CLL Increases the Frequency of CD4⁺HLADR⁺PD1⁺ T Cells That Demonstrate an Exhaustion Gene Signature and Potential Prognostic Value for Disease Progression

Alongside the expansion of leukaemic B cells, a common feature across chronic lymphocytic leukaemia (CLL) patients is widespread dysfunction in the T cell compartment. Such dysfunction can include failure to form competent immune synapses and a skewing of the T cell pool towards highly-differentiated memory cells. Previous work from our group has also demonstrated an inversion of the normal CD4⁺:CD8⁺ T cell ratio (such that CD8⁺ cells outnumber their CD4⁺ counterparts) in around a third of patients, correlating with a poor prognosis. In addition, we have defined a previously unreported CD4⁺ T cell subset, co-expressing HLA-DR and PD1, which occurred at higher frequencies in CLL patients compared to healthy controls and was associated with reduced progression-free survival.

In this study, a new larger cohort of over 190 untreated CLL patients has been investigated. The frequency of CD4⁺HLA-DR⁺PD1⁺ T cells was confirmed to be greater in CLL patients (range 0.2 - 42.4%) compared to age-matched healthy donors (range 0.6 - 5.6%), while CLL patients with an inverted CD4⁺:CD8⁺ ratio had higher frequencies than those with a normal ratio.

The nature and function of CD4⁺HLA-DR⁺PD1⁺ T cells was unknown, so phenotypic studies using multiparametric flow cytometry were undertaken. The CD4⁺HLA-DR⁺PD1⁺ T cell population was enriched for actively proliferative (Ki67⁺) and cytotoxic (Granzyme B⁺) cells compared to the total CD4⁺ T cell pool in CLL and showed no signs of cellular senescence (based on CD27 and CD28 expression). The overall patterns of co-expression of 7 phenotypic markers on CD4⁺HLA-DR⁺PD1⁺ T cells were highly complex and significantly different in CLL compared to age-matched healthy controls.

A gene expression analysis using Human Gene 2.0 ST Arrays was conducted with FACS-sorted T cells from 5 CLL patients and 4 age-matched healthy donors, sorted into CD4⁺HLA-DR^-PD1^-, CD4⁺HLA-DR^-PD1⁺ and CD4⁺HLA-DR⁺PD1⁺ populations. CD4⁺HLA-DR⁺PD1⁺ T cells showed enrichment of genes related to T cell exhaustion including TOX, recently reported as critical to exhaustion in CD8⁺ T cells (Alfei et al. 2019; Khan et al. 2019). Comparison of the 3 sorted T cell populations between CLL and healthy donors demonstrated enhanced expression of pathways related to cellular aging and protein turnover in CLL, as well as changes in metabolism.

Longitudinal follow-up of CLL patients using data collected over periods of up to 9 years revealed that the CD4⁺:CD8⁺ T cell ratio, particularly in those with an inverted ratio, was often highly stable. By contrast, the frequency of CD4⁺HLA-DR⁺PD1⁺ T cells was more dynamic, with changes >2 fold observed in the course of just a few weeks in some cases. Interestingly, there was no obvious change to the patterns of CD4⁺HLA-DR⁺PD1⁺ T cell frequency following treatment with ibrutinib over periods of up to 10 months, with these cells remaining a dynamically changing subset during therapy.

Overall, this work has shown that the CD4⁺HLA-DR⁺PD1⁺ T cell subset consists of a phenotypically heterogeneous population of T cells expressing genes associated with exhaustion but not senescence. The exact function of these T cells remains unclear, but CD4⁺HLA-DR⁺PD1⁺ T cells may represent a useful non-tumour biomarker for patients with increased risk of disease progression. This study also demonstrates the global impact of CLL on CD4⁺ T cells, with an effect of premature aging and altered metabolic processes. This may have important implications in the context of CLL for modern therapies which rely on the expansions of T cells, in particular chimeric antigen receptor (CAR) T cell therapy.