Blood Res.  2024;59:16. 10.1007/s44313-024-00017-7.

Abnormal frequency of the memory B cell subsets and plasmablasts in patients with congenital severe hemophilia A: correlation with “Inhibitor” formation

• Zekavat O
• Movahednezhad Y
• Shahsavani A
• Haghpanah S
• Shokrgozar N
• Golmoghaddam H
• Kalani M
• Bordbar M
• Arandi N

• KMID: 2562520
• DOI: http://doi.org/10.1007/s44313-024-00017-7

Abstract

Background
Development of antibodies against infused Factor VIII (FVIII) or "inhibitors" represents a major challenge following FVIII replacement therapy in patients with hemophilia A (HA). Recent studies have shown that certain cellular compartments of the immune system contribute to the production of such antibodies. Herein, we determined the frequency of class-switched ­CD19⁺IgD⁻CD27⁺ /non-class-switched ­CD19⁺IgD⁺CD27⁺ memory B cell subsets and ­CD19 + CD27^hiCD38^hi plasmablasts in patients with severe HA and their association with the development of inhibitors in these patients.
Methods
This cross-sectional case–control study enrolled 32 patients with severe HA, including 8 with and 24 without inhibitors, and 24 healthy individuals. The frequencies of the memory B cell subsets and plasmablasts were determined using flow cytometry.
Results
The frequency of ­­CD19⁺IgD⁺CD27⁺ non-class-switched memory B cells was significantly lower in patients with HA (including both patients with and without inhibitors) than in healthy controls. The percentages of both ­CD19⁺IgD⁻CD27⁺ class-switched and ­­CD19⁺IgD⁺CD27⁺ non-class-switched memory B cells did not differ significantly between patients with and without inhibitors. HA patients with inhibitors had significantly higher proportions of ­CD19⁺CD27^hiCD38^hi plasmablasts than the control group as well as the inhibitor (-) ones. No significant correlation was observed between the inhibitor levels with the percentages of memory B cell subsets and plasmablasts.
Conclusion
This study is the first to demonstrate a dysregulated proportion of ­CD19⁺IgD⁺CD27⁺ non-class-switched memory B cells and ­CD19⁺CD27^hiCD38^hi plasmablasts in patients with severe HA. Therefore, strategies targeting memory B-cell/plasmablast differentiation may have promising outcomes in the management of inhibitor formation in patients with severe HA.

Keyword

Severe hemophilia A; Inhibitors; Memory B cells; Plasmablasts

Figure

• 

  Fig. 1 Flow cytometry analysis of class-switched (CD19+IgD−CD27+) and non-class switched (CD19+IgD+CD27+) memory B cells

• 

  Fig. 2 Flow cytometry analysis of CD19+CD27hiCD38hi plasmablasts

• 

  Fig. 3 Comparison of the frequency of memory B cell subsets and plasmablasts between patients with HA and healthy controls (A) and between HA patients with/without inhibitors and healthy controls (B) Data are presented as mean ± SD; *P < 0.05, **P < 0.001

• 

  Fig. 4 Correlation between the inhibitor levels with the frequency of class-switched CD19+CD27+IgD− memory B cells (A), non-class-switched CD19+CD27+IgD+ memory B cells (B) and plasmablasts (C) in inhibitor (+) patients with HA

Reference

1. Ehrenforth S, Kreuz W, Scharrer I, et al. 1992; Incidence of development of factor VIII and factor IX inhibitors in haemophiliacs. Lancet. 339:594–8. DOI: 10.1016/0140-6736(92)90874-3. PMID: 1347102.

2. Graw J, Brackmann HH, Oldenburg J, Schneppenheim R, Spannagl M, Schwaab R. 2005; Haemophilia A: from mutation analysis to new therapies. Nat Rev Genet. 6:488–501. DOI: 10.1038/nrg1617. PMID: 15931172.

3. Hoyer LW, Hemophilia A. 1994; N Engl J Med. 330:38–47. DOI: 10.1056/NEJM199401063300108. PMID: 8259143.

4. Hoyer LW. 1995; The incidence of factor VIII inhibitors in patients with severe hemophilia A. Adv Exp Med Biol. 386:35–45. DOI: 10.1007/978-1-4613-0331-2_3. PMID: 8851013.

5. Mannucci PM, Tuddenham EG. 2001; The hemophilias-from royal genes to gene therapy. N Engl J Med. 344:1773–9. DOI: 10.1056/NEJM200106073442307. PMID: 11396445.

6. Oliveira CA, Velloso-Rodrigues C, Machado FC, et al. 2013; Cytokine profile and FVIII inhibitors development in haemophilia A. Haemophilia. 19:e139–42. DOI: 10.1111/hae.12096. PMID: 23387800.

7. Oldenburg J, Schröder J, Brackmann HH, Müller-Reible C, Schwaab R, Tuddenham E. 2004; Environmental and genetic factors influencing inhibitor development. Semin Hematol. 41(1 Suppl 1):82–8. DOI: 10.1053/j.seminhematol.2003.11.016. PMID: 14872427.

8. White GC 2nd, Kempton CL, Grimsley A, Nielsen B, Roberts HR. 2005; Cellular immune responses in hemophilia: why do inhibitors develop in some, but not all hemophiliacs? J Thromb Haemost. 3:1676–81. DOI: 10.1111/j.1538-7836.2005.01375.x. PMID: 16102033.

9. Arandi N, Zekavat OR, Shokrgozar N, Shahsavani A, Golmoghaddam H, Kalani M. 2023; Altered frequency of FOXP3+ regulatory T cells is associated with development of inhibitors in patients with severe hemophilia A. Int J Lab Hematol. 45:953–60. DOI: 10.1111/ijlh.14139. PMID: 37488961.

10. Ding KY, Ji WC, Wu JS, Li T, Sheng YY. 2014; Higher frequency of CD4(+) CD25(high) Treg cells in hemophilia patients with factor VIII inhibitor. Genet Mol Res. 13:1774–81. DOI: 10.4238/2014.March.17.5. PMID: 24668665.

11. El-Asrar MA, Hamed Ael-S, Darwish YW, Ismail EA, Ismail NA. 2016; Assessment of the frequency of regulatory T cells (CD4+CD25+CD127-) in children with hemophilia A: relation to factor VIII inhibitors and disease severity. Blood Coagul Fibrinolysis. 27:42–6. DOI: 10.1097/MBC.0000000000000377. PMID: 26226256.

12. Becker-Gotot J, Meissner M, Kotov V, et al. 2022; Immune tolerance against infused FVIII in hemophilia A is mediated by PD-L1+ Tregs. J Clin Invest. 132:e159925. DOI: 10.1172/JCI159925. PMID: 36107620. PMCID: PMC9663153.

13. Kurosaki T, Kometani K, Ise W. 2015; Memory B cells. Nat Rev Immunol. 15:149–59. DOI: 10.1038/nri3802. PMID: 25677494.

14. Seifert M, Küppers R. 2016; Human memory B cells. Leukemia. 30:2283–92. DOI: 10.1038/leu.2016.226. PMID: 27499139.

15. Inoue T, Moran I, Shinnakasu R, Phan TG, Kurosaki T. 2018; Generation of memory B cells and their reactivation. Immunol Rev. 283:138–49. DOI: 10.1111/imr.12640. PMID: 29664566.

16. Kurosaki T, Aiba Y, Kometani K, Moriyama S, Takahashi Y. 2010; Unique properties of memory B cells of different isotypes. Immunol Rev. 237:104–16. DOI: 10.1111/j.1600-065X.2010.00939.x. PMID: 20727032.

17. Irigoyen MB, Felippo ME, Primiani L, et al. 2012; Severe haemophilia A patients have reduced numbers of peripheral memory B cells. Haemophilia. 18:437–43. DOI: 10.1111/j.1365-2516.2011.02642.x. PMID: 21910787.

18. Verbruggen B, Novakova I, Wessels H, Boezeman J, van den Berg M, Mauser-Bunschoten E. 1995; The Nijmegen modification of the Bethesda assay for factor VIII: C inhibitors: improved specificity and reliability. Thromb Haemost. 73:247–51. DOI: 10.1055/s-0038-1653759. PMID: 7792738.

19. Patel SR, Lundgren TS, Baldwin WH, et al. 2022; Neutralizing antibodies against factor VIII can occur through a non-germinal center pathway. Front Immunol. 13:880829. DOI: 10.3389/fimmu.2022.880829. PMID: 35634288. PMCID: PMC9132091. PMID: 470104dc411c45558fc574d5808a9c73.

20. Nutt SL, Hodgkin PD, Tarlinton DM, Corcoran LM. 2015; The generation of antibody-secreting plasma cells. Nat Rev Immunol. 15:160–71. DOI: 10.1038/nri3795. PMID: 25698678.

21. Benson G, Auerswald G, Elezović I, et al. 2012; Immune tolerance induction in patients with severe hemophilia with inhibitors: expert panel views and recommendations for clinical practice. Eur J Haematol. 88:371–9. DOI: 10.1111/j.1600-0609.2012.01754.x. PMID: 22260405.

22. Nakar C, Shapiro A. 2019; Hemophilia A with inhibitor: Immune tolerance induction (ITI) in the mirror of time. Transfus Apher Sci. 58:578–89. DOI: 10.1016/j.transci.2019.08.008. PMID: 31447396.

23. Liu CL, Lyle MJ, Shin SC, Miao CH. 2016; Strategies to target long-lived plasma cells for treating hemophilia A inhibitors. Cell Immunol. 301:65–73. DOI: 10.1016/j.cellimm.2016.01.005. PMID: 26877251. PMCID: PMC4844017.

24. Reipert BM, Allacher P, Hausl C, et al. 2010; Modulation of factor VIII-specific memory B cells. Haemophilia. 16:25–34. DOI: 10.1111/j.1365-2516.2008.01962.x. PMID: 20536983.

25. Doshi BS, Rana J, Castaman G, et al. 2021; B cell-activating factor modulates the factor VIII immune response in hemophilia A. J Clin Invest. 131:e142906. DOI: 10.1172/JCI142906. PMID: 33651716. PMCID: PMC8262462.

26. Reipert BM. 2016; B-cell memory against factor VIII. Cell Immunol. 301:49–58. DOI: 10.1016/j.cellimm.2016.01.003. PMID: 26780996.