T Cells in CTCL Have an Exhausted Phenotype While Cutaneous Dendritic Cells Display a Normally Activated Mature Phenotype

Background: Mycosis fungoides (MF) and Sézary syndrome (SS) represent the most common types of cutaneous T-cell lymphoma (CTCL). CTCL develops from clonally-expanded, effector/central memory CD4+ T cells in a background of chronic inflammation. The eliciting Ag(s) and how this inflammation contributes to the development and progression of CTCL remain important unknowns. Despite tumor-infiltrating CD8+ T cells in skin, the malignant CD4+ T-cell population persists, indicating that these CD8+ T cells cannot exert an effective anti-tumor response, potentially due to T-cell exhaustion, which has been observed in chronic viral infections and is associated with disease progression in hematologic malignancies like ATLL and CLL. Exhausted T-cells are characterized by immunoinhibitory molecules, like programmed death-1 (PD-1). Engagement of PD-1 by PD-Ligand (L)-1/L-2 transduces a signal that leads to inhibition of T-cell functions, but the cells are not deleted. PD-L1 is expressed on T cells, dendritic cells (DCs), and many tumor cells, whereas PD-L2 expression is limited to antigen-presenting cells. Although malignant CD4+ T cells in Sézary syndrome lesions express PD-1, the relevance of the PD-1/PD-L1/2 pathway in CTCL and the immune cells in CTCL that express PD-L1/L2 are not known.

Methods: Epidermis and dermis of skin biopsies from consented CTCL pts (12) and healthy donors (3) were separated using dispase-II and incubated in RPMI-10% pooled human serum for 24 hrs. Migrated cutaneous T cells and DCs were phenotyped by flow cytometry for a variety of maturation markers and immune checkpoint molecules. In 3 pts skin findings were compared with matched PBMCs.

Results: Freshly isolated cutaneous CD8+ T cells displayed distinct effector and memory subsets with a higher frequency of effector-memory (CD28^-CD45RA^-CCR7^-) and terminally differentiated effector cells (CD28^-CD45RA⁺CCR7^-). In addition, CD8+ T-cells exhibited enhanced expression of PD-1, Lag-3, Tim-3, and ICOS. PD-1 was the receptor most overexpressed in CD8+ T cells compared with healthy controls. PD-1 expression on circulating CD8+ T cells was lower compared with skin-infiltrating CD8+ T-cells. In contrast, the malignant CD4+ T-cells expressed predominantly a central memory or transient memory subtype (CD28⁺CD45RA^-CCR7^+/-), with overexpression of PD-1, CTLA-4, Lag-3, and ICOS. Immune-suppressive CD3⁺CD4⁺FoxP3⁺CD127^-Tregs were not increased in PBMCs of CTCL pts. Migrated DCs displayed an activated, mature phenotype expressing HLA-DR⁺, CD1a^+/-, CD207 (Langerin)^+/-, CD14^+/-, CD11c⁺, CD83^low/med, CD86^low/med and PD-L1^+/- phenotype.

Conclusion: Both the malignant CD4+ T cells and tumor-infiltrating CD8+ T cells display an exhausted phenotype, while DC subsets display a normal activated phenotype. Our preliminary data imply a role for PD-1 and other checkpoint molecules like ICOS, Tim-3, Lag-3 in attenuating the anti-tumor response. Studies are underway in vitro to establish the function of PD-1, Lag-3, Tim-3, and ICOS on T cells in response to DC/LC stimulation to develop a rationale for therapeutic checkpoint blockade inhibitors to reverse the exhausted T cell phenotype and attendant lack of immune reactivity.