CLL Increases the Frequency of CD4+HLADR+PD1+ T Cells That Demonstrate an Exhaustion Gene Signature and Potential Prognostic Value for Disease Progression
Authors: Henley Peter M , Fegan Christopher , Man Stephen 
Published: November 2019

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.

Disclosures
Fegan: Abbvie: Consultancy, Other: Conference attendance sponsorship; Gilead: Honoraria; Janssen: Honoraria; Roche: Honoraria.