Paper Details

ABSTRACT

Introduction

Adult T-cell leukemia/lymphoma (ATLL) is a rare and fatal malignancy that develops in 3-5% of HTLV-1 carriers. In the U.S., two-thirds of ATLL patients are immigrants from the Caribbean islands. ATLL patients have limited treatment options especially those diagnosed outside of Japan. In more than 80% of the cases, the malignant T cells have a CD4⁺CD8^-CD7^-CD25^hiCADM1⁺ phenotype. These ATLL cells also bear some resemblance to regulatory T cells (Tregs). It is known that HTLV-1-specific CD8⁺ cytotoxic T lymphocytes (CTLs) play a critical role in restricting the expansion of virus-infected T cells and preventing ATLL development. Yet, their functional state after diagnosis and potential contribution to durable therapeutic response have yet to be investigated.

Methods

We developed a 30-marker spectral flow panel to immune profile patient PBMC samples at different clinical stages, including longitudinally collected samples from the highly promising Belinostat/ Zidovudine/Interferon-α combination therapy (NCT02737046). This Phase II consolidation trial produced an overall response rate of 57.1%, and a complete molecular response rate in the blood compartment of 33.3%, which are superior to most Phase I and Phase II trials in ATLL patients. To characterize phenotypic changes and clonal dynamics at single-cell resolution, we performed scRNA-seq, scTCR-seq, and CITE-seq on sort-purified CD8⁺ T cells. These cells were collected across three time points from two patients who achieved a complete response (CR).

Results

We confirmed that circulating ATLL cells present a phenotype resembling Tregs despite a high degree of heterogeneity. Most importantly, we discovered that PBMCs from treatment-naïve cases contain a unique population of phenotypically exhausted CD8⁺ T cells (PD-1⁺TIGIT⁺TOX⁺). Most interestingly, in patients who underwent a combined antiviral (ZDV)/ belinostat (a histone deacetylase inhibitor)/(IFN)a treatment (i.e.,the “Belinostat trial”) and achieved durable remission, this unique exhausted CD8⁺ T cell subset was decreased while a subset of CD45RA⁺CD62L^low T-BET⁺BCL6⁺ effector memory CD8⁺ T (T_EMRA) cells expanded and expressed high levels of cytolytic markers (Perforin⁺Granzyme B⁺). Single-cell transcriptomic analysis of circulating CD8⁺ T cells in two CR patients revealed discrete functional subsets of CD8⁺ T cells that include robust cytolytic, exhausted-proliferating, and NK-like effector cells. Moreover, longitudinal T cell clone fate mapping by TCR sequencing highlighted four distinct clonal behaviors, namely i) “early-dominant”, ii) persister, iii) transient expansion, and iv) “late boomer” clones, which largely maintain their phenotypes for up to 2 years, suggesting epigenetic and functional stability. Remarkably, in both patients, while ~half of expanded clones are persisters, the other half at the time of remission or second round of therapy are newly emergent, “late bloomer” cytolytic effector clones that may recognize new ATLL antigens.

Conclusions

Our study reveals for the first time a unique subset of cytotoxic CD8⁺ T cells at ATLL diagnosis that are highly proliferative yet phenotypically exhausted. Longitudinal analysis of two complete responders from the belinostat trial suggests that the anti-HTLV-1/ATLL CD8⁺ T-cell response actively drives durable clinical responses. Therefore, we propose that optimal ATLL therapeutic strategies must concurrently eliminate leukemic T cells and reinvigorate host CD8+ T-cell immunity to ensure lasting remission.