Immune Irregularities Detected in Patients With PNH

The development of paroxysmal nocturnal hemoglobinuria (PNH) has been linked to immune abnormalities, according to a recent study published in Saudi Medical Journal.¹

Immune Cells Fighting Disease Concept | image credit: Siarhei - stock.adobe.com

PNH is a disease of the hematopoietic stem cells (HSCs) that arises from an alteration in the phosphatidylinositol glycan biosynthesis class A (PIGA) gene. This mutation causes a deficiency in the expression of glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-AP): the cells lack GPI-AP altogether, including the essential proteins CD55 and CD59.² As a result, patients affected by PNH stand at an increased risk for intravascular hemolysis, bone marrow failure, and thrombosis. These PIGA mutations do occur in healthy individuals, the present authors add; however, in PNH the HSCs exhibit an abnormal reproduction rate of mutated PIGA, which consequently contributes to the hemolytic symptoms of the disease.¹

Furthermore, as Daria Babushok, Division of Hematology-Oncology, Hospital of University of Pennsylvania, expanded upon in a 2021 publication, “a PNH clone can provide an important diagnostic clue about the immune-mediated pathogenesis of marrow aplasia.”² This relationship demonstrates the growing value of unearthing PNH’s underlying mechanisms and associations. As, for example, bone marrow can be severely impacted in patients with PNH, developing a more comprehensive understanding of the disease can help guide and inform earlier interventions to combat the complications brought on by genetic alterations found in PNH.

The authors of the present study pointed to various literature that explores the mechanism of PNH clone proliferation. Currently this research has harkened on the role of immune cells in the autoimmune reactions observed in PNH, especially considering that the GPI-negative (GPI–) mutated HSCs have shown to evade immune responses, whereas GPI-positive (GPI+) cells are not as lucky.¹

To better understand the immunology of PNH, researchers conducted a study to assess different antibodies and the proportion of immune subsets in healthy individuals compared with those with PNH. From January 2021 until June 2023, samples were collected from a total of 25 patients with PNH from the Department of Hematology at Tianjin Medical University General Hospital, Tianjin, China. These samples were evaluated in comparison with those from 50 healthy controls.

The researchers proceeded by using heparin anticoagulation blood collection tubes to collect 2 mL blood samples from patient with PNH. Then, antibodies were added to the tube and incubated before 1 mL of hemolysin was added for further incubation. Afterwards, samples were placed in a centrifuge and subsequently resuspended in 1 mL of phosphate buffer solution (PBS). Following another centrifuge, another 200 μL PBS was added to the tubes and the Beckman CytoFLEX Flow Cytometer was used to detect resuscitated cells.

Results found that the percentage of T-cells (CD3⁺) was significantly higher in patients with PNH compared with healthy controls (median, 83.49% vs. 69.1%; P < .0001). In their analysis of T-cell subset differences, patient with PNH exhibited significantly higher percentages of CD3⁺CD8⁺ (median, 38.46% vs 22.2%) CD8⁺CD4⁺ (median, 0.76% vs 0.25%) compared with their healthy counterparts (P = .0004).

As the researchers conducted a further analysis on the significant subsets, it was observed that concentrations of CD3⁺ cells was positively associated with lactate dehydrogenase (P = .004), Flear cells in monocytes (P = .0303), and indirect bilirubin levels (P = .0379). Additionally, researchers found that patients with PNH had significantly reduced levels of B cells compared with healthy controls (median, 4.2% vs 14.2%; P < .0001), as well as naive B cells (median, 42.4% vs. 71.2%; P < .0001).

Cytokine secretion was assessed in addition to immune cells, with results showing that IL-6 was heightened in patients with PNH (median, 6.92 pg/mL vs 3.49 pg/mL; P = .0418), IL-17A was reduced (3.34 pg/mL vs 4.87 pg/mL; P = .0316), and that IL-17A was linked to anemia severity (P = .0112).

As the authors concluded, they highlighted how these findings suggest the involvement of immune cells in the immunological evasion of GPI-HSCs, which thus encourages PNH clone proliferation and contributes to disease progression. Still, they stated the need for future research to further expand available knowledge on the role of immune cells in the progression of PNH.

References

1. Wang G, Che M, Zeng L, et al. The immunologic abnormalities in patients with paroxysmal nocturnal hemoglobinuria are associated with disease progression. Saudi Med J. 2024;45(4):424-432. doi:10.15537/smj.2024.45.4.20231010

2. Babushok DV. When does a PNH clone have clinical significance? Hematology Am Soc Hematol Educ Program. 2021;2021(1):143-152. doi:10.1182/hematology.2021000245