Clonal, Exhausted and Activated Infiltrating T Lymphocytes in Langerhans Cell Histiocytosis Lesions

Background: Langerhans Cell Histiocytosis (LCH) is characterized by inflammatory lesions with pathologic CD207⁺ dendritic cells (DCs) with constitutively activated ERK. LCH lesions are associated with a local cytokine storm and exhibit a heterogeneous collection of inflammatory infiltrates (eosinophils, neutrophils and lymphocytes). The mechanisms behind tumor formation, both accumulation of pathologic CD207⁺ DCs as well as recruitment of inflammatory infiltrate, remain to be defined. Mutually exclusive somatic mutations in MAPK pathway genes have been identified in approximately 75% of LCH cases, including recurrent BRAF-V600Eand MAP2K1 mutations. However, the inflammatory infiltrate within LCH lesions is composed of cells without any BRAF mutation or ERK activation. We hypothesize that the pathologic inflammation in LCH is orchestrated by interactions between the pathologic MAPK-activated DCs and normal immune cells. Defining the nature and function of infiltrating T cells in LCH will not only provide insights into mechanisms of pathogenesis and potential therapeutic targets, but may also inform novel physiologic mechanisms of DC-mediated T cell function.

Methods: Clonality was investigated by exploring T cell receptor beta (TCRB) rearrangement in LCH patients. Quantitative analysis of different immune cell populations in the tumor microenvironment in 25 LCH patients was performed using spanning tree progression of density normalized events (SPADE).

Results: Our initial experiments identified clonal populations among LCH lesion-infiltrating CD3⁺ T cells. The complementarity-determining region 3 (CDR3) of the TCRBV07 gene demonstrated marked monoclonal expansion, suggesting a potential involvement of antigen selection in the pathogenesis of LCH. SPADE analysis of infiltrating CD3⁺ T cells revealed high expression of T cell immunoglobulin mucin (TIM-3), lymphocyte activated gene 3 (LAG3) and programmed cell death protein 1 (PD-1) compared to healthy controls, indicating that effector T cell exhaustion might be an important mechanism of immune dysregulation in LCH patients. In addition, in a cohort of LCH patients harboring wild-type BRAF, but not the BRAF-V600E mutation, a distinct subset of infiltrating CD3⁺ T cells were detected expressing both CD4 and CD8α receptors at high levels (double-positive, DP). Preliminary analysis showed high levels of IFNγ, but not TNF-α, after stimulation of these DP T cells.

Conclusions: The results from this study support a model in which LCH lesion DCs with activated MAPK signaling select and recruit activated T cells with potential for clonal expansion to LCH lesions. Multiple markers of T cell exhaustion were identified on infiltrating LCH lesion T cells. While the significance of double-positive T cells in LCH is uncertain, similar cells have been reported in the target organs of several auto-immune conditions and hematological and solid malignancies, consistent with double-positive T cells in sites of immune exhaustion. The function of this double-positive T cell population remains to be defined, but we postulate that these cells may participate in tumor immune responses in LCH due to their potential of having broad tumor reactivity. While the CD207⁺ DCs in LCH lesions is histologically uniform across the disease spectrum, the composition of the immune microenvironment is variable. The variability of the DP T cell population in BRAF-V600E vs BRAF wild-type DCs study suggests that specific mechanisms of MAPK activation may have some distinct impact on T cell recruitment and activation. While recent discoveries have enhanced understanding of the origin and aberrant MAPK activation of the pathologic DCs, very little is known about the role of lymphocytes or the inflammatory microenvironment. Characterizing the populations, function, and interactions of the LCH lesion immune cells may provide novel therapeutic opportunities, such as checkpoint inhibitors. Further, identifying the mechanisms that underlie the "cytokine storm" of LCH lesion may inform physiologic interactions between MAPK-activated DCs and effector immune cells.