Prior irradiation results in elevated programmed cell death protein 1 (PD-1) in T cells

<p><b>Purpose:</b> In this study we addressed the question whether radiation-induced adverse effects on T cell activation are associated with alterations of T cell checkpoint receptors.</p> <p><b>Materials and methods:</b> Expression levels of checkpoint receptors on T cell subpopulations were analyzed at multiple post-radiation time points ranging from one to four weeks in mice receiving a single fraction of 1 or 4 Gy of γ-ray. T cell activation associated metabolic changes were assessed.</p> <p><b>Results:</b> Our results showed that prior irradiation resulted in significant elevated expression of programmed cell death protein 1 (PD-1) in both CD4+ and CD8+ populations, at all three post-radiation time points. T cells with elevated PD-1 mostly were either central memory or naïve cells. In addition, the feedback induction of PD-1 expression in activated T cells declined after radiation.</p> <p><b>Conclusion:</b> Taken together, the elevated PD-1 level observed at weeks after radiation exposure is connected to T cell dysfunction. Recent preclinical and clinical studies have showed that a combination of radiotherapy and T cell checkpoint blockade immunotherapy including targeting the programmed death-ligand 1 (PD-L1)/PD-1 axis may potentiate the antitumor response. Understanding the dynamic changes in PD-1 levels in T cells after radiation should help in the development of a more effective therapeutic strategy.</p

Bmi1 protein expression is increased in immortalized and tumorigenic cervical cell lines and positively correlates with disease stage in cervical dysplasia and neoplasia <i>in vivo</i>.

<p>(<b>A</b>) Bmi1 protein levels were quantified by Western blot in primary HFKs and primary human ectocervical cells (HECs) expressing E6 or immortalized by E6/E7 and the cervical cancer cell line HeLa. Lysates were separated by 4–20% gradient SDS-PAGE. Antibodies were used to detect hTERT (1∶1000, Origene), Bmi1 (1∶200, F6, Millipore) and GAPDH (1∶2000, FL-335, Santa Cruz). (<b>B</b>)Tissue from a case of invasive cervical cancer was acquired. Representative images are shown containing cancerous lesions and adjacent normal, intact epithelium. Tissue staining with hematoxylin and eosin (<b>i</b>) and immunohistochemical stain with Bmi1 (1∶200, F6, Millipore) (<b>ii</b>) are shown. (<b>C</b>)Tissues of Cervical Intraepithelial Neoplasia Stage 1 (CIN1), CIN2, CIN3 or carcinoma <i>in situ</i>, and invasive cervical carcinoma were acquired. To quantify Bmi1 expression, stained slides were subjected to a randomized, blinded review by a board-certified clinical pathologist. A subset of slides was scored multiple times to demonstrate reproducibility. For each sample, the case number and diagnosis is provided with the corresponding an intensity score, the percentage of positive cells, the corresponding positivity score, and the combined score. Each case received an intensity score from 0–3 (0 = negative, 1 = weak, 2 = moderate, 3 = intense) and the percentage of positive cells was recorded, which was converted to a positivity score (0 = less than 10%, 1 = 11–49%, 2 = 50–74%, 3 = 75–100%). Combined scores were calculated by adding the intensity score and positivity scores. (<b>D</b>) Immunohistochemical staining with hematoxylin and eosin (<b>i, iii, v, vii, ix</b>) and for Bmi1 protein (1∶100, F6, Millipore) (<b>ii, iv, vi, viii, x</b>) was performed. Representative images are shown. Relevant controls are shown, staining with hematoxylin and eosin (<b>i</b>) and for Bmi1 protein (1∶100, F6, Millipore) (<b>ii</b>). Scale bar = 50 µm. (<b>E</b>) Mean and standard deviation of combined scores are shown.</p

HPV16 E7 Protein and hTERT Proteins Defective for Telomere Maintenance Cooperate to Immortalize Human Keratinocytes

<div><p>Previous studies have shown that wild-type human telomerase reverse transcriptase (hTERT) protein can functionally replace the human papillomavirus type 16 (HPV-16) E6 protein, which cooperates with the viral E7 protein in the immortalization of primary keratinocytes. In the current study, we made the surprising finding that catalytically inactive hTERT (hTERT-D868A), elongation-defective hTERT (hTERT-HA), and telomere recruitment-defective hTERT (hTERT N+T) also cooperate with E7 in mediating bypass of the senescence blockade and effecting cell immortalization. This suggests that hTERT has activities independent of its telomere maintenance functions that mediate transit across this restriction point. Since hTERT has been shown to have a role in gene activation, we performed microarray studies and discovered that E6, hTERT and mutant hTERT proteins altered the expression of highly overlapping sets of cellular genes. Most important, the E6 and hTERT proteins induced mRNA and protein levels of Bmi1, the core subunit of the Polycomb Group (PcG) complex 1. We show further that Bmi1 substitutes for E6 or hTERT in cell immortalization. Finally, tissue array studies demonstrated that expression of Bmi1 increased with the severity of cervical dysplasia, suggesting a potential role in the progression of cervical cancer. Together, these data demonstrate that hTERT has extra-telomeric activities that facilitate cell immortalization and that its induction of Bmi1 is one potential mechanism for mediating this activity.</p> </div

A telomere elongation-defective hTERT mutant cooperates with HPV E7 for immortalizing human keratinocytes (HFKs).

<p>Primary HFKs were transduced with pBABE-puro-based retroviruses containing hTERT or hTERT-HA and pLXSN-based retroviruses with HPV E7 or empty vector and selected with puromycin and G418 as previously described. Cultures were passed continuously in vitro, and growth curves were plotted. Cultures that did not proliferate and expand in 20 days were considered senescent and were terminated. This experiment was repeated more than three times with similar results. <u>(A) Telomerase activity.</u> Quantitative TRAP assays were done as described in the <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003284#s4" target="_blank">Materials and Methods</a>. Similar levels of telomerase activity were observed among cells expressing an hTERT construct. <u>(B) Telomere length.</u> Telomeres lengthened in hTERT/E7 cells, but shortened in hTERT-HA/E7 cells. Both wild-type hTERT and hTERT-HA immortalize HFKs in combination with HPV E7.</p

Catalytic-defective hTERT mutants cooperate with HPV E7 to immortalize HFKs.

<p><u>(A) Growth curves.</u> Primary HFKs were transduced with the indicated pBABE-puro based retroviruses containing wild-type hTERT, hTERT N+T, or hTERT-D868A and pLXSN-based retroviruses containing E7 or empty vector and then doubly selected with puromycin and G418 as previously described. Cultures were passed continuously in vitro and growth curves were plotted with population doubling over time in culture. Cultures that did not proliferate and expand in 20 days were considered senescent and were terminated. This experiment was repeated more than three times with similar results. Wild-type hTERT, hTERT-D868A, and hTERT N+T are all able to immortalize HFKs in combination with E7. <u>(B) Telomerase activity in early passage of the transduced cells.</u> CHAP lysates were harvested from early (p5) and telomerase activity was measured by quantitative real-time TRAP. <u>(C) Telomerase activity in late passage of the transduced cells.</u> Telomerase activity in late passage of cells was measured by quantitative real- time TRAP.</p

Bmi1 cooperates with E7 in cell immortalization.

<p>HFKs were doubly infected with pX-Bmi1 and pLXSN-E7, pLXSN-E7 alone, or empty vector. (A) Growth curve. Cells were passaged as described in the Methods section to determine the growth rate and lifespan of the cell populations. Bmi1, in cooperation with E7, induced cell immortalization equivalent to E6 or hTERT and E7. (B) Telomere length. A quantitative PCR-based technique (see Methods) was used to quantify the average telomere length in the indicated cell cultures.</p

Bmi1 protein increases in cell lines.

<p>Bmi1 protein levels were quantified by Western blot for (<b>A</b>) empty vector and E6 expressing cells and (<b>B</b>) hTERTwt or hTERT-D868A expressing cells. Bmi1+E7 lysate served as a positive control for Bmi1 protein. Lysates were separated by 4–20% gradient SDS-PAGE. Antibodies were used to detect hTERT (1∶1000, Origene) and Bmi1 (1∶200, F6, Millipore), normalized to GAPDH (1∶2000, FL-335, Santa Cruz) or ACTIN (1∶5000, Sigma). (<b>C</b>) Bmi1 protein levels were quantified by Western blot. Long-term serial passaging maintains increased levels of Bmi1 protein, as shown by the P102 hTERT+E7 lysate. Lysates were separated by 4–20% gradient SDS-PAGE. Antibodies were used to detect hTERT (1∶1000, Origene), Bmi1 (1∶200, F6, Millipore) and actin (1∶5000, Sigma). (<b>D</b>) Cell pellets were formalin fixed and paraffin embedded. Bmi1 protein levels were assayed by immunocytochemistry (1∶200, F6, Millipore). The images were captured with the Evos LX microscope. Scale bar = 100 µm.</p

hTERT wt and hTERT-D868A do not activate the hTERT promoter.

<p>Keratinocytes were transfected with either wt hTERT core promoter or the cyclin D1 promoter and either HPV16E6, hTERT wt, or hTERT-D8686A. The pRL-CMV <i>R. reniformis</i> reporter plasmid was also transfected into the cells to standardize for transfection efficiency. Luciferase activity was measured 24 hours after transfection using the Dual luciferase reporter assay system (Promega). Relative fold activation reflects the normalized luciferase activity induced by E6 and hTERT compared to the normalized activity of vector control. The value of pGL3B-hTERT activity with empty was set to 1. Error bars show the standard deviation for at least three independent experiments. Neither hTERT wt nor hTERT-D868A induce hTERT core promoter (A), while they are able to activate cyclin D1 promoter (B). HPV16 E6 was as a positive control for induction of hTERT promoter.</p

Telomerase activity does not correlate with telomere length during immortalization of human genital keratinocytes by the HPV E6 and E7 oncoproteins.

<p>Primary human foreskin keratinocytes (HFKs) and human ectocervical keratinocytes (HECs) were transduced with pLXSN-based retroviruses containing HPV E6, E7, E6/E7, or empty vector and selected as previously described. Cultures were passed continuously in vitro as described in the text and the number of cell doublings calculated and plotted versus time in culture. Cultures that did not proliferate and expand in 20 days were considered senescent and were terminated. This experiment was repeated more than five times with similar results. <u>(A) Immortalized cells exhibit similar levels of telomerase activity as in cervical cancer cells.</u> Quantitative TRAP assays as described in the <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003284#s4" target="_blank">Materials and Methods</a> were used to measure telomerase activity in E6/E7 immortalized HFKs, E6/E7 immortalized HECs, and SiHa (HPV-16 positive), HeLa (HPV-18 positive), and C33A (HPV negative) cervical cancer cell lines. <u>(B) Telomere length stabilizes in E6/E7 immortalized cells and cervical cancer cell lines.</u> Cellular DNAs were isolated from HFKs at indicated passages and cervical cancer cells, and relative telomere length (T/S ratio = telomere/single copy gene) was measured using real-time PCR, as described in the Material and Methods. Immortalized cells and cancer cells have relatively shorter telomeres. <u>(C) Telomere length shortens over cell passages during immortalization.</u> Cellular DNAs were isolated and subjected to real-time PCR-based telomere length measurement.</p

E6, hTERTwt, and hTERT-D868A alter the expression of overlapping gene sets, including chromatin remodeling genes such as <i>Bmi1</i>.

<p>Primary HFKs were stably transduced with either E6, hTERTwt, hTERT-D868A or puro babe control vector. Samples were submitted for whole genome expression array analysis (<b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003284#ppat.1003284.s005" target="_blank">Figure S3</a></b>). Expression profile changes shown by hTERTwt are compared to expression profile changes in E6 (<b>A</b>) and hTERT-D868A mutant (<b>B</b>). As expected, a significant amount of the expression changes seen in E6 (6991 total changes) were also altered by hTERT wt (1379, representing 20% of the E6 changes). Conversely, more than half of the hTERT changes (58%, 1379 of 2359 changes) were also seen by E6. Interestingly, of the 2359 genes altered by hTERTwt, 2077 of them (88%) were also altered by the hTERT^ci mutant (fold change >1.33 and <i>p</i> value <0.01). (<b>C</b>) 1258 changes were shared by E6, hTERTwt, and hTERT-D868A, including Bmi1. (<b>D</b>) Numerical fold change values are shown for all arrays as they correspond to two probes, NM_005180 and L13689. These accession numbers represent Bmi1 mRNA sequences that are 99% identical. Quantitative RT-PCR was performed on the E6 and hTERTwt or hTERT-D868A (<b>E</b>) with gene-specific primers for Bmi1 to validate the array results, normalized to GAPDH. n = 3. Bars represent mean ± SD.</p