T Cells from Chronic Lymphocytic Leukemia (CLL) Patients Display Dysregulated Expression of Immune Checkpoint Molecules and Activation Markers Mainly Restricted to CD4+ Cells and Correlated with Disease Activity
Authors: Palma Marzia , Gentilcore Giusy , Mozaffari Fariba , Heimersson Kia , Näsman-Glaser Barbro , Hansson Lotta , Österborg Anders , Mellstedt Håkan 
Published: October 2019

Abstract
Background
CLL patients (pts) have impaired humoral and cellular immune functions, which is largely due to profound defects of T-cells. Regulation and activation of T lymphocytes depend not only on T cell receptor signaling but also on co-signaling receptors delivering either inhibitory or stimulatory signals, known as immune checkpoints.
CTLA-4 (cytotoxic T lymphocyte-associated antigen-4) is transiently expressed on activated T cells, binding the same ligands as CD28, inhibiting T-cell activation. Similarly, programmed cell death protein 1 (PD-1) is expressed on activated CD4+ and CD8+ T cells inhibiting T-cell functions upon binding to the ligands B7-H1 (PD-L1, CD274) and B7-DC (PD-L2, CD273). CD137 is an inducible costimulatory receptor expressed by activated T cells.
Dysregulated expression of immune checkpoint receptors on T cells of CLL pts may have an impact on T-cell responsiveness and might be a mechanism for the immune deficiency in the disease.
Aim
To evaluate the expression of the immune checkpoint molecules CTLA-4, PD-1 and CD137 as well as of the cell proliferation marker Ki67, the activation marker CD69 and of CD103, a marker expressed on regulatory T cells, in T cells from CLL pts in different disease phases.
Methods
Peripheral blood samples were obtained from 69 CLL pts and 13 healthy control donors (HD). Pts were sub-grouped according to disease phase: indolent vs progressive (i.e. fulfilling criteria for active disease). The expression of CTLA-4, PD-1, PD-L1, CD69, CD103, CD137 and Ki-67 was assessed by flow-cytometry on CD4+ and CD8+ T cells. We also analysed the change in expression of these markers on T cells after 72 hours of PHA stimulation.
Results
CLL pts (n=17) had a significanty higher percentage of proliferating (Ki67+) CD3+ cells compared to HD (n=7) (median 3.7% in progressive vs 1.7% in indolent CLL vs 0.9% in HD, p=0.004 and p=0.04, respectively) (Fig.1). Progressive CLL pts had a significantly higher percentage Ki67+ CD4+ compared to indolent pts as well as HD (p=0.007 and p=0.001, respectively). Both indolent and progressive pts had higher percentage of Ki67+ CD8+ T cells compared to HD (p=0.01 and p=0.03, respectively).
The percentage of CTLA-4+ CD4+ and CTLA-4+ CD8+ cells was low in CLL pts as well as in HD. However, the percentage of PD-1+ CD4+ T cells was significantly higher in progressive (n=32) as compared to indolent (n=35) CLL pts (median 40.3% vs 23.3%, p<0.0001) and HD (n=13) (median 21.5%, p<0.0001) (Fig.2) and correlated positively to the white blood cell counts (WBC) at the time of testing (r=0.29, p=0.03), while no difference was found with regard to the percentage of PD-1+ CD8+ T cells. No difference was observed between CLL pts and HD regarding the expression of PD-L1 on T cells.
Both the percentage of CD69+ CD4+ and CD137+ CD4+ T cells were significantly higher in progressive as compared to indolent disease and correlated positively to WBC while no difference was found seen in CD8+ T cells.
The percentage of CD103+ T cells was significantly lower in progressive compared to and HD within both the CD4+ (p=0.02) and the CD8+ subpopulations (p=0.02).
After 72-hrs of PHA stimulation, PD-1 and CTLA-4 expression increased in CD4+ and CD8+ cells to a similar extent in CLL pts and HD, while PD-L1 increased in HD but not in progressive CLL pts (p=0.03 and p=0.007 for CD4+ and CD8+ cells, respectively). CD69 expression increased to a similar extent in CLL pts and HD, while CD137 expression increased more in T cells from progressive pts compared to HD (p=0.03 and 0.01 for the CD4+ and CD8+ cells, respectively). No increase in CD103 on CD8+ T-cells was observed in CLL pts compared to HD (p=0.04 and p=0.01 for the indolent and progressive pts, respectively).
Conclusions
Progressive CLL pts have more proliferating (Ki67+) T cells in both the CD4+ and CD8+ compartments compared to HD. CD4+ T-cells in progressive CLL pts display an activated phenotype (CD69+) and express the immune co-stimulatory molecule CD137 at a significantly higher level compared to indolent pts and HD. Nevertheless, the expression of the inhibitory immune checkpoint molecule PD-1 is so high that it is reasonable to assume that these cells are heavily impaired in their immune functions.
The differences observed in the expression of immune checkpoints and activation markers between CLL pts in different phases of the disease suggest that major changes occur in the CD4+ T-cell compartment during disease progression.
Figure 1. Figure 1. Figure 2. Figure 2.

Disclosures
Hansson: Jansse Cilag: Research Funding. Österborg:Janssen, Pharmacyclics, Gilead: Consultancy, Research Funding; Novartis: Research Funding.