Transcription Factors and Checkpoint Inhibitor Expression with Age: Markers of Immunosenescence?

Background: Aging is characterized by a progressive decline in immune surveillance that favors tumor development in older patients. One mechanism used by pre-malignant and/or malignant cells to escape immune surveillance is the upregulation of inhibitory immune checkpoint molecules, such as PD-1 and PD-L1. Another process associated with aging is genetic or epigenetic modifications of tumor suppressor genes (TSGs). We have reported a correlation between 6q deletion and progression into a T cell lymphoproliferative disease, identifying the BACH2 gene as a candidate TSG⁽¹⁾. We thus examined - on a molecular level - the expression of specific transcription factors (BACH2 and PRDM1) and checkpoint molecules (PD-1 and PD-L1) in the major lymphocytes subsets for their potential as predictive markers of immunosenescence.

Methods: Lymphocyte subpopulations (CD3+/CD4+; CD3+/CD8+ and CD19+) were isolated for subsequent molecular analyses using the MACS technology (Miltenyi), with the purity of each lymphocyte subpopulation between 95%-99%. PD-1 (PDCD1), PD-L1 (CD274), IL4, IFNG, BACH2 and PRDM1 (Blimp1) mRNA transcripts were quantified in the purified subpopulations using qRT-PCR. BACH2 and PRDM1 protein expression were examined by Western blotting. Peripheral blood mononuclear cells were treated by Etoposide (VP16) to induce intracellular oxidative stress. Apoptotic function was assessed by flow cytometry (Annexin-V and Propidium Iodide).

Results: Peripheral blood samples were obtained from 40 healthy donors (HDs) between the ages of 20 to 90 yrs, subdivided into <50 yrs (n=21; median: 36yo) and ³50 yrs (n=19; median: 61yo). And 40 blood samples from untreated patients with chronic lymphocytic leukemia (CLL; median: 67yo) were comparatively analyzed.

BACH2-deficient mice have been shown to have increased numbers of IL4-producing CD4⁺ T cells after activation⁽²⁾, with BACH2 known to repress PRDM1 expression in B andT cells^(2,3). We, therefore, examined BACH2 gene expression in the HD groups finding it was significantly downregulated in CD4⁺, CD8⁺ T cells and CD19⁺ B cells from the older HD group (p=0.001; 0.004 and 0.03, respectively). BACH2 expression was further reduced in CD4⁺, CD8⁺ T cells and CD19⁺ B cells from CLL patients compared to HD of similar age (p=0.001; <0.001 and 0.004). In contrast, PRDM1 was significantly upregulated in CD4⁺ and CD8⁺ T cells (p=0.003; 0.001) from CLL patients but not in their leukemic B cells. As expected, BACH2 expression was inversely correlated with PRDM1 in CD4⁺, CD8⁺ T cells and CD19⁺ B cells (r=0.57; 0.71 and 0.62, respectively). Western blot analysis demonstrated that BACH2 and Blimp1 (PRDM1) protein expressions in the T and B cell subpopulations were significantly correlated with transcript expression. We also observed that BACH2 downregulation is correlated with increased IL-4 gene expression (r=0.61) but not IFNγ in CD4⁺ T cells. These observations suggest that BACH2 downregulation in CD4⁺ T cells could enhance expressed Th2-related genes, such as IL-4 and PRDM1.

PD-1 gene expression was significantly upregulated in CD4⁺, CD8⁺ T cells (p=0.015 and 0.02) in the older HD group. PD-1 was also significantly upregulated in the same T cells subpopulations isolated from untreated CLL patients (p=0.001 and 0.002) when compared to a similar HD age group. High PD-L1 gene expression was correlated with increased age in HD B cells (p=0.04) with a further increase detected in leukemic B cells compared with the older HD group (p=0.001). We also observed an inverse correlation between BACH2 and PD-1 in CD4⁺, CD8⁺ T cells (r=0.59 and 0.68); and between BACH2 and PD-L1 in CD19⁺ B cells (r=0.66).

We further examined VP16-induced apoptosis in isolated lymphocytes subpopulations. As recently reported in BACH2-deficient mice⁽⁴⁾, our observation showed that BACH2 downregulation was strongly correlated with resistance to apoptosis in CD4⁺, CD8⁺ T cells and leukemic B cells (r=0.61; 0.75 and 0.69, respectively).

Conclusion: These data suggest that BACH2, PRDM1, PD-1 and PD-L1 gene expression is correlated with aging and age-related immunosuppression. These effects are even more pronounced in leukemic cells from CLL patients.

1. C. Sibille, et al.,(personal data) ; 2. S. Tsukumo, et al. Proc. Natl Acad Sci USA. 2013 ; 3. H. Tanaka et al. J Biol Chem. 2016 ; 4. Muto, A., et al. J Biol Chem. 2002;