-Blum, G.; Müller, G.A.; Koch, K.M.; Dinarello, C.A. Cytokines in human renal interstitial fibrosis. I. Interleukin-1 is a paracrine growth factor for cultured fibrosis-derived kidney fibroblasts. Kidney Int. 1995, 47, 837–844. [Google Scholar] [CrossRef] [Green Version]
- Burzynski, L.C.; Humphry, M.; Bennett, M.R.; Clarke, M.C.H. Interleukin-1α Activity in Necrotic Endothelial Cells Is Controlled by Caspase-1 Cleavage of Interleukin-1 Receptor-2: Implications for Allograft Rejection. J. Biol. Chem. 2015, 290, 25188–25196. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Leblanc, J.; Subrt, P.; Paré, M.; Hartell, D.; Sénécal, L.; Blydt-Hansen, T.; Cardinal, H. Practice Patterns in the Treatment and Monitoring of Acute T Cell–Mediated Kidney Graft Rejection in Canada. Can. J. Kidney Heal. Dis. 2018, 5, 2054358117753616. [Google Scholar] [CrossRef] [Green Version]
- Kim, C.H.; Broxmeyer, H.E. Chemokines: Signal lamps for trafficking of T and B cells for development and effector function. J. Leukoc. Biol. 1999, 65, 6–15. [Google Scholar] [CrossRef] [PubMed]
- Smyth, L.J.C.; Kirby, J.A.; Cunningham, A.C. Role of the mucosal integrin alpha(E)(CD103) beta (7) in tissue-restricted cytotoxicity. Clin. Exp. Immunol. 2007, 149, 162–170. [Google Scholar] [CrossRef]
- Rowshani, A.T.; Florquin, S.; Bemelman, F.; Kummer, J.A.; Hack, C.E.; Berge, I.J.T. Hyperexpression of the granzyme B inhibitor PI-9 in human renal allografts: A potential mechanism for stable renal function in patients with subclinical rejection. Kidney Int. 2004, 66, 1417–1422. [Google Scholar] [CrossRef] [Green Version]
- Heutinck, K.M.; Kassies, J.; Florquin, S.; Berge, I.J.T.; Hamann, J.; Rowshani, A.T. SerpinB9 expression in human renal tubular epithelial cells is induced by triggering of the viral dsRNA sensors TLR3, MDA5 and RIG-I. Nephrol. Dial. Transplant. 2012, 27, 2746–2754. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Lau, A.; Khan, K.; Pavlosky, A.; Yin, Z.; Huang, X.; Haig, A.; Liu, W.; Singh, B.; Zhang, Z.-X.; Jevnikar, A.M. Serine Protease Inhibitor-6 Inhibits Granzyme B–Mediated Injury of Renal Tubular Cells and Promotes Renal Allograft Survival. Transplantation 2014, 98, 402–410. [Google Scholar] [CrossRef]
- Heng, B.; Ding, H.; Ren, H.; Shi, L.; Chen, J.; Wu, X.; Lai, C.; Yu, G.; Xu, Y.; Su, Z. Diagnostic Performance of Fas Ligand mRNA Expression for Acute Rejection after Kidney Transplantation: A Systematic Review and Meta-Analysis. PLoS ONE 2016, 11, e0165628. [Google Scholar] [CrossRef] [Green Version]
Peritubular Capillaritis | |||
Scores | SGF (n = 10) | i-IFTA (n = 30) | p |
p0 | 9 (90%) | 25 (83.3%) | 0.52 |
p1 | 1 (10%) | 5 (16.6%) | |
p2 | 0 (0%) | 0 (0%) | |
p3 | 0 (0%) | 0 (0%) | |
Glomerulitis | |||
g0 | 9 (90%) | 23 (76.6%) | 0.62 |
g1 | 1 (10%) | 6 (20%) | |
g2 | 0 (0%) | 1 (3.3%) | |
g3 | 0 (0%) | 0 (0%) | |
Tubulitis | |||
t0 | 9 (90%) | 29 (96.6%) | 0.44 |
t1 | 1 (10%) | 1 (3.33%) | |
t2 | 0 (0%) | 0 (0%) | |
t3 | 0 (0%) | 0 (0%) | |
Interstitial fibrosis | |||
i0 | 10 (14.2%) | 0 (0%) | <0.001 |
i1 | 0 (0%) | 6 (20%) | |
i2 | 0 (0%) | 18 (60%) | |
i3 | 0 (0%) | 6 (20%) | |
Tubular atrophy | |||
t0 | 6 (60%) | 2 (6.6%) | <0.001 |
t1 | 4 (40%) | 7 (23.3%) | |
t2 | 0 (0%) | 15 (50%) | |
t3 | 0 (0%) | 6 (20%) | |
Interstitial inflammation | |||
i0 | 8 (80%) | 0 (0%) | 0.002 |
i1 | 2 (20%) | 18 (60%) | |
i2 | 0 (0%) | 9 (30%) | |
i3 | 0 (0%) | 3 (10%) | |
Intimal arteritis | |||
v0 | 9 (90%) | 11 (36.66%) | 0.014 |
v1 | 1 (10%) | 16 (53.3%) | |
v2 | 0 (0%) | 3 (10%) | |
v3 | 0 (0%) | 0 (0%) | |
Interstitial fibrosis and tubular atrophy (IFTA) | |||
IFTA0 | 9 (90%) | 0 (0%) | <0.001 |
IFTA1 | 1 (10%) | 10 (33.3%) | |
IFTA2 | 0 (0%) | 15 (50%) | |
IFTA3 | 0 (0%) | 5 (16.7%) |
Characteristics | SGF (Mean ± sd) | i-IFTA (Mean ± sd) | p Value |
---|---|---|---|
Pt. Gender (M:F) | 10:0 | 22:8 | 0.068 |
Do. Gender (M:F) | 2:8 | 8:22 | 0.673 |
Patient age (Years) | 44.36 ± 8.20 | 40.88 ± 8.37 | 0.257 |
Post-transplant biopsy interval (Months) | 46.70 ± 17.30 | 56.90 ± 25.37 | 0.246 |
eGFR (mL/min/1.73 m2) | 70.62 ± 22.14 | 44.13 ± 13.83 | <0.001 |
Tac level (ng/mL) | 4.82 ± 0.98 | 5.02 ± 1.51 | 0.689 |
Hemoglobin (gm/dL) | 12.94 ± 1.60 | 10.88 ± 1.61 | 0.001 |
TLC | 8.39 ± 2.12 | 8.50 ± 5.02 | 0.94 |
BUN | 25.79 ± 13.3 | 32.55 ± 8.84 | 0.074 |
Baseline creatinine (mg/dL) | 0.81 ± 0.47 | 0.92 ± 0.41 | 0.500 |
S. Creatinine (mg/dL) | 1.21 ± 0.18 | 2.22 ± 0.53 | <0.001 |
24 h urine protein (gm) | 0.16 ± 0.085 | 0.90 ± 0.61 | <0.001 |
HLA mismatch | 3.40 ± 0.69 | 3.10 ± 0.54 | 0.170 |
Induction regimen (Basiliximab) | 10 | 30 | 1.00 |
Baseline Immunosuppression Tacrolimus + MMF + Pred | 10 | 30 | 1.00 |
ESRD cause MN/HTN/NOS | 6/3/1 | 20/9/1 | 0.69 |
Characteristics | SGF | i-IF-TA | p Values |
---|---|---|---|
CD3+ T | 66.08 ± 6.8 | 65.18 ± 9.35 | 0.68 |
CD3+CD8+ T | 37.29 ± 4.11 | 34.68 ± 5.43 | 0.28 |
CD3+CD8+ granzyme-B T cell | 27.96 ± 4.86 | 23.19 ± 3.85 | 0.011 |
Proteinuria | Serum Creatinine | eGFR | |
---|---|---|---|
CTLCs (%) | R = −0.51 p < 0.001 | r = −0.28 p = 0.007 | r = −0.28 p = 0.037 |
Serum Granzyme B (pg/mL) | r = 0.343 p = 0.001 | r = 0.09 p = 0.3 | r = −0.18 p = 0.09 |
Supernatants Granzyme B (pg/mL) | r = −0.37 p < 0.001 | r = −0.31 p = 0.002 | r = 0.27 p = 0.011 |
Fold change in intragraft mRNA | r = 0.38 p < 0.001 | r = −0.12 p = 0.24 | r = −0.061 p = 0.58 |
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. |
© 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
Share and Cite
Yadav, B.; Prasad, N.; Agrawal, V.; Agarwal, V.; Jain, M. Lower Circulating Cytotoxic T-Cell Frequency and Higher Intragraft Granzyme-B Expression Are Associated with Inflammatory Interstitial Fibrosis and Tubular Atrophy in Renal Allograft Recipients. Medicina 2023, 59, 1175. https://doi.org/10.3390/medicina59061175
Yadav B, Prasad N, Agrawal V, Agarwal V, Jain M. Lower Circulating Cytotoxic T-Cell Frequency and Higher Intragraft Granzyme-B Expression Are Associated with Inflammatory Interstitial Fibrosis and Tubular Atrophy in Renal Allograft Recipients. Medicina. 2023; 59(6):1175. https://doi.org/10.3390/medicina59061175
Chicago/Turabian StyleYadav, Brijesh, Narayan Prasad, Vinita Agrawal, Vikas Agarwal, and Manoj Jain. 2023. "Lower Circulating Cytotoxic T-Cell Frequency and Higher Intragraft Granzyme-B Expression Are Associated with Inflammatory Interstitial Fibrosis and Tubular Atrophy in Renal Allograft Recipients" Medicina 59, no. 6: 1175. https://doi.org/10.3390/medicina59061175
APA StyleYadav, B., Prasad, N., Agrawal, V., Agarwal, V., & Jain, M. (2023). Lower Circulating Cytotoxic T-Cell Frequency and Higher Intragraft Granzyme-B Expression Are Associated with Inflammatory Interstitial Fibrosis and Tubular Atrophy in Renal Allograft Recipients. Medicina, 59(6), 1175. https://doi.org/10.3390/medicina59061175