B-Cell Distribution in Immune Thrombocytopenia and Its Relation to Ageing
Authors: Monzón Manzano Elena , Alvarez-Román María Teresa , del Pino Lucía , Acuña Paula , Kapur Rick , Porcelijn Leendert , Martin Salces Monica , G Arias-Salgado Elena , Rivas Pollmar Maria Isabel , Mendoza Ana , López Granados Eduardo , Jimenez Yuste Victor , Butta Nora 
Published: December 2024

Introduction
The spleen plays a critical role in immune thrombocytopenia (ITP), because it is a major site of platelet destruction in many patients and the major secondary organ of autoantibody production, with expansion of germinal centers (GC). After the age of 60, the incidence of ITP peaks.
Recent evidence shows striking shifts in B cell subsets in the elderly, suggesting an important role in the development of age-related immune dysfunction.
We aimed to investigate the distribution of B cells in patients with ITP compared to healthy controls (HC) of the same age range.
Methods
We included 40 patients with primary ITP ≤ 65 years old (yo), 62% female, 58.5±12 yo; platelet count 102 (5-502) platelets/µL; and 40 patients &gt;65 yo, 65% female, 76.5±7 yo, platelet count 117(3-556) platelets/µL; who had not received prior immunosuppressive treatment or B-cell depletion therapy. Sixty HC ≤ 65 yo, 51% female, 54±15 yo, 245 (105-431) platelets/µL; and 40 &gt;65 yo, 57% female, 78±9 yo, 244 (124-663) platelets/µL were included.
Anti-platelet antibodies (anti (a)-GPIIb/IIIa, a-GPV and a-GPIb/IX) were measured by MAIPA.
We characterised the pre-CG compartment of B cells, immature/transitional B-cells [including 3 maturation-associated populations of CD5- CD38++ CD21het CD24 ++ (CD5+ CD38+/++ CD21het CD24++, and CD5+ CD38het CD21+ CD24+ immature B-cells)]; 3 mature naïve B cells (CD21+ CD24+, CD21- CD24++, and CD21- CD24- subsets), together with unswitched IgMD+ memory B cells, switched memory B cells and plasmablasts/plasma cells] using standardized EuroFlow protocols and Infinicyt software.
B cells activating factor (BAFF) was measured in plasma with an ELISA kit (R&D, Spain).
Statistical analyses were performed using Graph Pad Prism 7.0 and two-way Kruskal-Wallis was used for multiple analyses.
Results
We observed significant differences between HC groups. Compared to HC ≤65 yo, HC &gt;65 yo had decreased immature B cell subsets (cells/µL, HC≤65: 34.4±19.7; HC&gt;65: 8.9±10.1, p&lt;0.001); in naïve B cell subsets (cells/µL, HC≤65: 93.6±53.8; HC&gt;65: 30.2±47.4, p&lt;0.001) and memory B cells (cells/µL, HC≤65: 76.8±39.1; HC&gt;65: 31.5±21.0, p&lt;0.001).
A full analysis of the pre-CG LB compartment showed distinct patterns for the subtypes of immature B cells when comparing ITP patients ≤65 yo with age-matched HC: there was no difference in the amount of CD5-CD38++CD21hetCD24+++ but reduced CD5+CD38+/++CD21het CD24++ (cells/µL, HC≤65:8.4±7.6; ITP≤65: 4.4±4.2, p&lt;0.01) and CD5+CD38+CD21+CD24+/++ (cells/µL, HC≤65:24.8±21.5; ITP≤65: 11.7±9.8, p&lt;0.001). A different distribution of naïve mature B cells was also observed: a decreased CD21+CD24+ compartment (cells/µL, HC≤65:92.2±56.1; ITP≤65: 55.3±47.8, p&lt;0.005) and an increase in CD21-CD24- (cells/µL, HC≤65:1.0±0.9; ITP≤65: 2.5±2.0, p&lt;0.001), while the number of CD21-CD24++ did not differ.
For post-CG B cells, ITP patients ≤65 yo had significant reductions in memory LBs (cells/µL, HC≤65:76.8±39.0; ITP≤65: 44.4±33.6, p&lt;0.005), with no differences in plasma cells.
There were no differences between HC&gt;65 and ITP patients &gt;65 in distribution of immature and in post-CG B cells, but a significant increase in CD21-CD24- (cells/µL, HC&gt;65: 1.7±1.3; ITP&gt;65: 2.6±1.9, p&lt;0.01) and CD21-CD24++ (cells/µL, HC&gt;65: 0.1±0.1; ITP&gt;65: 0.4±0.4, p&lt;0.05) in naïve mature B cells.
BAFF, essential to promote B cells maturation, was increased in both ITP groups (pg/mL; HC ≤65 yo: 405.4±78.8, ITP≤65 yo: 674.3±356.9, p&lt;0.001; HC &gt;65 yo: 500.4±193.3, ITP&gt;65 yo: 1100.1±675.0, p&lt;0.001); and no difference between both ITP groups.
Anti-platelet antibodies were detected in 44.0% of patients with ITP ≤65 yo (4.9% a-GPII/IIIa, 4.9% a-GPV, 4.9% a-GPIb/IX; 22.0% with 2 and 7.2% with 3 antibodies); and in 33% of those &gt;65 yo (3.2% a-GPII/IIIa, 6.6% a-GPV, 10.0% a-GPIb/IX; 3.2% with 2 and 10.0% with 3 antibodies).
Conclusion
Age modifies the distribution of B-cells in HC, so it is important to compare immunological data of patients with those from an age-matched HC. Naïve CD21-CD24- were higher in both ITP groups than in their respective HCs. LB subset with low CD21 expression is also expanded in other autoimmune diseases (Saadoun et al, PMID: 23279883). Our results highlight the need to identify patients who are more likely to respond to B-cell targeted therapies in order to tailor their individual treatment options.
Funded by ISCIII-European Union (PI22/01489), and by Beca Luis Álvarez 2022-IdiPAZ.