Immunologic Characterization and T cell Receptor Repertoires of Expanded Tumor-infiltrating Lymphocytes in Patients with Renal Cell Carcinoma.

The successful use of expanded tumor-infiltrating lymphocytes (TIL) in adoptive TIL therapies has been reported, but the effects of the TIL expansion, immunophenotype, function, and T cell receptor (TCR) repertoire of the infused products relative to the tumor microenvironment (TME) are not well understood. In this study, we analyzed the tumor samples (n = 58) from treatment-naïve patients with renal cell carcinoma (RCC), "pre-rapidly expanded" TILs (pre-REP TIL, n = 15) and "rapidly expanded" TILs (REP TIL, n = 25) according to a clinical-grade TIL production protocol, with single-cell RNA (scRNA)+TCRαß-seq (TCRαß sequencing), TCRß-sequencing (TCRß-seq), and flow cytometry. REP TILs encompassed a greater abundance of CD4+ than CD8+ T cells, with increased LAG-3 and low PD-1 expressions in both CD4+ and CD8+ T cell compartments compared with the pre-REP TIL and tumor T cells. The REP protocol preferentially expanded small clones of the CD4+ phenotype (CD4, IL7R, KLRB1) in the TME, indicating that the largest exhausted T cell clones in the tumor do not expand during the expansion protocol. In addition, by generating a catalog of RCC-associated TCR motifs from >1,000 scRNA+TCRαß-seq and TCRß-seq RCC, healthy and other cancer sample cohorts, we quantified the RCC-associated TCRs from the expansion protocol. Unlike the low-remaining amount of anti-viral TCRs throughout the expansion, the quantity of the RCC-associated TCRs was high in the tumors and pre-REP TILs but decreased in the REP TILs. Our results provide an in-depth understanding of the origin, phenotype, and TCR specificity of RCC TIL products, paving the way for a more rationalized production of TILs. Significance: TILs are a heterogenous group of immune cells that recognize and attack the tumor, thus are utilized in various clinical trials. In our study, we explored the TILs in patients with kidney cancer by expanding the TILs using a clinical-grade protocol, as well as observed their characteristics and ability to recognize the tumor using in-depth experimental and computational tools.

T and NK cell abundance defines two distinct subgroups of renal cell carcinoma.

Renal cell carcinoma (RCC) is considered as an immunogenic cancer. Because not all patients respond to current immunotherapies, we aimed to investigate the immunological heterogeneity of RCC tumors. We analyzedthe immunophenotype of the circulating, tumor, and matching adjacent healthy kidney immune cells from 52 nephrectomy patients with multi-parameter flow cytometry. Additionally, we studied the transcriptomic and mutation profiles of 20 clear cell RCC (ccRCC) tumors with bulk RNA sequencing and a customized pan-cancer gene panel. The tumor samples clustered into two distinct subgroups defined by the abundance of intratumoral CD3+ T cells (CD3high, 25/52) and NK cells (NKhigh, 27/52). CD3high tumors had an overall higher frequency of tumor infiltrating lymphocytes and PD-1 expression on the CD8+ T cells compared to NKhigh tumors. The tumor infiltrating T and NK cells had significantly elevated expression levels of LAG-3, PD-1, and HLA-DR compared to the circulating immune cells. Transcriptomic analysis revealed increased immune signaling (IFN-γ, TNF-α via NF-κB, and T cell receptor signaling) and kidney metabolism pathways in the CD3high subgroup. Genomic analysis confirmed the typical ccRCC mutation profile including VHL, PBRM1, and SETD2 mutations, and revealed PBRM1 as a uniquely mutated gene in the CD3high subgroup. Approximately half of the RCC tumors have a high infiltration of NK cells associated with a lower number of tumor infiltrating lymphocytes, lower PD-1 expression, a distinct transcriptomic and mutation profile, providing insights to the immunological heterogeneity of RCC which may impact treatment responses to immunological therapies.

Spatial immunoprofiling of the intratumoral and peritumoral tissue of renal cell carcinoma patients.

While the abundance and phenotype of tumor-infiltrating lymphocytes are linked with clinical survival, their spatial coordination and its clinical significance remain unclear. Here, we investigated the immune profile of intratumoral and peritumoral tissue of clear cell renal cell carcinoma patients (n = 64). We trained a cell classifier to detect lymphocytes from hematoxylin and eosin stained tissue slides. Using unsupervised classification, patients were further classified into immune cold, hot and excluded topographies reflecting lymphocyte abundance and localization. The immune topography distribution was further validated with The Cancer Genome Atlas digital image dataset. We showed association between PBRM1 mutation and immune cold topography, STAG1 mutation and immune hot topography and BAP1 mutation and immune excluded topography. With quantitative multiplex immunohistochemistry we analyzed the expression of 23 lymphocyte markers in intratumoral and peritumoral tissue regions. To study spatial interactions, we developed an algorithm quantifying the proportion of adjacent immune cell pairs and their immunophenotypes. Immune excluded tumors were associated with superior overall survival (HR 0.19, p = 0.02) and less extensive metastasis. Intratumoral T cells were characterized with pronounced expression of immunological activation and exhaustion markers such as granzyme B, PD1, and LAG3. Immune cell interaction occurred most frequently in the intratumoral region and correlated with CD45RO expression. Moreover, high proportion of peritumoral CD45RO+ T cells predicted poor overall survival. In summary, intratumoral and peritumoral tissue regions represent distinct immunospatial profiles and are associated with clinicopathologic characteristics.