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 (CD3(high), 25/52) and NK cells (NKhigh, 27/52). CD3(high) 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-gamma, TNF-alpha via NF-kappa B, and T cell receptor signaling) and kidney metabolism pathways in the CD3(high) 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 CD3(high) 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.Peer reviewe

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.Peer reviewe

Immunophenotyping and expanding tumor infiltrating lymphocytes in renal cell carcinoma

Syöpäkasvaimista löytyy useita erilaisia immuunipuolustuksen soluja, jotka tunkeutuvat kasvaimen mikroympäristöön, kuten kasvaimeen tunkeutuvia lymfosyyttejä (tumor infiltrating lymphocytes, TILs). Lisää tutkimusta tarvitaan eri imusolujen tehtävien ja kliinisen merkityksen ymmärtämiseen syövässä. Potilaiden välisten immunologisten erojen ymmärtäminen auttaa uusien hoitomuotojen kehittämisessä ja selvittämään, miksi vain osa potilaista saa myönteisen hoitovasteen. Munuaiskarsinooma on yleisin munuaisen syövistä. Sen ennuste on hyvä, silloin kun kasvaimen poisto on mahdollinen eikä etäpesäkkeitä ole muodostunut. Jos syöpäkasvain pääsee leviämään, ennuste munuaiskarsinoomalle heikkenee huomattavasti. Tämän maisteritutkielman tavoitteena oli karakterisoida kasvaimeen tunkeutuvia lymfosyyttejä munuaiskarsinoomapotilaiden näytteistä. Karakterisointiin käytettiin virtaussytometriaa, jossa vasta-aineet oli valittu tunnistamaan eri lymfosyyttipopulaatioita. Halusimme myös tutkia lymfosyyttejä tarkemmin kasvattamalla niitä potilasnäytteistä, ja tutkimalla lymfosyyttien toiminnallisuutta kasvattamalla niitä yhdessä syöpäsolujen kanssa. Tulosten perusteella näytteiden lymfosyyteissä on joitakin eroja eri munuaiskarsinooman alatyyppien välillä. Kuitenkin lisätutkimuksia tarvitaan näiden erojen tarkemmaksi määritykseksi. Suurin osa kasvatetuista lymfosyyteistä oli CD4+ T soluja, jotka ilmensivät muistisoluihin liittyviä CD45RO ja CCR7 antigeenejä. Osa kasvatetuista lymfosyyteistä ilmensi myös T solujen aktiivisuuteen ja terminaaliseen erilaistumiseen liittyviä antigeenejä. Yhteenvetona, tämä tutkielma tarjosi materiaalia ja näkökulmia tuleviin tutkimusprojekteihin koskien munuaiskarsinooman lymfosyyttejä.Tumors contain variable number of different immune cells that infiltrate the tumor microenvironment, such as tumor infiltrating lymphocytes (TILs). More research is needed to understand the functional and clinical importance of various TIL subgroups in cancer. Understanding the differences between individual cancer patients will help development of new treatment methods and discovering why only some patients respond to immunological treatments. Renal cell carcinoma (RCC) is the most common kidney cancer type with good overall survival prognosis when the tumor is surgically removed before it has metastasized. However, the prognosis of RCC is significantly decreased when the cancer has spread. The aim of this master’s thesis project was to characterize the tumor infiltrating lymphocyte populations in patient derived RCC samples. Characterization was done with flow cytometry and a custom antibody panel designed to detect various lymphocyte subpopulations. We also wanted to further study the TILs by expanding the lymphocytes from the tumor samples and test their function in an impendence-based assay against matched autologous tumor cells. Based on the flow cytometry results, the different RCC subtypes in the cohort showed some variation in TILs. Still, more research is needed to investigate these differences. We were able to culture the TILs from the RCC tumor samples, and most of them were CD4+ T cells expressing memory markers CD45RO and CCR7. Some expanded TILs expressed markers related to T cell activity and terminal differentiation. In conclusion, this thesis provided material and insights for future RCC TIL experiments as well as considerations for optimization needed in further studies

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-naive 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)+TCR alpha beta-seq (TCR alpha beta sequencing), TCR beta-sequencing (TCR beta-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+TCR alpha beta-seq and TCR beta-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.Peer reviewe

Table S4 from Immunologic Characterization and T cell Receptor Repertoires of Expanded Tumor-infiltrating Lymphocytes in Patients with Renal Cell Carcinoma

Supplementary Table S4 shows the publicly validated RCC-associated motifs, together with their antigen-specificities with TCRGP, and motifs found in the REP TILs using GLIPH2.</p

Table S1 from Immunologic Characterization and T cell Receptor Repertoires of Expanded Tumor-infiltrating Lymphocytes in Patients with Renal Cell Carcinoma

Supplementary Table S1 shows the RCC patient data including clinical parameters and status</p

Figure S15 from Immunologic Characterization and T cell Receptor Repertoires of Expanded Tumor-infiltrating Lymphocytes in Patients with Renal Cell Carcinoma

Figure S15 shows pathway enrichment analyses for the pre-REP TILs and REP TILs, as well as the abundance of the RCC-associated motifs found in the UMAP T-cell clusters for each tumor sample.</p

Figure S8 from Immunologic Characterization and T cell Receptor Repertoires of Expanded Tumor-infiltrating Lymphocytes in Patients with Renal Cell Carcinoma

Figure S8 shows the analyses of the different clonotype sizes and tracking their abundancies found in the bulk TCRb-seq data.</p

Figure S5 from Immunologic Characterization and T cell Receptor Repertoires of Expanded Tumor-infiltrating Lymphocytes in Patients with Renal Cell Carcinoma

Figure S5 shows representative gating strategies for the co-culture assays with different timepoints (6h, 48h) and conditions (baseline, unstimulated, T-cell stimulated).</p

Figure S2 from Immunologic Characterization and T cell Receptor Repertoires of Expanded Tumor-infiltrating Lymphocytes in Patients with Renal Cell Carcinoma

Figure S2 shows representative flow gating strategies for the immunophenotyping of various sample types (tumor, healthy kidney, pre-REP TILs and REP TILs), as well as the co-culture assays.</p