Characterizing Immunophenotypic and Functional Pseudo-Exhaustion in T Cells From CLL Patients: The Impact of Lenalidomide
Authors: Riches John C. , Davies Jeff K. , McClanahan Fabienne , Fatah Rewas , Iqbal Sameena , Agrawal Samir , Ramsay Alan G. , Gribben John G 
Published: October 2019

Abstract
Abstract 564
The ability to evade immune destruction is increasingly being recognised as a crucial feature of cancer cells. Chronic lymphocytic leukemia (CLL) is associated with profound defects in T-cell function, resulting in failure of anti-tumor immunity and increased susceptibility to infections. T cells from CLL patients exhibit functional defects and alterations in gene expression, that show similarities to exhausted T cells in chronic viral infections. However, it is unclear whether CLL T cells are truly exhausted, or whether these defects are restricted to expanded populations of CMV specific T cells. We investigated the phenotype and function of CD8+ T cells from CLL patients and controls matched for age and CMV-serostatus. We demonstrate an increased proportion of CCR7- effector T cells in both CLL patients and CMV-seropositive individuals (p<0.05). CD8+ and CD4+ T cells from CLL patients had increased expression of exhaustion markers CD160 and CD244 irrespective of CMV-serostatus (p<0.01), whereas increased PD1 expression on CD8+ T cells was limited to CMV-seronegative patients (p=0.002). CLL CD8+ T cells also showed functional defects in proliferation and cytotoxicity irrespective of CMV-serostatus, with the cytolytic defect caused by a combination of impaired granzyme B packaging into secretory vesicles and non-polarized degranulation. In contrast to virally-induced exhaustion, CLL T cells showed increased production of interferon-γ with increased T-BET expression (p<0.01), normal IL-2 production, and no downregulation of IL-7R. Therefore, while CLL CD8+ T cells exhibit some features of T-cell exhaustion, they show important differences (Table 1). These findings also exclude CMV as the sole cause of T cell defects in CLL.
Lenalidomide has recently been demonstrated to have significant clinical activity in CLL. Its mechanism of action in this disease is not well understood, but it thought to act primarily by a combination of CLL cell and immune cell activation. We therefore examined the ability of lenalidomide to repair the observed T cell defects by investigating the impact of this agent on the gene expression profiles and function of CLL T cells. Treatment of CLL CD8+ T cells with lenalidomide increased the expression of 137 genes, while 34 genes were downregulated. The most prominent changes in expression were of genes involved in cytoskeletal signaling including WASF1 (Wiskott-Aldrich syndrome protein, family member 1), and TPM2 (tropomyosin 2). There was also upregulation of genes involved in lymphocyte activation, including TNFSF4 (Tumor necrosis factor ligand superfamily, member 4: OX40L), LAG3 (Lymphocyte-activation gene 3), and TNF, and genes involved in cell proliferation such as IKZF1 (Ikaros) and GRN (Granulin). Although lenalidomide treatment or anti-CD3 stimulation alone had no impact on T-bet expression, co-treatment with both anti-CD3 stimulation and lenalidomide resulted in significantly enhanced T-bet expression and increased production of interferon-γ. In contrast, lenalidomide treatment alone was able to improve T cell cytotoxic function, associated with repair of trafficking of granzyme B into the immunological synapse.
In conclusion, T cells from CLL patients exhibit features of T-cell pseudo-exhaustion that are present irrespective of CMV serostatus. Treatment of CLL T cells with lenalidomide results in upregulation of genes involved in proliferation, activation, and cytoskeletal pathways, resulting in repair of the functional T cell defects.
Table 1. Comparison of the phenotypic and functional defects of T cells from CLL patients with T-cell “exhaustion” in chronic viral infections Exhausted T cells in chronic viral infections T cells from CLL patients Increased expression of inhibitory receptors Yes Yes Abnormal transcription factor profile Yes Yes Reduced proliferative potential Yes Yes Decreased expression of IL-7R (CD127) Yes No Decreased cytokine production ↓IL-2, ↓IFN-γ Yes No Impaired cytotoxicity Yes Yes

Disclosures:
Riches: Celgene: Research Funding. Gribben:Celgene: Honoraria; Roche: Honoraria; Pharmacyclics: Honoraria; GSK: Honoraria; Mundipharma: Honoraria; Gilead: Honoraria.