Flow cytometric analysis of Th1 and Th2 cytokines in PBMCs as a parameter of immunological dysfunction in patients of superficial transitional cell carcinoma of bladder

Abstract

Transitional cell carcinoma (TCC) is the commonest cancer of the bladder. Although majority of TCC can be diagnosed at an early stage and removed easily by transurethral resection of tumor (TURT), the management of this carcinoma is complicated due to frequent recurrences usually within 6 months to one-year period. An imbalance between the Th1 and Th2 immune responses has been attributed to immune dysregulation in various malignancies. The present study aims to evaluate the Th1 and Th2 balance in Peripheral Blood Mononuclear Cells of 41 TCC patients (20 recurrent and 21 non-recurrent) using flow cytometry. It also further assesses immunological and cellular factors influencing the anti-neoplastic activity of the TCC patients and in 21 normal healthy subjects in terms of their cytokine expression and various cell surface markers. The findings of the study revealed that the cell surface markers CD3+, CD4+ and CD8+ along with NK cells were found to be significantly lower in patients than healthy controls (p < 0.01). The mean percent expression of CD4+ was significantly lower in patients showing recurrence (23.9 +/- 9.84) as compared to patients with non-recurrence (31.1 +/- 12.27). The percentage of CD4+T-cells (mean +/- SD) producing IFN-gamma, IL-2 and TNF-alpha were statistically significantly reduced in patients (19.1 +/- 4.94, 52.3 +/- 20.86 and 12.8 +/- 4.49) as compared to healthy controls (23.3 +/- 3.67, 67.5 +/- 12.0 and 17.6 +/- 5.96 respectively), (p < 0.01, 0.018, 0.001). On the contrary, the mean levels of IL-4, IL-6 and IL-10 in patients (63.8+/-17.01, 60.4+/-14.79 and 65.7 +/- 14.84 respectively) were significantly higher as compared to healthy controls (24.4 +/- 8.77, 26.5 +/- 5.28 and 20.6 +/- 3.81 respectively), (p < 0.001). No statistically significant difference was observed in the cytokine expression between patients showing recurrence and non-recurrence. Patients with bladder cancer seem to develop a Th2 dominant status with a deficient type1 immune response. The lymphocyte evaluation along with cytokine measurement can provide a sensitive and valuable tool for evaluating the function of cell-mediated immunity in these patients and can also find application in therapeutic monitoring of bladder cancer patients as new targets for immunotherapy.

Fig. 1

Mean and standard deviation of the phenotypic characterization and the frequency of CD4+T-cells producing Th1 and Th2 cytokines in healthy controls and bladder cancer patients

Fig. 2

Mean and standard deviation of the phenotypic characterization and the frequency of CD4+T-cells producing Th1 and Th2 cytokines in recurrent and non-recurrent bladder cancer patients

Fig. 3

Dot plots of Flow cytometric analysis in a bladder cancer patient and a healthy control subject showing the a phenotypic characterization and the b frequency of CD4+ cytokine-positive T-lymphocytes by flow cytometry. 10,000 lymphocytes were analyzed with cell-size gating. The frequencies of CD4+IFN-γ+, IL-2+, and TNF-α+ T-cells were reduced whereas; CD4+IL-4+, IL-6+ and IL-10+ were higher in patients with TCCs than in healthy controls

Fig. 3

Dot plots of Flow cytometric analysis in a bladder cancer patient and a healthy control subject showing the a phenotypic characterization and the b frequency of CD4+ cytokine-positive T-lymphocytes by flow cytometry. 10,000 lymphocytes were analyzed with cell-size gating. The frequencies of CD4+IFN-γ+, IL-2+, and TNF-α+ T-cells were reduced whereas; CD4+IL-4+, IL-6+ and IL-10+ were higher in patients with TCCs than in healthy controls