T-Cells From Patients with CLL Exhibit Phenotypic and Transcription Factor Profiles of Exhaustion Independent of CMV Serostatus
Authors: Riches John C. , Davies Jeff , Iqbal Sameena , Fatah Rewas , Agrawal Samir , Ramsay Alan G. , Gribben John G. 
Published: October 2019

Abstract
Abstract 1780
Cancer is associated with immune dysfunction, contributing to the failure to mount an effective anti-tumor immune response. T-cell exhaustion, a state of acquired T-cell dysfunction initially described in the context of chronic viral infections, has recently been described in human solid tumors. We have previously demonstrated alterations in gene expression and defects in function in T-cells from patients with chronic lymphocytic leukemia (CLL) and noted similarities to those described in exhausted T-cells in murine models. Therefore, we used multiparameter flow cytometry to determine if T-cells from patients with untreated CLL (n=23) had surface phenotypic and transcription factor profiles of exhaustion. When compared with healthy controls (n=10), an increased proportion of circulating CD8+ T-cells showed an effector-memory phenotype (p=0.018), with a shift towards a greater proportion of CCR7-CD45RA+ terminally differentiated cells. We found increased expression of markers of exhaustion including CD279 (PD-1) (p=0.0057), CD160 (p=0.0006), CD244 (p=0.0057), and CD57 (p=0.011), and decreased expression of CD28 (p=0.0058) on CLL CD8+ T-cells compared with healthy CD8+ T-cells. Increased expression of PD-1 (p=0.0019), CD160 (p=0.0011), CD57 (p=0.007), and LAG-3 (p=0.038) was also noted on CLL CD4+ T-cells. We next determined the level of the transcription factors T-bet and eomesodermin and the transcriptional repressor Blimp-1, as these proteins have been implicated in CD8+ effector-memory differentiation and development of T-cell exhaustion. Importantly, CD8+ T-cells from CLL patients showed increased expression of T-bet (p=0.038), eomesodermin (p=0.0037), and Blimp-1 (p=0.0002), compared with CD8+ T-cells from healthy donors. Furthermore, PD-1 expression identified a subset of exhausted CD8+ T-cells with high intranuclear staining of Blimp-1 that was markedly expanded in patients with CLL (p=0.0002).
Previous studies have identified expanded populations of CMV specific CD8+ T-cells in CMV seropositive CLL patients. As chronic viral infection is a known cause of T-cell exhaustion, we determined whether our findings were limited to CMV seropositive patients. We observed that increased expression of T-bet and decreased expression of CD28 was seen only in CD8+ T-cells from CMV seropositive patients. T-bet represses expression of PD-1 and sustains CD8+ T-cell responses during chronic viral infection, and we noted relatively lower expression of PD-1 on CD8+ T-cells in seropositive compared with seronegative patients (p=0.049), although PD-1 expression was still higher than in healthy controls. However, CD8+ T-cells from both CMV seronegative and seropositive patients had significantly higher expression of CD160, CD244, and CD57 compared to CD8+ T-cells from healthy donors (Table 1). Furthermore, CD8+ T-cells from both CMV seronegative and seropositive patients had increased expression of Blimp-1 and eomesodermin.
Table 1. Surface phenotype/transcription factor profile of CLL according to CMV serostatus Healthy CLL: CMV IgG negative (n=8) CLL: CMV IgG positive (n=8) Phenotype PD-1 LOW INCREASED** INCREASED* CD160 LOW INCREASED** INCREASED** CD57 LOW INCREASED* INCREASED** CD244 LOW INCREASED* INCREASED*** CD28 HIGH NO CHANGE DECREASED** Transcription factor T-bet LOW NO CHANGE INCREASED** Eomes LOW INCREASED* INCREASED*** Blimp-1 LOW INCREASED*** INCREASED*** * p<0.05, ** p<0.01, *** p<0.001
In conclusion, T-cells from patients with CLL show phenotypic and transcription factor profiles of T-cell exhaustion that is not limited to the CMV-seropositive group. These findings may explain the acquired immune deficiency in patients with CLL, and provide potential therapeutic targets to reverse immune dysfunction in this disease.

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