Evaluation of T Cell Compartment By Mass Cytometry Reveals Distinct Patterns of Expression of Exhaustion Markers in Chronic Lymphocytic Leukemia

T cell exhaustion is characterized by the coordinated expression of a series of inhibitory receptors such as programmed death-1 (PD-1), 2B4, CD160 and TIGIT, arising in the context of chronic infection and cancer. It is a progressive process characterized by the stepwise acquisition of inhibitory receptors and a corresponding decrease in effector function. The recent interest in strategies to invigorate tumor specific T cells and the use of checkpoint inhibitors have provided a paradigm-shift in cancer treatment. However, not all patients achieve a favorable clinical response to checkpoint blockade. Thus, there is an urgent need to understand the underlying mechanisms that lead to immunotherapy resistance.

To this end, we designed a Cytometry by Time-of-Flight (CyTOF) panel of 40 monoclonal antibodies recognizing maturation markers, exhaustion markers, homing and chemokine receptors, and performed a detailed single cell analysis of the T cell repertoire in peripheral blood samples collected from 46 untreated CLL patients and 21 age-matched healthy controls. CD8+ T-cells from patients with CLL had significantly higher expression of a number of checkpoint molecules and differentiation markers, including 2B4 (75.7%±2.2% vs 46.9%±3.6%, p<0.0001), TIGIT (59.3%±2.5% vs 41.05%±2.7%, p<0.0001), PD-1 (42.8%±2.6% vs 32.4%±2.07% p<0.05), CD160 (33.6%±2.2% vs 25.4%±2.7% p<0.05), CD57 (49.1%±3.2% vs 26.4%±3.2%, p<0.0001) and KLRG1 (74.7%±2.9% vs 46.1%±5.2%, p:0.0005) compared to healthy controls. Similarly, CD4+ T cells in CLL expressed significantly higher levels of inhibitory molecules, including TIGIT (28.6%±2.07% vs 18.1%±0.7% p=0.0005), CD39, a marker of exhaustion involved in the generation of the immunosuppressive molecule adenosine (13.7%±2.4% vs 5.5%±0.6% p<0.005) and PD-1 (43.5%±2.3% vs 32.3%±1.4%, p<0.05) compared to healthy donors.

To further interrogate the CD8+ and CD4+ T-cell compartments in CLL, we performed high-dimensional visualization and unsupervised hierarchical clustering of the CyTOF data. The majority of CD8+ T cells from CLL patients expressed multiple checkpoint molecules per cell when compared to healthy donors, with co-expression of 2 receptors seen in 23.6%±1.1% vs. 18.6%±1.2%; p<0.05, 3 receptors in 31.07%±1.6% vs. 17.73%±1.4%; p<0.0001 and 4 inhibitory receptors in 13.5%±1.4% vs. 6.7%±0.9%; p:0.0005, respectively. A significantly greater proportion of CD4+ and CD8+ T-cells co-expressed TIGIT and PD-1. Interestingly, the inhibitory molecule CD160 was always co-expressed in conjunction with 2B4, TIGIT, CD57 and/or KLRG1, with very few single CD160 expressing CD8+ T-cells and negligible expression in the absence of other checkpoint molecules, indicating that CD160 expression is a late event during T cell exhaustion in CLL.

Focusing on key T cell subsets expressing combinations of inhibitory receptors allowed greater characterization of CLL T cells and led us to conclude that CD4+ T cells expressing inhibitory receptors are exhausted while CD8+ T cells are both exhausted and senescent, based on high expression of CD57 and KLRG1. The exhausted/senescent CD8+ T cell compartment in CLL was heterogeneous and was characterized by altered and reduced expression of chemokine and homing receptors, including CCR6, CCR4, CCR2 and CXCR3. Notably, CD8+ exhausted/senescent T cells expressed significantly reduced levels of the co-stimulatory molecules, CD28 and CD27, with reduced expression of homing receptors and activating receptors. In contrast, CD127 expression was significantly higher on exhausted/senescent T cells, suggesting that IL-7 could be important in the maintenance of this subset.

Taken together, our findings indicate a remarkable heterogeneity in the expression patterns of inhibitory molecules on CD8+ and CD4+ T-cells in CLL. On a per cell basis, CLL CD8+ T cells expressed more inhibitory receptors compared to healthy controls, suggesting that certain patients may benefit from combinational use of checkpoint molecules. In-depth dissection of the CD8 T cell compartment revealed a dominantly senescent/exhausted T cell profile with limited cell proliferation capacity, suggesting that checkpoint inhibition is less likely to invigorate the endogenous antitumor T-cell response in such patients.