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Despite the extensive catalogue of recurrent mutations in chronic lymphocytic leukaemia (CLL), the diverse molecular driving events and the resulting range of disease phenotypes remain incompletely understood. To study the molecular heterogeneity of CLL, we performed RNA-sequencing on 184 CLL patient samples. Unsupervised analysis revealed two major independent axes of gene expression variation: the first one aligned with the mutational status of the immunoglobulin heavy variable (IGHV) genes, and concomitantly, with the three-group stratification of CLL by global DNA methylation pattern, and affected biological functions including B- and T-cell receptor signaling. The second one aligned with trisomy 12 status and affected chemokine signaling. Furthermore, we searched for differentially expressed genes associated with gene mutations and copy-number aberrations and detected strong signatures for TP53, BRAF and SF3B1, as well as for del(11)(q22.3), del(17)(p13) and del(13)(q14) beyond the dosage effect. We discovered strong non-additive effects (i.e., genetic interactions, or epistasis) of IGHV mutation status and trisomy 12 on multiple phenotypes, including the expression of 893 genes. Multiple types of epistasis were observed, including synergy, buffering, suppression and inversion. Our study reveals previously underappreciated gene expression signatures for (epi)genomic variants in CLL and the presence of epistasis between them. The findings will serve as a reference for a functional resolution of CLL molecular heterogeneity.

The transcription factor Eomesodermin (EOMES) promotes IL-10 production of CD4+T-cells, which has been linked to immunosuppressive and cytotoxic activities. We detected EOMES-expressing CD4+T-cells in lymph node samples of patients with chronic lymphocytic leukemia (CLL) or diffuse large B-cell lymphoma. This was in line with an observed expansion of EOMES-positive CD4+T-cells in leukemic Eµ-TCL1 mice, a well-established model of CLL, and upon adoptive transfer of TCL1 leukemia in mice. Transcriptome and flow cytometry analyses revealed that EOMES does not only drive the transcription of IL-10, but rather controls a unique differentiation program in CD4+T-cells. Moreover, EOMES was necessary for the accumulation of a specific CD4+T-cell subset that expresses IFNγ and IL-10, as well as inhibitory receptors, like PD-1 and LAG3. T-cell transfer studies in leukopenicRag2-/-mice showed that EOMES-deficient CD4+T-cells were inferior in controlling TCL1 leukemia development compared to wildtype T-cells, even though expansion ofEomes-/-CD4+T-cells was observed. We further showed that control of TCL1 leukemia was driven by IL-10 receptor-mediated signals, asIl10rb-deficient CD4+T-cells showed impaired anti-leukemia activity. Altogether, our data suggest that IL-10 producing PD-1+CD4+T-cells contribute to CLL control in an EOMES- and IL-10R-dependent manner.