CD8+ T Lymphocytes Immune Depletion and LAG-3 Overexpression in Hodgkin Lymphoma Tumor Microenvironment Exposed to Anti-PD-1 Immunotherapy

Abstract

Background: Resistance to anti-PD-1 remains a considerable clinical challenge for the treatment of patients with classical Hodgkin lymphoma (cHL), and mechanisms of anti-PD-1 resistance remain unknown. This pilot study aims to investigate the tumor microenvironment in patients with cHL relapsing after anti-PD-1. Methods: This study investigated tumor samples of eight patients with cHL, including four patients exposed to anti-PD-1 with a paired longitudinal histological analysis before and after anti-PD-1, and four patients not exposed to anti-PD-1 who served as control for the cellular biological investigations. Fresh cells tumor microenvironment analysis included phenotypic characterization of their T cell surfaces immune checkpoint markers PD-1, PD-L1, ICOS, TIM-3, LAG-3, 41-BB and BTLA. Tumor tissues immunohistochemistry staining included CD30, CD4, CD8, CD68, CD163, PD-L1, PD-1, LAG-3 and TIM-3. Findings: Paired longitudinal tumor tissues analysis in the tumor microenvironment found a CD8+ lymphocytes tumor depletion and an increase of LAG-3 staining after anti-PD-1 exposure. The fresh cells analysis of the tumor microenvironment in patients exposed to anti-PD-1 found CD8+ lymphocyte depletion, with an elevated CD4+/CD8+ lymphocytes ratio (median ratio 9.77 in exposed anti-PD-1 versus 2.39 in not-exposed anti-PD-1 patients; p = 0.0943). On the cell surfaces of CD4+ lymphocytes, the median positive expression of LAG-3 was significantly higher in the samples exposed to anti-PD-1 compared to the controls (15.05 [IQR:17.91–10.65] versus 3.84 [IQR 1.87–6.57]; p = 0.0376). Interpretation: This pilot study proposes hypotheses for understanding the resistance to immunotherapies in patients with Hodgkin lymphoma. Hodgkin lymphoma exposed to anti-PD-1 correlated in tumor microenvironment with an immune depletion of CD8+ T lymphocytes and overexpression of LAG-3 on CD4+ helper T lymphocytes.

A unique expression pattern of LAG3 distinct from that of other immune checkpoints in diffuse large B‐cell lymphoma

“…Contrary to our observation that the expressions of PD‐L1 and LAG‐3 were negatively correlated, LAG‐3 was modestly correlated with PD‐L1 expression in this study. In contrast, in a paired longitudinal tumor tissue analysis performed on cHL, CD8‐positive T‐cell depletion and increased LAG‐3 expression were observed after anti‐PD‐1 therapy 28 . In the cell surface analysis of CD4‐positive T‐cells, LAG‐3 expression was significantly higher in the patient samples treated with PD‐1 than in the control group.…”

Advancement of anti‐LAG‐3 in cancer therapy

“…LAG‐3 receptors are fundamentally expressed on nearly all T lymphocytes, including activated CD4 + T cells, 13,14 CD8 + T cells, 37 Foxp3 + regulatory T cells, 38–41 naïve T cells, 27 and some activated CD4 + differentiated subsets (Th1, Th0) 12,42 . The primary biological function of LAG‐3‐expressing T lymphocytes is immune suppression.…”

Resistance mechanisms of immune checkpoint inhibition in lymphoma: Focusing on the tumor microenvironment

“…The role of LAG-3 in resistance to PD-1 blockade has been indicated by several studies in HL. First of all, overexpression of LAG-3 was observed on CD4 + T lymphocytes in TME after exposing to anti-PD-1 therapy ( Michot et al, 2021 ). Moreover, using single cell RNA sequencing, a cluster of type 1 T regulatory (Treg) cells was identified to highly express LAG-3 but not PD-1, exhibiting a significant immunosuppressive effect on T cell ( Aoki et al, 2020 ).…”