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Lymphocyte Subgroups and KREC Numbers in Common Variable Immunodeficiency: A Single Center Study

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Common variable immunodeficiency (CVID) results in defective B cell differentiation and impaired antibody production and is the most common symptomatic primary immunodeficiency. Our aim was to evaluate the correlation among B cell subgroups, κ-deleting recombination excision circle (KREC) copy numbers, and clinical and immunological data of the patients with CVID, and evaluate the patients according to classifications currently available to define the role of KREC copy numbers in the diagnosis of CVID. KREC analysis was performed using a quantitative real-time polymerase chain reaction assay, and B cell subgroups were measured by flow cytometry. The median age of the patients (n = 30) was 25 (6–69) years. Parental consanguinity ratio was 33%. The median age at diagnosis was 15 (4–59), and follow-up period was 6 (1–37) years. CD19+ and CD4+ cell counts at the time of diagnosis were low in 66.7% and 46.7% of the patients, respectively. CD19+ cell counts were positively correlated with KREC copy numbers in patients and healthy controls. CD19+ cell counts and KREC copy numbers were significantly reduced in CVID patients compared to healthy controls as expected. KRECs are quantitative markers for B cell defects. We found low CD4+ cell numbers, recent thymic emigrants, and lymphopenia in some of the patients at diagnosis, which reminds the heterogeneity of CVID’s etiology. In this study, a positive correlation was shown between CD19+ cell counts and KREC copy numbers. Low KREC copy numbers indicated B cell deficiency; however, high KREC copy numbers were not sufficient to rule out CVID.

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  1. 1.

    Yong PF, Thaventhiran JE, Grimbacher B. “A rose is a rose is a rose,” but CVID is not CVID. Common variable immune deficiency (CVID), what do we know in 2011? Adv Immunol. 2011;111:47–107. https://doi.org/10.1016/B978-0-12-385991-4.00002-7.

  2. 2.

    Warnatz K, Denz A, Dra R, Braun M, Groth C, Wolff-vorbeck G, et al. Severe deficiency of switched memory B cells (CD27+IgM−IgD−) in subgroups of patients with common variable immunodeficiency: a new approach to classify a heterogeneous disease. Blood. 2002;99:1544–51.

  3. 3.

    Piqueras B, Galicier L, Cruyssen FBDER. Common variable immunodeficiency patient classification based on impaired B cell memory differentiation correlates with clinical aspects. J Clin Immunol. 2003;23:385–400.

  4. 4.

    Wehr C, Kivioja T, Schmitt C, Ferry B, Witte T, Eren E, et al. The EUROclass trial: defining subgroups in common variable immunodeficiency. Blood. 2008;111:77–86. https://doi.org/10.1182/blood-2007-06-091744.

  5. 5.

    Driessen GJ, Van Zelm MC, Van Hagen PM, Hartwig NG, Trip M, Warris A, et al. B-cell replication history and somatic hypermutation status identify distinct pathophysiologic backgrounds in common variable immunodeficiency. Blood. 2011;118:6814–23. https://doi.org/10.1182/blood-2011-06-361881.

  6. 6.

    Serana F, Chiarini M, Zanotti C, Sottini A, Bertoli D, Bosio A, et al. Use of V(D)J recombination excision circles to identify T- and B-cell defects and to monitor the treatment in primary and acquired immunodeficiencies. J Transl Med. 2013;11:1–11. https://doi.org/10.1186/1479-5876-11-119.

  7. 7.

    Chiarini M, Zanotti C, Serana F, Sottini A, Bertoli D, Caimi L, et al. T-cell receptor and K-deleting recombination excision circles in newborn screening of T-and B-cell defects: review of the literature and future challenges. J Public Health Res. 2013;2:9–16. https://doi.org/10.4081/jphr.2013.e3.

  8. 8.

    Sottini A, Serana F, Bertoli D, Chiarini M, Valotti M, Vaglio Tessitore M, et al. Simultaneous quantification of T-cell receptor excision circles (TRECs) and K-deleting recombination excision circles (KRECs) by real-time PCR. J Vis Exp. 2014:1–10. https://doi.org/10.3791/52184.

  9. 9.

    van Zelm MC, Szczepański T, van der Burg M, van Dongen JJM. Replication history of B lymphocytes reveals homeostatic proliferation and extensive antigen-induced B cell expansion. J Exp Med. 2007;204:645–55. https://doi.org/10.1084/jem.20060964.

  10. 10.

    ESID, ESID Registry – working definitions for clinical diagnosis of PID, (2015) 1–15. 732.

  11. 11.

    Goksuluk D, Korkmaz S, Zararsiz G, Karaagaoglu AE. EasyROC: an interactive web-tool for roc curve analysis using r language environment. The R J. 2016;8:213–30. https://doi.org/10.32614/rj-2016-042.

  12. 12.

    Ravkov E, Slev P, Heikal N. Thymic output: assessment of CD4+ recent thymic emigrants and T-cell receptor excision circles in infants. Cytometry B Clin Cytom. 2017;92:249–57. https://doi.org/10.1002/cyto.b.21341.

  13. 13.

    Schatorjé EJH, Gemen EFA, Driessen GJA, Leuvenink J, van Hout RWNM, de Vries E. Paediatric reference values for the peripheral T cell compartment. Scand J Immunol. 2012;75:436–44. https://doi.org/10.1111/j.1365-3083.2012.02671.x.

  14. 14.

    Björk AH, Óskarsdóttir S, Andersson BA, Friman V. Antibody deficiency in adults with 22q11.2 deletion syndrome. Am J Med Genet A. 2012;158 A:1934–40. https://doi.org/10.1002/ajmg.a.35484.

  15. 15.

    Oksenhendler E, Gérard L, Fieschi C, Malphettes M, Mouillot G, Jaussaud R, et al. Infections in 252 patients with common variable immunodeficiency. Clin Infect Dis. 2008;46:1547–54. https://doi.org/10.1086/587669.

  16. 16.

    Al Kindi M, Mundy J, Sullivan T, Smith W, Kette F, Smith A, et al. Utility of peripheral blood B cell subsets analysis in common variable immunodeficiency. Clin Exp Immunol. 2012;167:275–81. https://doi.org/10.1111/j.1365-2249.2011.04507.x.

  17. 17.

    Bright P, Grigoriadou S, Kamperidis P, Buckland M, Hickey A, Longhurst HJ. Changes in B cell immunophenotype in common variable immunodeficiency: cause or effect - is bronchiectasis indicative of undiagnosed immunodeficiency? Clin Exp Immunol. 2013;171:195–200. https://doi.org/10.1111/cei.12010.

  18. 18.

    Yazdani R, Seify R, Ganjalikhani-Hakemi M, Abolhassani H, Eskandari N, Golsaz-Shirazi F, et al. Comparison of various classifications for patients with common variable immunodeficiency (CVID) using measurement of B-cell subsets. Allergol Immunopathol (Madr). 2017;45:183–92. https://doi.org/10.1016/j.aller.2016.07.001.

  19. 19.

    Ben-Yehuda A, Weksler ME. Immune senescence: mechanisms and clinical implications. Cancer Investig. 1992;10:525–31. https://doi.org/10.3109/07357909209024815.

  20. 20.

    Miller RA. The aging immune system: primer and prospectus. Science (80-. ). 1996;273:70–4.

  21. 21.

    Ginaldi L, Loreto MF, Corsi MP, Modesti M, De Martinis M. Immunosenescence and infectious diseases. Microbes Infect. 2001;3:851–7. https://doi.org/10.1016/s1286-4579(01)01443-5.

  22. 22.

    Cancro MP, Hao Y, Scholz JL, Riley RL, Frasca D, Blomberg BB. B cells and aging: molecules and mechanisms. Trends Immunol. 2009;30:313–8. https://doi.org/10.1016/j.it.2009.04.005.B.

  23. 23.

    Frasca D, Landin AM, Lechner SC, Ryan JG, Schwartz R, Riley RL, et al. Aging down-regulates the transcription factor E2A, activation-induced cytidine deaminase, and Ig class switch in human B cells. J Immunol. 2008;180:5283–90. https://doi.org/10.4049/jimmunol.180.8.5283.

  24. 24.

    Frasca D, Diaz A, Romero M, Landin AM, Blomberg BB. Age effects on B cells and humoral immunity in humans. Ageing Res Rev. 2011;10:330–5. https://doi.org/10.1016/j.arr.2010.08.004.

  25. 25.

    Kamae C, Nakagawa N, Sato H, Honma K, Mitsuiki N, Ohara O, et al. Common variable immunodeficiency classification by quantifying T-cell receptor and immunoglobulin κ-deleting recombination excision circles. J Allergy Clin Immunol. 2013;131:1437–40. https://doi.org/10.1016/j.jaci.2012.10.059.

  26. 26.

    Serana F, Airò P, Chiarini M, Zanotti C, Scarsi M, Frassi M, et al. Thymic and bone marrow output in patients with common variable immunodeficiency. J Clin Immunol. 2011;31:540–9. https://doi.org/10.1007/s10875-011-9526-6.

  27. 27.

    Atschekzei F, Ahmad F, Witte T, Jacobs R, Schmidt RE. Limitation of simultaneous analysis of T-cell receptor and κ-deleting recombination excision circles based on multiplex real-time polymerase chain reaction in common variable immunodeficiency patients. Int Arch Allergy Immunol. 2016;171:136–40. https://doi.org/10.1159/000450950.

  28. 28.

    Mensen A, Ochs C, Stroux A, Wittenbecher F, Szyska M, Imberti L, et al. Utilization of TREC and KREC quantification for the monitoring of early T- and B-cell neogenesis in adult patients after allogeneic hematopoietic stem cell transplantation. J Transl Med. 2013;11:6–9. https://doi.org/10.1186/1479-5876-11-188.

  29. 29.

    Serana F, Sottini A, Chiarini M, Zanotti C, Ghidini C, Lanfranchi A, et al. The different extent of B and T cell immune reconstitution after hematopoietic stem cell transplantation and enzyme replacement therapies in SCID patients with adenosine Deaminase deficiency. J Immunol. 2010;185:7713–22. https://doi.org/10.4049/jimmunol.1001770.

  30. 30.

    Korsunskiy I, Blyuss O, Gordukova M, Davydova N, Gordleeva S, Molchanov R, et al. TREC and KREC levels as a predictors of lymphocyte subpopulations measured by flow cytometry. Front Physiol. 2019;10:1–8. https://doi.org/10.3389/fphys.2018.01877.

  31. 31.

    Barbaro M, Ohlsson A, Borte S, Jonsson S, Zetterström RH, King J, et al. Newborn screening for severe primary immunodeficiency diseases in Sweden—a 2-year pilot TREC and KREC screening study. J Clin Immunol. 2017;37:51–60. https://doi.org/10.1007/s10875-016-0347-5.

  32. 32.

    Nourizadeh M, Shakerian L, Borte S, Fazlollahi M, Badalzadeh M, Houshmand M, et al. Newborn screening using TREC/KREC assay for severe T and B cell lymphopenia in Iran. Scand J Immunol. 2018;88:1–9. https://doi.org/10.1111/sji.12699.

  33. 33.

    Bateman EAL, Ayers L, Sadler R, Lucas M, Roberts C, Woods A, et al. T cell phenotypes in patients with common variable immunodeficiency disorders: associations with clinical phenotypes in comparison with other groups with recurrent infections. Clin Exp Immunol. 2012;170:202–11. https://doi.org/10.1111/j.1365-2249.2012.04643.x.

  34. 34.

    Nechvatalova J, Pavlik T, Litzman J, Vlkova M. Terminally differentiated memory T cells are increased in patients with common variable immunodeficiency and selective IgA deficiency. Cent Eur J Immunol. 2017;42:244–51. https://doi.org/10.5114/ceji.2017.70966.

  35. 35.

    Giovannetti A, Pierdominici M, Mazzetta F, Marziali M, Renzi C, Mileo AM, et al. Unravelling the complexity of T cell abnormalities in common variable immunodeficiency. J Immunol. 2007;178:3932–43. https://doi.org/10.4049/jimmunol.178.6.3932.

  36. 36.

    Bonilla FA, Barlan I, Chapel H, Costa-Carvalho BT, Cunningham-Rundles C, de la Morena MT, et al. International Consensus Document (ICON): common variable immunodeficiency disorders. J Allergy Clin Immunol Pract. 2016;4:38–59. https://doi.org/10.1016/j.jaip.2015.07.025.

  37. 37.

    van Schouwenburg PA, Davenport EE, Kienzler AK, Marwah I, Wright B, Lucas M, et al. Application of whole genome and RNA sequencing to investigate the genomic landscape of common variable immunodeficiency disorders. Clin Immunol. 2015;160:301–14. https://doi.org/10.1016/j.clim.2015.05.020.

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We appreciate Dr. Luisa Imberti and Dr. Alessandra Sottini from Laboratorio CREA, Spedali Civili di Brescia, Italy, for kindly supplying the TREC-KREC-TCRAC plasmid to our laboratory. The authors thank to the patients and their families for their collaboration and participation. This project was supported by Scientific and Technological Research Council of Turkey (Project Number: 315S125) and also by Hacettepe University Scientific Research Projects Coordination Unit (Project number: N17128).

Author information

Ismail Yaz, MSc, performed experiments, collected the data, contributed data or analysis tools, interpreted the data, and wrote the paper.

Begum Ozbek, MSc, performed experiments, contributed data collection, and interpreted the data.

Yuk Yin NG, PhD, contributed experiments and analysis tools.

Pinar Gur Cetinkaya, MD, contributed data collection.

Sevil Oskay Halacli, PhD, contributed data collection.

Cagman Tan, MD, contributed data collection and interpreted the data.

Merve Kasikci, MSc, analyzed the data.

Can Kosukcu, MSc, analyzed the data.

Ilhan Tezcan, MD, PhD, supervised the study.

Deniz Cagdas Ayvaz, MD, PhD, conceived, designed, and supervised the study.

Correspondence to Deniz Cagdas.

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Yaz, I., Ozbek, B., Ng, Y.Y. et al. Lymphocyte Subgroups and KREC Numbers in Common Variable Immunodeficiency: A Single Center Study. J Clin Immunol (2020). https://doi.org/10.1007/s10875-020-00761-2

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  • CVID
  • KRECs
  • B cells
  • qRT-PCR
  • Flow cytometry