Proliferation of a Novel CD3neg Hyperactivated T-Cell Population Characterizes Adult Hemophagocytic Lymphohistiocytosis and Associates with Disease Activity
Authors: Rocco Joseph M. , Laidlaw Elizabeth , Yates Bonnie , Schroeder Brett , Limerick Emily , Johnson William T. , Oved Joseph H. , Galindo Frances , Ewing Whitney , Fitzhugh Courtney , Manion Maura , Lisco Andrea , Holland Steven , Shah Nirali N. , Sereti Irini 
Published: December 2024

Introduction: Pathologic T-cell activation occurs in children with hemophagocytic lymphohistiocytosis (HLH) leading to increased IFNγ production which drives its pathogenesis. However, HLH in adults can occur due to diverse etiologies with significant variability in disease severity. The pathogenesis of adult HLH remains undefined and characterizing mechanisms of disease and key prognostic factors are a high priority.

Methods: We performed high-dimensional flow cytometry on peripheral blood (PB) samples from 30-patients meeting either or both HLH-2004/H-Score criteria. PB was collected at acute HLH presentation in 22-patients and after HLH-directed treatment in 8-patients. Potential triggers were diverse including malignancy (n=8 [post-CD22 CAR T-cell, n=4]), genetic (n=3), rheumatologic (n=3), post-allogeneic stem cell transplant (n=2), and infection-associated (n=14). Eight-individuals with infection-associated HLH improved with treatment of infection alone and were further separated into an HLH-mimic subgroup. A 30-marker flow panel including monocyte and T-/NK-cell markers of differentiation, activation, and function was performed. Comparisons were made between the full HLH cohort (n=30) and individuals with severe coronavirus disease-2019 (COVID-19, n=6) and healthy volunteers (HVs, n=12). Additional comparisons were made within the HLH cohort between those sampled prior to treatment (Acute group, n=14), post-treatment (Post-Tx group, n=8), and the HLH-mimic group (n=8). A follow-up PB sample, collected 1-week after treatment initiation, was also evaluated in 8-patients within the Acute group. Unsupervised clustering and differential abundance analyses were performed.

Results: 30-patients meeting HLH criteria with a median age of 38-years (range: 19-70) were analyzed. Thirteen (43%) were female and 22 (73%) were non-White. CXCL9 (median: 43508pg/mL [IQR: 11412-56783) and soluble CD25 (median: 4002pg/mL [IQR: 3035-8506]) were elevated in all patients. Activated (CD38hiHLADR+) CD4 T-cells (median: 26% [IQR: 10-42]) and CD8 T-cells (median: 44% [IQR: 28-58]) were significantly increased in HLH compared to severe COVID19 (CD4: 2.4% [1.7-3.1]; CD8: 3.4 [3.3-5.6]) and HVs (CD4: 1.2% [0.8-2.1]; CD8: 2.1 [1.4-3.2]) (p&lt;0.01). There were no significant differences in activated T-cells between the HLH subgroups (Acute vs Post-Tx vs HLH-mimics). Unsupervised clustering analysis identified three unique activated T-cell populations (CD38hiHLADR+) which expressed high levels of cell cycling (Ki-67hi), Th1 differentiation (EOMES+), and STAT1 phosphorylation. Interestingly, two of these hyperactivated T-cell subsets demonstrated loss of CD3, CD25, and CD28 expression despite preserved CD4 and CD8 expression. These CD3neg populations also showed greater levels of activation and trajectory analysis indicated they developed from the CD3+ activated T-cell subset. Additionally, the CD3+ T-cells had increased PD1 and IL18R expression, however these markers were lost on the CD3neg hyper-activated T-cells which also expressed both naïve/memory (CD127+) and terminal differentiation (CD57+) markers. Comparisons within the HLH cohort revealed significantly increased CD3neg hyperactivated T-cells in the Acute subgroup (median: 5.5% of CD45+ cells [2.8-11.1]) compared to the Post-Tx (median: 0.63% [0.38-0.93]) (p=0.005) and HLH mimic (median: 0.45% [0.18-1.0]) (p&lt;0.001) subgroups. In longitudinally sampled HLH-patients, this CD3neg T-cell subset significantly decreased during treatment response.

Conclusions: A unique CD3neg hyperactivated T-cell population is expanded in adult HLH. This T-cell subset was found specifically in individuals with severe hyperinflammation requiring more aggressive treatment irrespective of the etiologic trigger. Mechanisms driving the expansion and functional characterization of this novel population require further study as it could provide new insights into the pathogenesis of HLH and may signify an adult population that can benefit from HLH-targeted immunosuppressive therapy.