Immune signature of tumor infiltrating immune cells in renal cancer

Abstract

Tumor-associated immune cells have been discussed as an essential factor for the prediction of the outcome of tumor patients. Lymphocyte-specific genes are associated with a favorable prognosis in colorectal cancer but with poor survival in renal cell carcinoma (RCC). Flow cytometric analyses combined with immunohistochemistry were performed to study the phenotypic profiles of tumor infiltrating lymphocytes (TIL) and the frequency of T cells and macrophages in RCC lesions. Data were correlated with clinicopathological parameters and survival of patients. Comparing oncocytoma and clear cell (cc)RCC, T cell numbers as well as activation-associated T cell markers were higher in ccRCC, whereas the frequency of NK cells was higher in oncocytoma. An intratumoral increase of T cell numbers was found with higher tumor grades (G1:G2:G3/4 = 1:3:4). Tumor-associated macrophages slightly increased with dedifferentiation, although the macrophage-to-T cell ratio was highest in G1 tumor lesions. A high expression of CD57 was found in T cells of early tumor grades, whereas T cells in dedifferentiated RCC lesions expressed higher levels of CD69 and CTLA4. TIL composition did not differ between older (>70 y) and younger (<58 y) patients. Enhanced patients' survival was associated with a higher percentage of tumor infiltrating NK cells and Th1 markers, e.g. HLA-DR+ and CXCR3+ T cells, whereas a high number of T cells, especially with high CD69 expression correlated with a worse prognosis of patients. Our results suggest that immunomonitoring of RCC patients might represent a useful tool for the prediction of the outcome of RCC patients.

Keywords: CD13; CD57; CD69; MDSC, myeloid-derived suppressor cells; MFI, mean fluorescence intensity; NK, natural killer cells; PE, phycoerythrine; RCC, renal cell carcinoma; TIL, tumor-infiltrating lymphocytes; flow cytometry; immunohistochemistry; immunophenotype; mAb, monoclonal antibody; oncocytoma; renal cell carcinoma; survival; tumor infiltrating lymphocytes (TIL); tumor-associated macrophages (TAM).

Figure 1.

The distribution of T-, B- and NK cells differs in oncocytoma and clear cell (cc) renal cell carcinoma (RCC). Tumor infiltrating lymphocytes (TIL) were stained directly after mechanical tumor dissociation as described in Materials and Methods with antibodies (T cells: CD3, B cells: CD19, natural killer (NK) cells: CD3negCD56) and analyzed by 4-color flow cytometry. While T cells and NK cells exert an approximately equal proportion in oncocytoma (n = 6), NK cells represent only 20% of TIL in ccRCC (n = 92). Results are given as percentage of lymphocytes.

Figure 2.

Intratumoral T cells and macrophages increase during renal tumor dedifferentiation. T cells (CD3+) and macrophages (CD68+) were counted in 2.5 mm² (average of 10 representative fields) after immunohistochemical staining (peroxidase) of 2-μm paraffin-embedded sections of oncocytoma (OCT; n = 6) and renal cell carcinoma (clear cell histology) of different nuclear grading (n = 62). Absolute intratumoral and peritumoral cell numbers are shown for T cells (A) and macrophages (B). The highest ratio of “macrophages to T cells” was found in G1 tumor grades, as illustrated in (C). Results are given as mean value ± SE.

Figure 3.

The phenotype of tumor infiltrating lymphocytes differs with dedifferentiation of renal tumor lesions. Tissues of oncocytoma (OCT, n = 6) and renal cell carcinoma (clear cell type) of different nuclear grading (n = 7, 46, 9 for G1, G2, G3/4) were treated directly after mechanical tumor dissociation with antibody staining and analyzed by 4-color flow cytometry as described in materials and methods. Tumor dedifferentiation goes along with the enrichment of T cells (CD3+) and a parallel decrease of NK cell percentages (CD56+CD3neg). The percentage of CD8+ T cells does not correlate with tumor grading. Whereas CD69 expression of T cells clearly increases with tumor dedifferentiation, the percentage of CD57+ T cells and of CD4+CD7negative T cells rather decrease. Results are given as mean value ± SE. Asterisks mark significant differences (ANOVA, Bonferroni) with the respective reference parameter shown as an open circle.

Figure 4.

Relationship between the phenotype of tumor infiltrating lymphocytes of renal cell carcinoma (clear cell type) and patient's survival. Patients with higher T cell and lower NK cell percentage in tumor tissue were closely correlated with poorer overall survival. A better survival was correlated with a high percentage of TH1-associated markers, such as HLA-DR expression or CXCR3 intensity, as well as a lower CD69 expression of T cells. A tendency to a better overall survival was found for a low CD13 expression of T cells. Kaplan–Meier analysis is shown, patients with censored survival times are denoted by tick marks.