Increased Cytotoxic CD4+ T Cells with Reduced Cytotoxic Gene Profile Expression in Cytomegalovirus Reactivated Kidney Transplant Patients
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Abstract
Cytotoxic CD4+ T cells eliminate human cytomegalovirus (HCMV)-infected cells through direct cytotoxic granules exocytosis. We aimed to evaluate the functional cytotoxic gene profile of CD4+ T cells alongside the frequency of the cytotoxic phenotype in renal transplant recipients with cytomegalovirus reactivation.
Blood samples were collected from twenty renal recipients with and without HCMV reactivation (HCMV+ and HCMV- groups) and ten healthy adults (control group). CD4+ T cells were isolated to assess the frequency of cytotoxic CD4+ T cells via CD107a surface staining using flow cytometry and to evaluate gene expression of perforin, granzyme B, Runt-related transcription factor 3 (RUNX3), and Eomesodermin (Eomes) by quantitative PCR.
The frequency of CD4+ CD107a+ T cells was higher in the HCMV+ group compared to the HCMV- group and significantly higher than in the control group (22.69±3.47 vs. 16.41±2.24 and 11.60±1.17, respectively). Perforin gene levels were reduced in the HCMV+ group compared to the other two groups, while granzyme B gene levels were similar between HCMV+ and HCMV- groups but lower than in the control group (0.63±1.24 vs. 0.67±2.27 and 1.00±0.00, respectively).
This study demonstrated an increased frequency of cytotoxic CD4+ T cells with potentially reduced functionality in kidney transplant patients with HCMV infection. It also suggests that these cells might employ other mechanisms, such as death receptor-mediated killing, or the production of other granules.
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2. Afshari A, Yaghobi R, Golshan M. Cytomegalovirus microRNAs level determination in kidney recipients post transplantation. Virol J. 2022;19(1):147.
3. Soleimanian S, Yaghobi R, Karimi MH, Geramizadeh B, Roozbeh J. The Direct Influence of Cytomegalovirus Lysate on the Natural Killer Cell Receptor Repertoire. Iran J Allergy Asthma Immunol. 2021;20(6):721-33.
4. Hassanzadeh Y, Yaghobi R, Pakzad P, Geramizadeh B. Decreased frequency of Th22 cells and IL- 22 cytokine in kidney transplant patients with active cytomegalovirus infection. BMC Immunol. 2023;24(1):18.
5. Juno JA, van Bockel D, Kent SJ, Kelleher AD, Zaunders JJ, Munier CML. Cytotoxic CD4 T Cells—Friend or Foe during viral infection? Front Immunol. 2017;8:19.
6. Cenerenti M, Saillard M, Romero P, Jandus C. The Era of Cytotoxic CD4 T Cells. Front Immunol. 2022;13:867189.
7. Hassanzadeh Y, Yaghobi R, Pakzad P, Geramizadeh B. Risk assessment of human cytomegalovirus infection in solid organ transplantation: Insight into CD4+ T cell subsets. Scand J Immunol. 2022:96(5):e13212.
8. Cohnen A, Chiang SC, Stojanovic A, Schmidt H, Claus M, Saftig P, et al. Surface CD107a/LAMP-1 protects natural killer cells from degranulation-associated damage. Blood. 2013;122(8):1411-1418.
9. Verschoor CP, Picard E, Andrew MK, Haynes L, Loeb M, Pawelec G, et al. NK- and T-cell granzyme B and K expression correlates with age, CMV infection and influenza vaccine-induced antibody titres in older adults. Front Aging. 2023;3:1098200.
10. Takeuchi A, Saito T. CD4 CTL, a Cytotoxic Subset of CD4+ T Cells, Their Differentiation and Function. Front Immunol. 2017;8(2):194.
11. Soleimanian S, Yaghobi R, Karimi MH, Geramizadeh B, Roozbeh J, Hossein Aghdaie M, et al. Circulating NKG2C + NK cell expressing CD107a/LAMP-1 subsets at the onset of CMV reactivation in seropositive kidney transplant recipients. Transpl Immunol. 2021;69:101460.
12. Hasegawa T, Oka T, Son HG, Oliver-Garcı´a VS, Azin M, Eisenhaure TM, et al. Cytotoxic CD4+ T cells eliminate senescent cells by targeting cytomegalovirus antigen. Cell. 2023;186(7):1417–31.
13. Gordon CL, Miron M, Thome JJC, Matsuoka N, Weiner J, Rak MA, et al. Tissue reservoirs of antiviral T cell immunity in persistent human CMV infection. J Exp Med. 2017;214(3):651–67
14. Almanan M, Raynor J, Sholl A, Wang M, Chougnet C, Cardin RD, et al. Tissue-specific control of latent CMV reactivation by regulatory T cells. PLoS Pathog. 2017;13(8):e1006507. 15. Verma S, Weiskopf D, Gupta A, McDonald B, Peters B, Sette A, et al. Cytomegalovirus-specific CD4 T cells are cytolytic and mediate vaccine protection. J Virol. 2016;90(2): 650 -8.
16. Perera MR, Greenwood EJD, Crozier TWM, Elder EG, Schmitt J, Crump CM, et al. Human Cytomegalovirus Infection of Epithelial Cells Increases SARS-CoV-2 Superinfection by Upregulating the ACE2 Receptor. J Infect Dis. 2023;227(4):543–53.
17. Xiong N, Fu Y, Hu S, Xia M, Yang J. CCR10 and its ligands in regulation of epithelial immunity and diseases. Protein Cell. 2012;3(8):571–80.
18. Murphy PM. Chemokines and Chemokine Receptors, in: Drs. Robert R. Rich, Thomas A. Fleisher, William T. Shearer, Harry W. Schroeder, A.J. Frew, C.M. Weyand (Eds.), Clinical Immunology (Fifth Edition): Principles and Practice, Elsevier Ltd, 2019, p157-70. https://doi.org/10.1016/C2015-0-00344-6.
19. Higdon LE, Ahmad AA, Schaffert S, Margulies KB, Maltzman JS. CMV-Responsive CD4 T Cells Have a Stable Cytotoxic Phenotype Over the First Year Post-Transplant in Patients Without Evidence of CMV Viremia. Front Immunol. 2022;13:904705.
20. Kallemeijn MJ, Boots AMH, van der Klift MY, Brouwer E, Abdulahad WH, Verhaar JAN, et al. Ageing and latent CMV infection impact on maturation, differentiation and exhaustion profiles of T-cell receptor gammadelta T-cells. Sci Rep. 2017;7(1):5509.
21. Chanouzas D, Sagmeister M, Faustini S, Nightingale P, Richter A, Ferro CJ, et al. Subclinical Reactivation of Cytomegalovirus Drives CD4+CD28null T-Cell Expansion and Impaired Immune Response to Pneumococcal Vaccination in Antineutrophil Cytoplasmic Antibody–Associated Vasculitis. J Infect Dis. 2019;219(2):234–44.
22. Tamura Y, Yamane K, Kawano Y, Bullinger L, Wirtz T, Weber T, et al. Concomitant Cytotoxic Effector Differentiation of CD4+ and CD8+ T Cells in Response to EBV-Infected B Cells. Cancers. 2022;14(17):4118.
23. Pachnio A, Ciaurriz M, Begum J, Lal N, Zuo J, Beggs A, et al. Cytomegalovirus Infection Leads to Development of High Frequencies of Cytotoxic Virus-Specific CD4+ T Cells Targeted to Vascular Endothelium. PLoS Pathog. 2016;12(9):e1005832
24. Soleimanian S, Yaghobi R, Karimi MH, Geramizadeh B, Roozbeh J. Altered Signatures of Plasma Inflammatory Proteins and Phonotypic Markers of NK Cells in Kidney Transplant Patients upon CMV Reactivation. Curr Microbiol. 2023;80(1):9.
25. Casazza JP, Betts MR, Price MA, Precopio ML, Ruff LE, Brenchley JM, et al. Acquisition of direct antiviral effector functions by CMV-specific CD4+ T lymphocytes with cellular maturation. J Exp Med. 2006;203(13):2865–77.
26. Luo XH, Meng Q, Rao M, Liu Z, Paraschoudi G, Dodoo E, et al. The impact of inflationary cytomegalovirus-specific memory T cells on anti-tumour immune responses in patients with cancer. Immunology. 2018;155(3):294–308.
27. Malyshkina A, Littwitz-Salomon E, Sutter K, Zelinskyy G, Windmann S, Schimmer S, et al. Fas Ligand-mediated cytotoxicity of CD4+ T cells during chronic retrovirus infection. Sci Rep. 2017;7(8):7785.
28. Serroukh Y, Gu-Trantien C, Hooshiar Kashani B, Defrance M., Manh TPV, Azouz A, et al. The transcription factors Runx3 and ThPOK cross-regulate acquisition of cytotoxic function by human Th1 lymphocytes. eLife. 2018;7:e30496.
29. Hoeks C, van Puijfelik F, Koetzier SC, Rip J, Corsten CEA, Wierenga-Wolf AF, et al. Differential Runx3, Eomes, and T-bet expression subdivides MS-associated CD4+ T cells with brain-homing capacity. Eur J Immunol. 2024;54(2):e2350544.
30. Cachot A, Bilous M, Liu YC, Li X, Saillard M, Cenerenti M, et al. Tumor-specific cytolytic CD4 T cells mediate immunity against human cancer. Sci Adv. 2021;7(9):eabe3348.
31. Eshima K, Misawa K, Ohashi C, Iwabuchi K. Role of T-bet, the master regulator of Th1 cells, in thecytotoxicity of murine CD4+ T cells. Microbiol Immunol. 2018;62(5):348–56.
32. Phetsouphanh C, Aldridge D, Marchi E, Munier ML, Meyerowitz J, Murray L, et al. Maintenance of Functional CD57+ Cytolytic CD4+ T Cells in HIV+ Elite Controllers. Front Immunol. 2019;10:1844.
33. Takeuchi A, El Sherif Gadelhaq Badr M, Miyauchi K, Ishihara C, Onishi R, Guo Z, et al. CRTAM determines the CD4+ cytotoxic T lymphocyte lineage. J Exp Med. 2016;213(1):123–38.
34. Wang Y, Chen Z, Wang T, Guo H, Liu Y, Dang N, et al. A novel CD4+ CTL subtype characterized by chemotaxis and inflammation is involved in the pathogenesis of Graves’ orbitopathy. Cell Mol Immunol. 2021;18(3):735–45.
35. Mazzoni A, Maggi L, Siracusa F, Ramazzotti M, Rossi MC, Santarlasci V, et al. Eomes controls the development of Th17-derived (non-classic) Th1 cells during chronic inflammation. Eur J Immunol. 2019;49(1):79–95.
36. Al-Banna NA, Vaci M, Slauenwhite D, Johnston B, Issekutz TB. CCR4 and CXCR3 play different roles in the migration of T cells to inflammation in skin, arthritic joints, and lymph nodes. Eur J Immunol. 2014;44(6):1633–43.
37. Shabir S, Smith H, Kaul B, Pachnio A, Jham S, Kuravi S, et al. Cytomegalovirus-Associated CD4+CD28null Cells in NKG2D-Dependent Glomerular Endothelial Injury and Kidney Allograft Dysfunction. Am J Transplant. 2016;16(4):1113–28.
38. Hassouneh F, Goldeck D, Pera A, van Heemst D, Slagboom PE, Pawelec G, et al. Functional Changes of T-Cell Subsets with Age and CMV Infection. Int J Mol Sci. 2021;22(18):9973.
39. Huygens A, Lecomte S, Tackoen M, Olislagers V, Delmarcelle Y, Burny V, et al. Functional Exhaustion Limits CD4+ and CD8+ T-Cell Responses to Congenital Cytomegalovirus Infection. J Infect Dis. 2015;212(3):484-94.
2. Afshari A, Yaghobi R, Golshan M. Cytomegalovirus microRNAs level determination in kidney recipients post transplantation. Virol J. 2022;19(1):147.
3. Soleimanian S, Yaghobi R, Karimi MH, Geramizadeh B, Roozbeh J. The Direct Influence of Cytomegalovirus Lysate on the Natural Killer Cell Receptor Repertoire. Iran J Allergy Asthma Immunol. 2021;20(6):721-33.
4. Hassanzadeh Y, Yaghobi R, Pakzad P, Geramizadeh B. Decreased frequency of Th22 cells and IL- 22 cytokine in kidney transplant patients with active cytomegalovirus infection. BMC Immunol. 2023;24(1):18.
5. Juno JA, van Bockel D, Kent SJ, Kelleher AD, Zaunders JJ, Munier CML. Cytotoxic CD4 T Cells—Friend or Foe during viral infection? Front Immunol. 2017;8:19.
6. Cenerenti M, Saillard M, Romero P, Jandus C. The Era of Cytotoxic CD4 T Cells. Front Immunol. 2022;13:867189.
7. Hassanzadeh Y, Yaghobi R, Pakzad P, Geramizadeh B. Risk assessment of human cytomegalovirus infection in solid organ transplantation: Insight into CD4+ T cell subsets. Scand J Immunol. 2022:96(5):e13212.
8. Cohnen A, Chiang SC, Stojanovic A, Schmidt H, Claus M, Saftig P, et al. Surface CD107a/LAMP-1 protects natural killer cells from degranulation-associated damage. Blood. 2013;122(8):1411-1418.
9. Verschoor CP, Picard E, Andrew MK, Haynes L, Loeb M, Pawelec G, et al. NK- and T-cell granzyme B and K expression correlates with age, CMV infection and influenza vaccine-induced antibody titres in older adults. Front Aging. 2023;3:1098200.
10. Takeuchi A, Saito T. CD4 CTL, a Cytotoxic Subset of CD4+ T Cells, Their Differentiation and Function. Front Immunol. 2017;8(2):194.
11. Soleimanian S, Yaghobi R, Karimi MH, Geramizadeh B, Roozbeh J, Hossein Aghdaie M, et al. Circulating NKG2C + NK cell expressing CD107a/LAMP-1 subsets at the onset of CMV reactivation in seropositive kidney transplant recipients. Transpl Immunol. 2021;69:101460.
12. Hasegawa T, Oka T, Son HG, Oliver-Garcı´a VS, Azin M, Eisenhaure TM, et al. Cytotoxic CD4+ T cells eliminate senescent cells by targeting cytomegalovirus antigen. Cell. 2023;186(7):1417–31.
13. Gordon CL, Miron M, Thome JJC, Matsuoka N, Weiner J, Rak MA, et al. Tissue reservoirs of antiviral T cell immunity in persistent human CMV infection. J Exp Med. 2017;214(3):651–67
14. Almanan M, Raynor J, Sholl A, Wang M, Chougnet C, Cardin RD, et al. Tissue-specific control of latent CMV reactivation by regulatory T cells. PLoS Pathog. 2017;13(8):e1006507. 15. Verma S, Weiskopf D, Gupta A, McDonald B, Peters B, Sette A, et al. Cytomegalovirus-specific CD4 T cells are cytolytic and mediate vaccine protection. J Virol. 2016;90(2): 650 -8.
16. Perera MR, Greenwood EJD, Crozier TWM, Elder EG, Schmitt J, Crump CM, et al. Human Cytomegalovirus Infection of Epithelial Cells Increases SARS-CoV-2 Superinfection by Upregulating the ACE2 Receptor. J Infect Dis. 2023;227(4):543–53.
17. Xiong N, Fu Y, Hu S, Xia M, Yang J. CCR10 and its ligands in regulation of epithelial immunity and diseases. Protein Cell. 2012;3(8):571–80.
18. Murphy PM. Chemokines and Chemokine Receptors, in: Drs. Robert R. Rich, Thomas A. Fleisher, William T. Shearer, Harry W. Schroeder, A.J. Frew, C.M. Weyand (Eds.), Clinical Immunology (Fifth Edition): Principles and Practice, Elsevier Ltd, 2019, p157-70. https://doi.org/10.1016/C2015-0-00344-6.
19. Higdon LE, Ahmad AA, Schaffert S, Margulies KB, Maltzman JS. CMV-Responsive CD4 T Cells Have a Stable Cytotoxic Phenotype Over the First Year Post-Transplant in Patients Without Evidence of CMV Viremia. Front Immunol. 2022;13:904705.
20. Kallemeijn MJ, Boots AMH, van der Klift MY, Brouwer E, Abdulahad WH, Verhaar JAN, et al. Ageing and latent CMV infection impact on maturation, differentiation and exhaustion profiles of T-cell receptor gammadelta T-cells. Sci Rep. 2017;7(1):5509.
21. Chanouzas D, Sagmeister M, Faustini S, Nightingale P, Richter A, Ferro CJ, et al. Subclinical Reactivation of Cytomegalovirus Drives CD4+CD28null T-Cell Expansion and Impaired Immune Response to Pneumococcal Vaccination in Antineutrophil Cytoplasmic Antibody–Associated Vasculitis. J Infect Dis. 2019;219(2):234–44.
22. Tamura Y, Yamane K, Kawano Y, Bullinger L, Wirtz T, Weber T, et al. Concomitant Cytotoxic Effector Differentiation of CD4+ and CD8+ T Cells in Response to EBV-Infected B Cells. Cancers. 2022;14(17):4118.
23. Pachnio A, Ciaurriz M, Begum J, Lal N, Zuo J, Beggs A, et al. Cytomegalovirus Infection Leads to Development of High Frequencies of Cytotoxic Virus-Specific CD4+ T Cells Targeted to Vascular Endothelium. PLoS Pathog. 2016;12(9):e1005832
24. Soleimanian S, Yaghobi R, Karimi MH, Geramizadeh B, Roozbeh J. Altered Signatures of Plasma Inflammatory Proteins and Phonotypic Markers of NK Cells in Kidney Transplant Patients upon CMV Reactivation. Curr Microbiol. 2023;80(1):9.
25. Casazza JP, Betts MR, Price MA, Precopio ML, Ruff LE, Brenchley JM, et al. Acquisition of direct antiviral effector functions by CMV-specific CD4+ T lymphocytes with cellular maturation. J Exp Med. 2006;203(13):2865–77.
26. Luo XH, Meng Q, Rao M, Liu Z, Paraschoudi G, Dodoo E, et al. The impact of inflationary cytomegalovirus-specific memory T cells on anti-tumour immune responses in patients with cancer. Immunology. 2018;155(3):294–308.
27. Malyshkina A, Littwitz-Salomon E, Sutter K, Zelinskyy G, Windmann S, Schimmer S, et al. Fas Ligand-mediated cytotoxicity of CD4+ T cells during chronic retrovirus infection. Sci Rep. 2017;7(8):7785.
28. Serroukh Y, Gu-Trantien C, Hooshiar Kashani B, Defrance M., Manh TPV, Azouz A, et al. The transcription factors Runx3 and ThPOK cross-regulate acquisition of cytotoxic function by human Th1 lymphocytes. eLife. 2018;7:e30496.
29. Hoeks C, van Puijfelik F, Koetzier SC, Rip J, Corsten CEA, Wierenga-Wolf AF, et al. Differential Runx3, Eomes, and T-bet expression subdivides MS-associated CD4+ T cells with brain-homing capacity. Eur J Immunol. 2024;54(2):e2350544.
30. Cachot A, Bilous M, Liu YC, Li X, Saillard M, Cenerenti M, et al. Tumor-specific cytolytic CD4 T cells mediate immunity against human cancer. Sci Adv. 2021;7(9):eabe3348.
31. Eshima K, Misawa K, Ohashi C, Iwabuchi K. Role of T-bet, the master regulator of Th1 cells, in thecytotoxicity of murine CD4+ T cells. Microbiol Immunol. 2018;62(5):348–56.
32. Phetsouphanh C, Aldridge D, Marchi E, Munier ML, Meyerowitz J, Murray L, et al. Maintenance of Functional CD57+ Cytolytic CD4+ T Cells in HIV+ Elite Controllers. Front Immunol. 2019;10:1844.
33. Takeuchi A, El Sherif Gadelhaq Badr M, Miyauchi K, Ishihara C, Onishi R, Guo Z, et al. CRTAM determines the CD4+ cytotoxic T lymphocyte lineage. J Exp Med. 2016;213(1):123–38.
34. Wang Y, Chen Z, Wang T, Guo H, Liu Y, Dang N, et al. A novel CD4+ CTL subtype characterized by chemotaxis and inflammation is involved in the pathogenesis of Graves’ orbitopathy. Cell Mol Immunol. 2021;18(3):735–45.
35. Mazzoni A, Maggi L, Siracusa F, Ramazzotti M, Rossi MC, Santarlasci V, et al. Eomes controls the development of Th17-derived (non-classic) Th1 cells during chronic inflammation. Eur J Immunol. 2019;49(1):79–95.
36. Al-Banna NA, Vaci M, Slauenwhite D, Johnston B, Issekutz TB. CCR4 and CXCR3 play different roles in the migration of T cells to inflammation in skin, arthritic joints, and lymph nodes. Eur J Immunol. 2014;44(6):1633–43.
37. Shabir S, Smith H, Kaul B, Pachnio A, Jham S, Kuravi S, et al. Cytomegalovirus-Associated CD4+CD28null Cells in NKG2D-Dependent Glomerular Endothelial Injury and Kidney Allograft Dysfunction. Am J Transplant. 2016;16(4):1113–28.
38. Hassouneh F, Goldeck D, Pera A, van Heemst D, Slagboom PE, Pawelec G, et al. Functional Changes of T-Cell Subsets with Age and CMV Infection. Int J Mol Sci. 2021;22(18):9973.
39. Huygens A, Lecomte S, Tackoen M, Olislagers V, Delmarcelle Y, Burny V, et al. Functional Exhaustion Limits CD4+ and CD8+ T-Cell Responses to Congenital Cytomegalovirus Infection. J Infect Dis. 2015;212(3):484-94.
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How to Cite
1.
Hassanzadeh Y, Yaghobi R, Pakzad P, Geramizadeh B. Increased Cytotoxic CD4+ T Cells with Reduced Cytotoxic Gene Profile Expression in Cytomegalovirus Reactivated Kidney Transplant Patients. Iran J Allergy Asthma Immunol. 2024;:1-13.
