D-1 Gene Polymorphism in Salivary Gland Tumors

Objectives This study aimed to assess PD-1gene polymorphism in salivary gland tumors in patients referred to Khalili Hospital in Shiraz. Methods This case-control study evaluated 48 patients with salivary gland tumors and 100 age- and sex-matched healthy controls. First, 5cc blood samples were obtained from patients and transferred to vials containing anti-coagulated EDTA. DNA was extracted, and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was performed on the samples. The PD-1 gene genotype was determined using the Fermentas kit. After 24 hours of incubation, all the samples were electrophoresed. The genotypes were reported based on the size of bands, and the chi-square test was applied. To compare the alleles, the Fisher’s Exact test was applied. The Yates correction was used to compare the genotype and genotypic alleles based on the tumor grade. Results The mean age was 44.81±15.69 years in patients and 46.54± 13.86 years in controls. Statistical analysis did not show any significant difference in PD1 gene polymorphism between the two groups (P=0.098). No significant correlation was found between the genotype frequency and lymph node involvement (P=0.06), tumor genotype (P=0.12), side (right or left) (P=0.22), tumor location (P=0.27), and size or invasion of the tumor to the surrounding tissue (P=0.14). PD1.3 genotype frequency did not differ significantly between malignant and benign tumors (P=0.6). Conclusion This study did not reveal any significant difference in genotype frequency of PD1.3 in the patient and control groups; however, further studies are needed with a larger sample size to obtain more accurate results

Killer cell immunoglobulin-like receptors (KIRs) genotype and haplotype analysis in Iranians with non-melanoma Skin Cancers

Background: The innate immune system against malignancies is mainly orchestrated by natural killer cells, which carry out killing mechanisms by using their receptors, such as killer immunoglobulin-like receptors (KIRs). This study was designed to determine the diversity of KIR genes in non-melanoma skin cancers. Methods: A total of 160 subjects with skin cancer, including 60 cases of squamous cell carcinoma and 100 cases of basal cell carcinoma (BCC), and 270 healthy subjects formed the study groups. The sequence-specific polymerase chain reaction was carried out to detect the presence or absence of 16 KIR genes. Results: KIR3DL1 (p = 0.0381, OR = 4.78, 95% CI = 1.108 to 20.62) increased in BCC patients compared to healthy controls. Conclusion: We concluded that the higher frequency of KIR3DL1 in BCC patients compared with healthy controls may increase the probability of developing BCC in Iranians. keywords:Basal cell carcinoma KIR Natural killer cells Receptors Squamous cell carcinom

Triggering of lymphocytes by CD28, 4-1BB, and PD-1 checkpoints to enhance the immune response capacities

Tumor infiltrating lymphocytes (TILs) usually become exhausted and dysfunctional owing to chronic contact with tumor cells and overexpression of multiple inhibitor receptors. Activation of TILs by targeting the inhibitory and stimulatory checkpoints has emerged as one of the most promising immunotherapy prospectively. We investigated whether triggering of CD28, 4-1BB, and PD-1 checkpoints simultaneously or alone could enhance the immune response capacity of lymphocytes. In this regard, anti-PD-1, CD80-Fc, and 4-1BBL-Fc proteins were designed and produced in CHO-K1 cells as an expression host. Following confirmation of the Fc fusion proteins’ ability to bind to native targets expressed on engineered CHO-K1 cells (CHO-K1/hPD-1, CHO-K1/hCD28, CHO-K1/hCTLA4, and CHO-K1/h4-1BB), the effects of each protein, on its own and in various combinations, were assessed in vitro on T cell proliferation, cytotoxicity, and cytokines secretion using the Mixed lymphocyte reaction (MLR) assay, 7-AAD/CFSE cell-mediated cytotoxicity assay, and a LEGENDplex™ Human Th Cytokine Panel, respectively. MLR results demonstrated that T cell proliferation in the presence of the combinations of anti-PD-1/CD80-Fc, CD80-Fc/4-1BBL-Fc, and anti-PD-1/CD80-Fc/4-1BBL-Fc proteins was significantly higher than in the untreated condition (1.83-, 1.91-, and 2.02-fold respectively). Furthermore, anti-PD-1 (17%), 4-1BBL-Fc (19.2%), anti-PD-1/CD80-Fc (18.6%), anti-PD-1/4-1BBL-Fc (21%), CD80-Fc/4-1BBL-Fc (18.5%), and anti-PD-1/CD80-Fc/4-1BBL-Fc (17.3%) significantly enhanced cytotoxicity activity compared to untreated condition (7.8%). However, concerning the cytokine production, CD80-Fc and 4-1BBL-Fc alone or in combination significantly increased the secretion of IFN‐γ, TNF-α, and IL-2 compared with the untreated conditions. In conclusion, this research establishes that the various combinations of produced anti-PD-1, CD80-Fc, and 4-1BBL-Fc proteins can noticeably induce the immune response in vitro. Each of these combinations may be effective in killing or destroying cancer cells depending on the type and stage of cancer

Western blot analysis of purified Fc fusion proteins by probing with goat anti-human IgG (Fc specific)-peroxidase antibody under reducing conditions.

M: Protein ladder, Lane 1: anti- PD-1, Lane 2: CD80-Fc, and Lane 3: 4-1BBL-Fc.</p

Minimal dataset.

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Potential of mono and combination treatments of anti-PD-1, CD80-Fc, and 4-1BBL-Fc proteins on cytokine release.

An allogeneic MLR was performed and supernatants were harvested on day 4 for cytokine analysis in triplicates. This figure shows concentration of 6 cytokines: (A) IFN‐γ (B) TNF-α (C) IL-2 (D) IL-10 (E) IL-17A (F) IL-17F. Data is reported as mean ± SEM (*p≤ 0.05, **p≤0.01, ***P≤0.0001, ****P≤0.0001).</p

T cells cytotoxicity activity in the presence and absence of anti-PD-1, CD80-Fc, and 4-1BBL-Fc proteins in two types of treatment, namely, mono and combination.

CFSE-target cells and effector cells from 6 healthy donors were co-cultured (A) at 1:1 and 10:1 ratios (two-way ANOVA was applied for the data collected) (B) At 10:1 ratio, all of the conditions were compared with the untreated using one-way ANOVA. (*p≤ 0.05, **p≤0.01, ***P≤0.0001). The column height represents the average percentage of the 7-AAD-positive CFSE-target cells and error bars are ± SEM.</p

The flow cytometry binding analysis of Fc fusion proteins with engineered CHO-K1 cells in 0, 0.2, 1, 5, and 10 μg/mL concentrations.

Binding capacity was detected by FITC mouse anti-human IgG and using stable cell lines engineered A) human PD-1 (CHO-K1/hPD-1) B) human CD28 (CHO-K1/hCD28) C) human CTLA4 (CHO-K1/hCTLA4), and D) human 4-1BB (CHO-K1/h4-1BB).</p

T cell proliferation induction of anti-PD-1, CD80-Fc, and 4-1BBL-Fc proteins in mono and combination treatments.

For an allogeneic MLR, responder cells from 6 healthy donors were stained with CFSE and mixed with γ-irradiated PBMCs at a 1:1 ratio. PBMCs were harvested after 5 days and analyzed by flow cytometry. (A) Histogram plots depict examples of two populations used in this study for MLR analysis: M1 (divided) and M2 (highly divided) populations of untreated (Light gray) and PHA conditions (dark gray). M2 is a subset of the M1 population that contains cells with a high division rate. (B) Percentage of CFSE intensity was measured in different conditions and statistical analysis was performed using a one-way ANOVA test (*p≤ 0.05, **p≤0.01). The column height represents the average percentage of the CFSE- labeled cells and error bars are ± SEM.</p

Original image of western blot.

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