Hemophagocytic lymphohistiocytosis (HLH) is a severe and life-threatening syndrome characterized by dysregulated immune response and excessive cytokine release. The disease is associated with rapid onset, rapid progression, and high mortality. The underlying immune dysfunction plays a crucial role in the pathogenesis and outcome of HLH [16]. Although the introduction of the HLH-1994 treatment regimen has reduced HLH-related mortality, the long-term survival rate remains unsatisfactory. In recent years, extensive research on the complex immunological properties of HLH has led to the development of clinical trials investigating targeted immunotherapies, significantly impacting the treatment landscape for HLH patients [17, 18]. A comprehensive and systematic evaluation of the host immune status in HLH is essential for better understanding its pathogenesis and early identification of disease occurrence and risk stratification. This, in turn, can guide clinicians to implement timely and effective targeted interventions, ultimately improving patient prognosis. While some studies have reported on lymphocyte subset analysis and immunophenotypic profiles in primary HLH (pHLH) [11, 12] or mixed pHLH and acquired HLH (aHLH) populations [13] data specifically focused on aHLH patients are limited [14, 15]. Therefore, our study aims to provide a more comprehensive assessment of the host immune status in hemophagocytic lymphohistiocytosis, including analysis of lymphocyte subsets, immunophenotypes, and cytokine levels.
Our study shows that the lymphocyte subsets in the HLH group were dysregulated. The percentage of CD8 + T cells was increased but with decreased percentages of CD4 + T cells and NK cells, which were consistent with previous research findings [19, 20]. In the early stage of adult secondary HLH patients, T cells that exert cellular immunity function are mainly CD8 + T cells that are activated and proliferated under antigen stimulation [21], which significantly increases the percentages of T cells and CD8 + T cells. Decreased or absent NK activity is a hallmark and one of the diagnostic criterions of HLH [16]. This criterion relates to NK function [22], but the percentage of NK is also affected. Meanwhile, the absolute numbers of whole lymphocyte subsets were significantly impaired in HLH group. The reasons may be various, including blood thirst caused by macrophage activation, senescence and apoptosis caused by T cell activation, and secondary diseases, such as the immunosuppressive state of tumor, sepsis and other diseases [23–26]. Additionally, specific immune cell phenotypes in HLH patients were investigated. The expression of HLA-DR on CD8 + T cells was found to be increased, indicating an activated immune response in HLH patients. Analysis of T cell phenotypes revealed an increased percentage of central memory (CM) subtype (CCR7 + CD45RA-) in CD8 + T cells, along with a decreased percentage of terminally differentiated effector CD8 + T cells (TEMRA subtype, CCR7-CD45RA+). These findings suggest altered T cell differentiation and activation patterns in HLH.
The dysregulation of B cell subpopulations in HLH patients is a significant observation, as it provides insights into the underlying immune dysfunctions in this syndrome. The alterations in B cell subsets indicate a disturbance in the maturation and differentiation processes of B cells, which may contribute to the pathogenesis of HLH. In HLH patients, the percentages of unswitched B cells (CD27 + IgD+) and double negative B cells (CD27-IgD-) were found to be significantly decreased compared to healthy controls. Unswitched B cells are considered to be naïve or early memory B cells, while double negative B cells are typically associated with transitional or exhausted B cell phenotypes. The reduction in these B cell subsets suggests impaired B cell development or survival in HLH. Conversely, the percentages of memory B cells (CD27 + IgD-) and plasma B cells (CD27 + CD38+) were markedly increased in HLH patients. Memory B cells are involved in the generation of specific immune responses upon re-exposure to antigens, while plasma cells are responsible for antibody production. The expansion of these subsets indicates an active immune response and potential hyperactivation of B cells in HLH.
Our findings revealed notable changes in proinflammatory cells, including monocytes, Th17 cells, and Tfh17 cells, as well as anti-inflammatory cells, such as Treg cells and Th2 cells, in HLH patients. These alterations suggest an imbalance in immune responses and dysregulated cytokine profiles in the disease in HLH. Interestingly, we also observed increased expressions of perforin and granzyme B on NK cells in HLH patients compared to disease controls. Additionally, the expressions of granzyme B on NKT cells were also elevated. This finding is consistent with the notion that mutations in cytotoxic-related genes, such as perforin or granzyme, in NK cells are often considered the primary cause of primary HLH in patients [27]. Perforin plays an crucial role in cytotoxic activity of NK and cytotoxic CD8 T cells [28]. NK cells exhibit spontaneous cytotoxicity toward target cells, which relies on the release of perforin and granzyme B [29]. Previous studies have reported that perforin expression, degranulation, and cytotoxic function of NK cells are generally normal in HLH patients. However, activation markers such as CD69, HLA-DR, and ICAM-1 are highly expressed on the surface of NK cells, suggesting their highly activated state [30, 31]. This contradicts our findings, indicating that there may be variations within the HLH patient population or differences in immune damage severity in the disease control group.
Multivariate Cox analysis and K-M analysis revealed significant correlations between the levels of IL-6 and the percentage of CD3 + T cells and the 28-day overall survival (OS) in patients with HLH. Prognostic analyses focusing on lymphocyte subsets and phenotypes in HLH patients are limited in previous studies, and the conclusions drawn from these studies vary greatly. The discrepancies between our findings and previous studies could be attributed to the high heterogeneity among HLH patients and the varying degree of immune damage observed in the disease control group. Further investigations are needed to elucidate the underlying mechanisms and gain a better understanding of the functional implications of altered immune cell subsets in the context of HLH.