Immune signature of tumor infiltrating immune cells in renal cancer

Introduction

RCC accounts for approximately 5% of all human malignancies in the Western world and its incidence is steadily increasing. It is a heterogeneous disease consisting of the most frequent clear cell carcinoma (ccRCC) (75%) and the renal papillary carcinoma with an incidence of 10–15%, both arising from epithelial cells of the proximal tubuli. In contrast, chromophobe RCC (5%) and the benign oncocytoma (3%–7%) arise from the intercalating cells of the collecting ducts of the kidney.¹

The clinical outcome of RCC patients is highly variable. Due to the lack of symptoms approximately one-third RCC patients have already an advanced disease at the time of diagnosis.² Furthermore, 25–30% of RCC patients treated for local disease develop metastasis. Radical or partial nephrectomy remain the only effective method of cure for localized disease.³ The outcome of RCC patients with metastatic disease is poor with a 5 y survival rate of less than 5%. Treatment options have been limited due to the resistance to chemotherapy and radiation. Recently, the treatment of metastatic RCC has been revolutionized by the development of targeted therapies, in particular by tyrosine kinase inhibitors (sunitinib, sorafenib, pazopanib, axitinib), antiangiogenic agents such as bevacizumab, and mTOR inhibitors, which have been shown to increase the progression-free survival of RCC patients.⁴

Despite considerable progress in our understanding of the pathogenesis, genetics, and pathology of RCC, the prediction of the clinical outcome for individual cases remains difficult. RCC represents an immunosensitive tumor due to the relatively high spontaneous regression rate and the high levels of tumor infiltrating immune cells, including lymphocytes, macrophages and dendritic cells. Tumor-infiltrating lymphocytes (TIL) are influenced by the local cellular and soluble components of the tumor microenvironment.⁵ Despite antigen-specific HLA-restricted TIL clones can be found after culture of RCC tumor tissues,^6,7,8 the function of these T cells in RCC is often impaired – due to their incomplete activation or anergic status (for a review see ref.⁹). Tumor cells have developed different strategies to escape immune surveillance, such as loss or downregulation of MHC class I expression, expression of co-inhibitory molecules as well as secretion of cytokines and factors, which create an immune suppressive microenvironment.⁵ These processes lead to a failure of the local antitumor response and thus contribute to tumor progression. Recent studies demonstrated that the density of CD8+ T cells correlated with poor prognosis in RCC patients.¹⁰ Furthermore, high numbers of immune suppressive cells, such as regulatory T cells or myeloid derived suppressor cells (MDSC) in the peripheral blood and/or tumor lesions of RCC patients, were associated with a poor clinical outcome. ^11,12 In order to increase the knowledge about the control and dynamics of immune responses directed against RCC, the immune cell phenotype of renal tumor lesions was characterized in this study. Flow cytometric analysis combined with immunohistochemistry was performed and correlated with tumor histology, Fuhrmann grading, tumor size, age of patients as well as patients’ survival.

Results

Discussion

Immune cell infiltration is a common feature of RCC, but the contribution of lymphocytes to tumor elimination remains unclear. RCC are predominantly infiltrated by T- and NK cells expressing markers of activation and of memory suggesting a tissue-specific T cell activation in RCC. ^15,16 Despite convincing evidence of immune reactions to tumor cells exists, these immune responses remain ineffective, and the tumor infiltrated by immune cells continues to grow. The mechanisms that cause tumor escape include suppression of effector cells or immune cell dysfunction which could be mediated by immunosuppressive factors of the tumor microenvironment, including TGF-β, IL-10, gangliosides, adenosine, products of oxidative stress and thrombospondin, ⁵ or by the tumor cells itself expressing e.g., the non-classical HLA-G molecules and co-inhibitory molecules of the B7-H family. ^17,18 Due to the interaction with KIR2DL4, ILT2 and/or ILT4, HLA-G negatively interferes with NK and T cell immune responses (for review see ¹⁹), while B7-H1 binds to PD1 thereby blocking T cell responses.¹⁸

The phenotypic and functional profile of tumor-infiltrating immune cells varies depending on the tumor subtype and the tumor microenvironment and may influence prognosis and disease outcome. During the last years, the analysis of the microenvironment has kindled interest since the nature, the density and localization of cells of the adaptive immune system directly influence the risk of relapse and can be superior to the TNM classification in some cancers, in particular colorectal carcinoma.²⁰ Using Cox regression analyses, the prognostic value of markers specifically expressed in B and T cells was correlated with overall survival. Whereas lymphocyte-specific genes were associated with a favorable prognosis in colorectal cancer, this was not the case for RCC.²¹ In this study, a combination of flow cytometry und immunohistochemistry revealed, that (i) a high NK cell percentage as well as Th1-associated markers, such as HLA-DR and CXCR3 were associated with better survival in ccRCC, (ii) the percentage of macrophages to T cells was highest in G1 tumor grades as is the percentage of CD57+ T cells, (iii) the T cell percentage and the absolute number of T cells increased with dedifferentiation of ccRCC, (iv) a poor prognosis was associated with special T cell molecules, such as CD13 and CD69.

Using flow cytometry, we compared data of the benign oncocytoma with ccRCC. Oncocytoma are rare renal epithelial neoplastic lesions that exhibit antigenic characteristics of distal nephron intercalated cells.²² Data characterizing lymphocytic infiltration of oncocytoma are lacking. In 1992 Störkel and co-authors described a low lymphocytic infiltration density in oncocytoma compared to ccRCC,²³ which was confirmed by us and extended to NK cells and markers of T cell activation. The frequency of NK cells was nearly 50 % in oncocytoma, since T cells are rare. TIL in oncocytoma compared to renal cancer differed with respect to the expression of function-associated markers, with a lower T cell expression of CD13, CD26, CD28, CD69 and HLA-DR and a high percentage of CD57+ T cells. A transition from markers expressed in oncocytoma, in good-differentiated and in poor-differentiated ccRCC tumor lesions was found in some markers, in particular in the percentage of NK cells, in T cell count or in the percentage of CD69+ T cells. The latter increased with grading and staging and correlated with poor patient's survival. Tumor-induced CD69+CD4+CD25neg T cells have been claimed to be a new subset of regulatory T cells, since they are not Foxp3+, but express membrane-bound TGF-ß1 and can suppress T cell proliferation in a cell–cell contact manner.²⁴ Therefore, CD69+ T cells could play a major role in the transition from a renal tumor controlled by immune cells to a tumor which evades immune control. A high expression of CD69+CD25neg T cells was found in chronic pancreatitis as well as in pancreatic ductal adenocarcinoma (associated with nodal invasion and higher grading) and L1CAM/CD171 expression of tumor cells reduced proliferation, diminished CD25 expression and elevated CD69 expression in effector T cells.²⁵

We found that the percentage of NK cells decreased in ccRCC with higher grading, while the number of T cells increased. Since the absolute numbers of T cells are four-fold higher in G3/4 tumors, the decrease in NK cells might be only in the percentage of lymphocytes and not in absolute cell numbers. However, a higher percentage of NK cells predicts a better survival of RCC patients in our analysis, which is in line with other reports.¹⁰ As recently shown, NK cells from TIL with a high percentage of NK cells (>20%) are CD16^bright and non-cytolytic ex vivo, but developed cytotoxicity after a short-term activation with IL-2.²⁶ Only a few CD16^bright NK cells and lower perforin expression were found in tumors with low NK cell frequencies. These data suggest a functional difference according to NK cell density as well as an influence of T cells on NK cell function, which has to be clarified in future work.

This study confirms the high expression level of markers of T cell exhaustion in TIL of RCC, such as CD57. However, both the accumulation of CD57+ T cells as well as of CD7neg/CD4+ T cells was more pronounced in low grade and not in high grade RCC lesions and even T cells of oncocytoma showed a high expression of CD57. Though CD57 has been claimed to be a general marker of proliferative instability (for a review see ref.²⁷), CD57 expression correlates strongly with simultaneous expression of granzymes A, B, and perforin.²⁸ Therefore, our data might implement a high cytotoxic potential of CD57+ T cells in oncocytoma and early grade ccRCC, but not in poor-differentiated tumors. However, in melanoma CD8+CD57+ T cells have been described as incompletely-differentiated cytotoxic T cells with hybrid phenotypic and functional properties of both an early effector-memory type and a terminally-differentiated effector cell.²⁹ These T cells co-expressed CD27, CD28, CD57, and granzyme B, but little or no perforin. They could be induced to proliferate, produce a high level of IFNγ, and to differentiate into perforin (high) mature cytotoxic T cells in vitro, but addition of TGF-β1 prevented further differentiation. Our data give no hint to an association of CD57 expression and the patients’ survival.

Using T-cell receptor clonotype mapping of expanded TIL, low numbers of distinct expanded T-cell clonotypes were found in RCC as compared with melanoma lesions.⁸ Comparing TIL in melanoma and RCC a more pronounced T-cell differentiation was reported in RCC (e.g., a higher percentage of effector memory T cells).³⁰ The authors discuss that CD70 expression as a peculiarity of RCC cells can result in T-cell costimulation and subsequently in proliferative exhaustion of T cells. However, no association was found between CD70 expression and staging or grading of RCC by other authors.³¹ A comparison of TIL from RCC and lung cancer demonstrated a higher frequency of B cells in lung cancer (18% in lung adenocarcinoma vs. 6% in RCC), but more NK cells in RCC.¹³. In addition, a RCC-specific T-cell activation with more CD26, CD13, and HLA-DR was found, while more CD25 and CD69 was detected on lung cancer TIL.¹³ Lung cancer patients with the lowest percentages of CD13+ tumor-infiltrating T cells show a better survival,³² similarly to the situation in ccRCC patients described in this study. Whereas in lung cancer 14% of T cells expressed CD13,³² 32% of T cells are CD13+ in ccRCC (and 2% in oncocytoma). CD13/Aminopeptidase N is a ubiquitously expressed ectopeptidase and involved in multiple cellular functions, such as the modulation of kinins, neuropeptides, and chemotactic mediators as well as in adhesion, cell–cell interactions and signal transduction (for a review see ref.³³). CD13+CD4+CD25+ T cells were shown to increase in the blood of lung cancer patients in a stage-dependent way and to exert a high suppressive capacity.³⁴ The antibody WM15 can inhibit enzymatic aminopeptidase N activity and can suppress the expression of FoxP3, TGFß or CTLA4 on regulatory T cells, pointing to a role of the enzymatic activity of CD13 in the T cell suppression.³⁴ Up to now we cannot explain why especially the middle range of CD13 expression is associated with a poor patient's survival. However, CD13+CD69+ T cells might play a key role in the immune evasion of ccRCC, which has to be addressed in the future.

Using immunohistochemistry, an enrichment of macrophages was found by us especially in G1 tumor lesions, where the ratio of macrophages to T cells was 3.9 to 1. The presence of macrophages in early tumor grades of ccRCC might precede the accumulation of T cells. During the initial stages of tumor progression tumor cells produce migration factors for monocytes. Several reports connect the presence of macrophages with T cell suppression, since cells of the myeloid lineage can produce immunosuppressive factors, including prostaglandin E2, indoleamine 2,3-dioxygenase and IL-10 (for a review see ref.³⁵). The contact of MDSC with T cells rapidly depletes arginine from the microenvironment, causing a profound inhibition of T cell proliferation, cytokine production and the expression of the CD3ζ chain of the T cell receptor. Therefore, T cells stimulated by tumor antigens in this arginine-depleted microenvironment possibly develop defects and become anergic.³⁶ A recent study on tumor-associated macrophages in RCC describes an association of the number of CD68+ macrophages with a shorter survival as well as an accumulation of M2 type macrophages expressing the scavenger receptor CD163 and the interferon regulatory factor 4 in ccRCC.³⁷ Other authors investigated the intratumoral balance of polarized tumor-associated macrophages and showed that especially CD206/mannose receptor-positive macrophages (M2 type) are associated with reduced survival in RCC patients.³⁸ Recently, markers for human MDSC have been developed (e.g., CD14+HLA-DRlow) therefore the inclusion of myeloid cells in flow cytometric analyses would be a worthwhile approach in future studies. A higher amount of HLA-DRlow (CD13high) monocytes in the blood of RCC patients has been described already especially in late tumor stages ³⁹ and might be the cause for poor results obtained with cancer vaccines of monocyte-derived dendritic cells.⁴⁰

In conclusion, this study demonstrated peculiarities of tumor-infiltrating immune cells in renal tissues. Further understanding of the dynamics of intra-tumoral NK cells, T cells and macrophages would provide key insights into the process of immune suppression in renal cancer and help to develop successful immunotherapies, urgently needed especially for metastatic RCC.

Materials and Methods

Supplementary Material

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Supplemental Material

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