Expanded CD8+ T cells of murine and human CLL are driven into a senescent KLRG1+ effector memory phenotype

Abstract

Altered numbers and functions of T cells have previously been demonstrated in chronic lymphocytic leukemia (CLL) patients. However, dynamics and specific T-cell subset alterations have not been studied in great detail. Therefore, we studied CLL blood lymphocyte subsets of individual patients in a longitudinal manner. Dynamic expansions of blood CD4 + and CD8 + T-cell numbers were consistently associated with a progressively increasing CLL leukemic compartment. Interestingly, the T-cell subset expansion over time was more pronounced in CD38 + CLL. Additionally, we performed gene expression profiling of CD3 + T cells of CLL patients and normal donors. Using gene set enrichment analysis, we found significant enrichment of genes with higher expression in CLL T cells within CD8+ effector memory and terminal effector T-cell gene signatures. In agreement with these data, we observed a marked expansion of phenotypic CD8 + effector memory T cells in CLL by flow cytometry. Moreover, we observed that increments of CD8 + effector memory T cells in human CLL and also mouse CLL (Eμ-TCL1 model) were due to an expansion of the inhibitory killer cell lectin-like receptor G1 (KLRG1) expressing cellular subset. Furthermore, higher plasma levels of the natural KLRG1 ligand E-cadherin were detected in CLL patients compared to normal donor controls. The predominance of KLRG1+ expression within CD8+ T cells in conjunction with increased systemic soluble E-cadherin might significantly contribute to CLL immune dysfunction and might additionally represent an important component of the CLL microenvironment.

Fig. 1

Longitudinal kinetics of the accessory T-cell compartment in relation to disease progression and CD38 status. a Levels of total CD3⁺, CD8⁺ and CD4⁺ T cells, NK cells (defined as CD56⁺CD16⁺ cells), absolute lymphocytes and CD19⁺ B cells of individual CLL patients (n = 46) were determined over time. The slope of the regression line was determined for each lymphocyte subset of every patient. An increment of ≥500 lymphocytes/μl per month was arbitrarily defined as progressive disease (n = 23 patients). An increment <500 lymphocytes/μl per month was regarded as stable disease (n = 23 patients). Representative graphs of a patient with stable (left) and a patient with progressive disease (right) are shown. b Comparison of lymphocyte subset increments of stable and progressive disease patients. c Lymphocyte subset increments per month of CD38⁺ are significantly higher than increments of CD38⁻ progressive CLL patients. d Absolute numbers of peripheral blood accessory lymphocyte subsets including CD3⁺ total T cells, CD4⁺ T cells, CD8⁺ T cells and NK cells were analyzed according to Binet stage and CD38 expression status. Subgroups: Binet A, CD38⁻, n = 46; CD38⁺, n = 14; Binet B, CD38⁻, n = 14, CD38⁺, n = 12; Binet C, CD38⁻, n = 5; CD38⁺, n = 11. n.d., not determined; PB, peripheral blood; NK, natural killer cells. *P < 0.05; **P < 0.01; ***P < 0.001

Fig. 2

Genes with higher expression in CLL T cells are significantly enriched within gene signatures of CD8⁺ effector memory and terminal effector T cells. Gene set enrichment analysis (GSEA) showing enrichment of genes with higher expression in CLL T cells in published gene signatures of sorted normal donor CD8⁺ effector memory (T _em)/terminal effector (T _eff) T-cell populations (n = 8) in comparison with those of sorted CD8⁺ naive (T _naive)/central memory (T _cm) T-cell populations (n = 8) [14]. The GSEA enrichment plot (right) and the heat map of the top ranking leading edge genes (left) are shown. In the GSEA plot, the x-axis represents the ranked list of genes from the highest to the lowest probe signal between T _em/eff versus T _naive/cm CD8⁺ T-cell populations. The black lines relate to the position of genes with higher expression in CLL T cells. The height of the green enrichment curve illustrates the degree of enrichment. A strong correlation was observed between normal donor T _em/eff genes and the CLL T-cell gene signature. In the heat map (left), signal intensities of leading edge genes (left) are shown by shades of red (upregulation) and blue (downregulation). NES normalized enrichment score; FDR false discovery rate

Fig. 3

The CD8⁺ effector memory T-cell compartment of CLL patients is expanded. a Peripheral blood mononuclear cells were stained with antibodies against CD8, CD45RA, CCR7 and with propidium iodide (PI). Subsets of CD8⁺ T cells were defined as naive (T _naive; CD45RA⁺, CCR7⁺), central memory (T _cm; CD45RA⁻, CCR7⁺), effector memory (T _em, CD45RA⁻, CCR7⁻) and terminal effector (T _eff, CD45RA⁺,CCR7⁻) cells. Representative plots of normal donor (ND) and CLL samples are shown. b Quantification of the flow cytometric CD8⁺ T-cell subset analysis. The relative proportion of naive CD8⁺ T cells is decreased, while the relative proportion of CD8⁺ T _em cells is increased. c Absolute cell numbers of CLL CD8⁺ T _cm and T _em cells were increased. FSC forward scatter; SSC side scatter; PB peripheral blood. *P < 0.05; **P < 0.01

Fig. 4

The high numbers of CD8⁺ T cells in CLL are due to an expansion of KLRG1⁺ cellular subsets. a KLRG1 expression within total CD8⁺ T cells and within CD8⁺ subsets according to the gating strategy depicted in Fig. 3 a. Representative plots of a normal donor and a CLL sample are shown. b The proportion of KLRG1⁺ cells within CD8⁺ T cells was compared between CLL patients and normal donor controls (left panel). Absolute numbers of KLRG1⁺CD8⁺ T cells were increased in the peripheral blood (PB) of CLL patients compared to normal donors (ND), while the numbers of KLRG1⁻CD8⁺ cells did not differ between the two groups (right panel). c Significantly increased proportions of KLRG1⁺ cells were found in CD8⁺ T _naïve, T _cm and T _em cell subsets. d KLRG1⁺CD8⁺ T _cm and T _eff cell numbers were significantly increased in CLL patients compared to normal donors. e CLL plasma levels of soluble E-cadherin (sE-cadherin), ligand of KLRG1, are increased compared to normal donors. ND normal donor.*P < 0.05; **P < 0.01

Fig. 5

Peritoneal cavity and spleen CD8⁺ T cells of TCL1 transgenic mice are shifted toward a KLRG1⁺ effector memory phenotype. a Representative flow cytometric analysis of wild-type versus TCL1 peritoneal CD8⁺ T-cell subsets is displayed. Definition of murine CD8⁺ T-cell subsets: naïve (T _naive: CD62L⁺, CD44^low), central memory (T _cm: CD62L⁺, CD44⁺) and effector memory (T _em: CD62L^low, CD44⁺). Within the CD8⁺ T _em compartment, KLRG1 expression was analyzed. b Compared to wild-type mice, the peritoneal CD8⁺ T-cell compartment of TCL1 transgenic mice was characterized by a decreased fraction of naive cells and an increased fraction of effector memory cells. c Within peritoneal TCL1 CD8⁺ T _em compartment, the proportion of KLRG1⁺ cells was significantly increased. d Representative flow cytometric analysis of splenic CD8⁺ T-cell subsets. e TCL1 transgenic mice displayed a significant increase in CD8⁺ T-cell numbers (left). Significantly elevated KLRG1⁺CD8⁺ T _em cell numbers were present in the spleens of TCL1 transgenic mice (right). *P < 0.05; **P < 0.01; ***P < 0.001. WT wild-type