Characterization of the T-cell landscape and its functional role in Chronic Lymphocytic Leukemia

Chronic lymphocytic leukemia (CLL) is a lymphoid neoplasm characterized by an accumulation of mature B lymphocytes in blood and peripheral lymphoid organs which highly depend on a tumor-supportive microenvironment. Altered T-cell distribution and function have since long been observed in the CLL microenvironment, but the exact pathological role of the different T-cell subsets remains uncertain. In the present work, the spectrum of CLL-associated T-cell phenotypes were investigated by using leading-edge single-cell technologies. Mass cytometry analyses of lymph nodes (LN), peripheral blood and bone marrow of CLL patients together with reactive lymph nodes (rLNs) of donors without cancer identified the CLL LN as a distinct niche, where CD8+ effector memory T-cells with an exhausted phenotype accumulate. Single-cell transcriptome and TCR-clonality analyses of LN T-cells further revealed a clonal expansion restricted to effector memory CD8+ T-cells, and enabled the characterization of the specific cross-talk between CLL cells and T- cell subsets. Besides, the single-cell transcriptome of T-cells from the Eμ-TCL1 mouse model of CLL was examined and shown to be similar to that of T-cells from CLL patients. Since genome-wide association studies have identified that a single-nucleotide polymorphism affecting the T-cell master regulator EOMES is associated with CLL development, the role of this transcription factor in the disease was investigated. Epigenetic and single-cell RNA sequencing analyses revealed that EOMES is not expressed in CLL cells but in T-cells, and that its levels are highest in exhausted CD8+ T-cells. Interestingly, Eomes deficiency in CD8+ T-cells prevented their expansion and led to a decreased leukemia control in the TCL1 mouse model, providing a novel layer of evidence for an anti-tumor role of CD8+ T-cells in CLL. Furthermore, mass cytometry and single-cell RNA-sequencing analyses revealed an increase of T regulatory type 1 (TR1) CD4+ T-cells in CLL LNs compared to rLNs. Such accumulation was likewise confirmed in spleen of Eμ-TCL1 mice using flow cytometry. Strikingly, TR1 cells failed to expand from Eomes-deficient CD4+ T-cells adoptively transferred in leukemic mice, and consequently were less capable of controlling leukemia development. Moreover, TR1 cell-mediated CLL control required IL-10 receptor signaling, as Il10rb-/- CD4+ T- cells showed impaired anti-leukemia activity. Taken together, the data generated herein comprehensively and deeply characterize the composition and phenotype of the T-cell compartment of CLL patients in comparison to individuals without cancer, and significantly improve our understanding of the function of distinct T-cell subsets in CLL

EOMES and IL-10 regulate antitumor activity of T regulatory type 1 CD4 + T cells in chronic lymphocytic leukemia

The transcription factor eomesodermin (EOMES) promotes interleukin (IL)-10 expression in CD4(+) T cells, which has been linked to immunosuppressive and cytotoxic activities. We detected cytotoxic, programmed cell death protein-1 (PD-1) and EOMES co-expressing CD4(+) T cells in lymph nodes (LNs) of patients with chronic lymphocytic leukemia (CLL) or diffuse large B-cell lymphoma. Transcriptome and flow cytometry analyses revealed that EOMES does not only drive IL-10 expression, but rather controls a unique transcriptional signature in CD4(+) T cells, that is enriched in genes typical for T regulatory type 1 (T(R)1) cells. The T(R)1 cell identity of these CD4(+) T cells was supported by their expression of interferon gamma and IL-10, as well as inhibitory receptors including PD-1. T(R)1 cells with cytotoxic capacity accumulate also in Eµ-TCL1 mice that develop CLL-like disease. Whereas wild-type CD4(+) T cells control TCL1 leukemia development after adoptive transfer in leukopenic Rag2(−/)(−) mice, EOMES-deficient CD4(+) T cells failed to do so. We further show that T(R)1 cell-mediated control of TCL1 leukemia requires IL-10 receptor (IL-10R) signaling, as Il10rb-deficient CD4(+) T cells showed impaired antileukemia activity. Altogether, our data demonstrate that EOMES is indispensable for the development of IL-10-expressing, cytotoxic T(R)1 cells, which accumulate in LNs of CLL patients and control TCL1 leukemia in mice in an IL-10R-dependent manner

EOMES is essential for antitumor activity of CD8+ T cells in chronic lymphocytic leukemia

Genome-wide association studies identified a single-nucleotide polymorphism (SNP) affecting the transcription factor Eomesodermin (EOMES) associated with a significantly increased risk to develop chronic lymphocytic leukemia (CLL). Epigenetic analyses, RNA sequencing, and flow cytometry revealed that EOMES is not expressed in CLL cells, but in CD

Interleukin-10 receptor signaling promotes the maintenance of a PD-1^int TCF-1⁺ CD8⁺ T cell population that sustains anti-tumor immunity

T cell exhaustion limits anti-tumor immunity and responses to immunotherapy, Here, we explored the microenvironmental signals regulating T cell exhaustion using a model of chronic lymphocytic leukemia (CLL). Single-cell analyses identified a subset of PD-1(hi), functionally impaired CD8(+) T cells that accumulated in secondary lymphoid organs during disease progression and a functionally competent PD-1(int) subset. Frequencies of PD-1(i)(nt) TCF-1(+) CD8(+) T cells decreased upon Il10rb or Stat3 deletion, leading to accumulation of PD-1(hi) cells and accelerated tumor progression. Mechanistically, inhibition of IL-10R signaling altered chromatin accessibility and disrupted cooperativity between the transcription factors NFAT and AP-1, promoting a distinct NFAT-associated program. Low IL10 expression or loss of IL-10R-STAT3 signaling correlated with increased frequencies of exhausted CD8(+) T cells and poor survival in CLL and in breast cancer patients, Thus, balance between PD-1(hi), exhausted CD8(+) T cells and functional PD-1(i)(nt) TCF-1(+) CD8(+) T cells is regulated by cell-intrinsic IL-10R signaling, with implications for immunotherapy

Interleukin-10 receptor signaling promotes the maintenance of a PD-1int TCF-1+ CD8+ T cell population that sustains anti-tumor immunity

T cell exhaustion limits anti-tumor immunity and responses to immunotherapy, Here, we explored the microenvironmental signals regulating T cell exhaustion using a model of chronic lymphocytic leukemia (CLL). Single-cell analyses identified a subset of PD-1(hi), functionally impaired CD8(+) T cells that accumulated in secondary lymphoid organs during disease progression and a functionally competent PD-1(int) subset. Frequencies of PD-1(i)(nt) TCF-1(+) CD8(+) T cells decreased upon Il10rb or Stat3 deletion, leading to accumulation of PD-1(hi) cells and accelerated tumor progression. Mechanistically, inhibition of IL-10R signaling altered chromatin accessibility and disrupted cooperativity between the transcription factors NFAT and AP-1, promoting a distinct NFAT-associated program. Low IL10 expression or loss of IL-10R-STAT3 signaling correlated with increased frequencies of exhausted CD8(+) T cells and poor survival in CLL and in breast cancer patients, Thus, balance between PD-1(hi), exhausted CD8(+) T cells and functional PD-1(i)(nt) TCF-1(+) CD8(+) T cells is regulated by cell-intrinsic IL-10R signaling, with implications for immunotherapy