Targeting of multiple tumor-associated antigens by individual T cell receptors during successful cancer immunotherapy.

The T cells of the immune system can target tumors and clear solid cancers following tumor-infiltrating lymphocyte (TIL) therapy. We used combinatorial peptide libraries and a proteomic database to reveal the antigen specificities of persistent cancer-specific T cell receptors (TCRs) following successful TIL therapy for stage IV malignant melanoma. Remarkably, individual TCRs could target multiple different tumor types via the HLA A∗02:01-restricted epitopes EAAGIGILTV, LLLGIGILVL, and NLSALGIFST from Melan A, BST2, and IMP2, respectively. Atomic structures of a TCR bound to all three antigens revealed the importance of the shared x-x-x-A/G-I/L-G-I-x-x-x recognition motif. Multi-epitope targeting allows individual T cells to attack cancer in several ways simultaneously. Such "multipronged" T cells exhibited superior recognition of cancer cells compared with conventional T cell recognition of individual epitopes, making them attractive candidates for the development of future immunotherapies.

Proteomics of Melanoma Response to Immunotherapy Reveals Mitochondrial Dependence.

Immunotherapy has revolutionized cancer treatment, yet most patients do not respond. Here, we investigated mechanisms of response by profiling the proteome of clinical samples from advanced stage melanoma patients undergoing either tumor infiltrating lymphocyte (TIL)-based or anti- programmed death 1 (PD1) immunotherapy. Using high-resolution mass spectrometry, we quantified over 10,300 proteins in total and ∼4,500 proteins across most samples in each dataset. Statistical analyses revealed higher oxidative phosphorylation and lipid metabolism in responders than in non-responders in both treatments. To elucidate the effects of the metabolic state on the immune response, we examined melanoma cells upon metabolic perturbations or CRISPR-Cas9 knockouts. These experiments indicated lipid metabolism as a regulatory mechanism that increases melanoma immunogenicity by elevating antigen presentation, thereby increasing sensitivity to T cell mediated killing both in vitro and in vivo. Altogether, our proteomic analyses revealed association between the melanoma metabolic state and the response to immunotherapy, which can be the basis for future improvement of therapeutic response.

CD8+ T cell activation in cancer comprises an initial activation phase in lymph nodes followed by effector differentiation within the tumor.

Improvements in tumor immunotherapies depend on better understanding of the anti-tumor T cell response. By studying human tumor-draining lymph nodes (TDLNs), we found that activated CD8+ T cells in TDLNs shared functional, transcriptional, and epigenetic traits with TCF1+ stem-like cells in the tumor. The phenotype and TCR overlap suggested that these TDLN cells were precursors to tumor-resident stem-like CD8+ T cells. Murine tumor models revealed that tumor-specific CD8+ T cells were activated in TDLNs but lacked an effector phenotype. These stem-like cells migrated into the tumor, where additional co-stimulation from antigen-presenting cells drove effector differentiation. This model of CD8+ T cell activation in response to cancer is different from that of canonical CD8+ T cell activation to acute viruses, and it proposes two stages of tumor-specific CD8+ T cell activation: initial activation in TDLNs and subsequent effector program acquisition within the tumor after additional co-stimulation.

Landscape of Infiltrating T Cells in Liver Cancer Revealed by Single-Cell Sequencing.

Systematic interrogation of tumor-infiltrating lymphocytes is key to the development of immunotherapies and the prediction of their clinical responses in cancers. Here, we perform deep single-cell RNA sequencing on 5,063 single T cells isolated from peripheral blood, tumor, and adjacent normal tissues from six hepatocellular carcinoma patients. The transcriptional profiles of these individual cells, coupled with assembled T cell receptor (TCR) sequences, enable us to identify 11 T cell subsets based on their molecular and functional properties and delineate their developmental trajectory. Specific subsets such as exhausted CD8+ T cells and Tregs are preferentially enriched and potentially clonally expanded in hepatocellular carcinoma (HCC), and we identified signature genes for each subset. One of the genes, layilin, is upregulated on activated CD8+ T cells and Tregs and represses the CD8+ T cell functions in vitro. This compendium of transcriptome data provides valuable insights and a rich resource for understanding the immune landscape in cancers.

Spatial maps of T cell receptors and transcriptomes reveal distinct immune niches and interactions in the adaptive immune response.

T cells mediate antigen-specific immune responses to disease through the specificity and diversity of their clonotypic T cell receptors (TCRs). Determining the spatial distributions of T cell clonotypes in tissues is essential to understanding T cell behavior, but spatial sequencing methods remain unable to profile the TCR repertoire. Here, we developed Slide-TCR-seq, a 10-µm-resolution method, to sequence whole transcriptomes and TCRs within intact tissues. We confirmed the ability of Slide-TCR-seq to map the characteristic locations of T cells and their receptors in mouse spleen. In human lymphoid germinal centers, we identified spatially distinct TCR repertoires. Profiling T cells in renal cell carcinoma and melanoma specimens revealed heterogeneous immune responses: T cell states and infiltration differed intra- and inter-clonally, and adjacent tumor and immune cells exhibited distinct gene expression. Altogether, our method yields insights into the spatial relationships between clonality, neighboring cell types, and gene expression that drive T cell responses.

Immunosurveillance in clinical cancer management.

The progression of cancer involves a critical step in which malignant cells escape from control by the immune system. Antineoplastic agents are particularly efficient when they succeed in restoring such control (immunosurveillance) or at least establish an equilibrium state that slows down disease progression. This is true not only for immunotherapies, such as immune checkpoint inhibitors (ICIs), but also for conventional chemotherapy, targeted anticancer agents, and radiation therapy. Thus, therapeutics that stress and kill cancer cells while provoking a tumor-targeting immune response, referred to as immunogenic cell death, are particularly useful in combination with ICIs. Modern oncology regimens are increasingly using such combinations, which are referred to as chemoimmunotherapy, as well as combinations of multiple ICIs. However, the latter are generally associated with severe side effects compared with single-agent ICIs. Of note, the success of these combinatorial strategies against locally advanced or metastatic cancers is now spurring successful attempts to move them past the postoperative (adjuvant) setting to the preoperative (neoadjuvant) setting, even for patients with operable cancers. Here, the authors critically discuss the importance of immunosurveillance in modern clinical cancer management.

The Transcription Factor Bhlhe40 Programs Mitochondrial Regulation of Resident CD8+ T Cell Fitness and Functionality.

Tissue-resident memory CD8+ T (Trm) cells share core residency gene programs with tumor-infiltrating lymphocytes (TILs). However, the transcriptional, metabolic, and epigenetic regulation of Trm cell and TIL development and function is largely undefined. Here, we found that the transcription factor Bhlhe40 was specifically required for Trm cell and TIL development and polyfunctionality. Local PD-1 signaling inhibited TIL Bhlhe40 expression, and Bhlhe40 was critical for TIL reinvigoration following anti-PD-L1 blockade. Mechanistically, Bhlhe40 sustained Trm cell and TIL mitochondrial fitness and a functional epigenetic state. Building on these findings, we identified an epigenetic and metabolic regimen that promoted Trm cell and TIL gene signatures associated with tissue residency and polyfunctionality. This regimen empowered the anti-tumor activity of CD8+ T cells and possessed therapeutic potential even at an advanced tumor stage in mouse models. Our results provide mechanistic insights into the local regulation of Trm cell and TIL function.

Enhanced Proteomic Coverage in Tissue Microenvironment by Immune Cell Subtype Library-Assisted DIA-MS.

Immune cells that infiltrate the tumor microenvironment (TME) play crucial roles in shaping cancer development and influencing clinical outcomes and therapeutic responses. However, obtaining a comprehensive proteomic snapshot of tumor-infiltrating immunity in clinical specimens is often hindered by small sample amounts and a low proportion of immune infiltrating cells in the TME. To enable in-depth and highly sensitive profiling of microscale tissues, we established an immune cell-enriched library-assisted strategy for data-independent acquisition mass spectrometry (DIA-MS). Firstly, six immune cell subtype-specific spectral libraries were established from sorted cluster of differentiation markers, CD8+, CD4+ T lymphocytes, B lymphocytes, natural killer cells, dendritic cells, and macrophages in murine mesenteric lymph nodes (MLNs), covering 7815 protein groups with surface markers and immune cell-enriched proteins. The feasibility of microscale immune proteomic profiling was demonstrated on 1 µg tissue protein from the tumor of murine colorectal cancer (CRC) models using single-shot DIA; the immune cell-enriched library increased coverage to quantify 7419 proteins compared to directDIA analysis (6978 proteins). The enhancement enabled the mapping of 841 immune function-related proteins and exclusive identification of many low-abundance immune proteins, such as CD1D1, and CD244, demonstrating high sensitivity for immune landscape profiling. This approach was used to characterize the MLNs in CRC models, aiming to elucidate the mechanism underlying their involvement in cancer development within the TME. Even with a low percentage of immune cell infiltration (0.25-3%) in the tumor, our results illuminate downregulation in the adaptive immune signaling pathways (such as C-type lectin receptor signaling, and chemokine signaling), T cell receptor signaling, and Th1/Th2/Th17 cell differentiation, suggesting an immunosuppressive status in MLNs of CRC model. The DIA approach using the immune cell-enriched libraries showcased deep coverage and high sensitivity that can facilitate illumination of the immune proteomic landscape for microscale samples.

The spread of interferon-γ in melanomas is highly spatially confined, driving nongenetic variability in tumor cells.

Interferon-γ (IFNγ) is a critical antitumor cytokine that has varied effects on different cell types. The global effect of IFNγ in the tumor depends on which cells it acts upon and the spatial extent of its spread. Reported measurements of IFNγ spread vary dramatically in different contexts, ranging from nearest-neighbor signaling to perfusion throughout the entire tumor. Here, we apply theoretical considerations to experiments both in vitro and in vivo to study the spread of IFNγ in melanomas. We observe spatially confined niches of IFNγ signaling in 3-D mouse melanoma cultures and human tumors that generate cellular heterogeneity in gene expression and alter the susceptibility of affected cells to T cell killing. Widespread IFNγ signaling only occurs when niches overlap due to high local densities of IFNγ-producing T cells. We measured length scales of ~30 to 40 µm for IFNγ spread in B16 mouse melanoma cultures and human primary cutaneous melanoma. Our results are consistent with IFNγ spread being governed by a simple diffusion-consumption model and offer insight into how the spatial organization of T cells contributes to intratumor heterogeneity in inflammatory signaling, gene expression, and immune-mediated clearance. Solid tumors are often viewed as collections of diverse cellular "neighborhoods": Our work provides a general explanation for such nongenetic cellular variability due to confinement in the spread of immune mediators.

Cancer germline antigens and tumor-agnostic CD8+ T cell evasion.

Cancer germline antigens (CGAs) are expressed in immune-privileged germline tissues, while epigenetically silenced in somatic tissues. CGAs become re-expressed in tumors and can promote oncogenesis. Tumors prominently exploit mechanisms similar to those in germline tissues to shield from immunosurveillance. We hypothesize that CGAs contribute towards tumor escape from immune effector CD8+ T cells. For illustrative purposes, we assessed the co-presence or -absence of CGAs with these cells in multiple tumor types. Considering a broad array of CD8+ T cell evasive mechanisms, we exemplify the co-occurrence of gene transcripts of eight CGAs with those of adhesion molecules, endothelial cells, and/or the Wnt pathway. We present a novel concept of CGAs and their association with CD8+ T cell evasion, which may be relevant for future immunotherapeutic interventions.

Tumor-infiltrating lymphocyte therapy: Clinical aspects and future developments in this breakthrough cancer treatment.

Tumor-infiltrating lymphocyte (TIL) therapy is a promising approach for treating refractory or advanced solid cancers by using autologous TILs harvested from cancer tissues. Despite the heterogeneity of cancer, TIL therapy can potentially produce a positive therapeutic response, including complete remission. After decades of research on lymphocyte functions, culture/expansion methods, therapeutic protocols, and multiple clinical trials, TIL therapy has finally reached a stage where it can be formally approved for clinical use. TIL therapy is expected to hold a unique position among anti-cancer therapeutic options as a standard intervention. To successfully introduce TIL therapy into clinical settings, there is a need to expand therapeutic indications and set up the best protocols for cancer tissue sampling and manufacturing, and related clinical trials. Moreover, studies on next-generation TIL therapy have already begun, and post-approval real-world data will promote and support further research.

IMMUNOREACT 7: Regular aspirin use is associated with immune surveillance activation in colorectal cancer.

BACKGROUND: Long-term daily use of aspirin reduces incidence and mortality due to colorectal cancer (CRC). This study aimed to analyze the effect of aspirin on the tumor microenvironment, systemic immunity, and on the healthy mucosa surrounding cancer. METHODS: Patients with a diagnosis of CRC operated on from 2015 to 2019 were retrospectively analyzed (METACCRE cohort). Expression of mRNA of immune surveillance-related genes (PD-L1, CD80, CD86, HLA I, and HLA II) in CRC primary cells treated with aspirin were extracted from Gene Expression Omnibus-deposited public database (GSE76583). The experiment was replicated in cell lines. The mucosal immune microenvironment of a subgroup of patients participating in the IMMUNOREACT1 (ClinicalTrials.gov NCT04915326) project was analyzed with immunohistochemistry and flow cytometry. RESULTS: In the METACCRE Cohort, 12% of 238 patients analyzed were aspirin users. Nodal metastasis was significantly less frequent (p = .008) and tumor-infiltrating lymphocyte infiltration was higher (p = .02) among aspirin users. In the CRC primary cells and selected cell lines, CD80 mRNA expression was increased following aspirin treatment (p = .001). In the healthy mucosa surrounding rectal cancer, the ratio of CD8/CD3 and epithelial cells expressing CD80 was higher in aspirin users (p = .027 and p = .034, respectively). CONCLUSIONS: These data suggested that regular aspirin use may have an active role in enhancing immunosurveillance against CRC.

Negative prognostic behaviour of PD-L1 expression in tongue and larynx squamous cell carcinoma and its significant predictive power in combination with PD-1 expression on TILs.

BACKGROUND: Biomarkers that can predict outcome will improve the efficacy of treatment for HNSCC patients. In this regard, we retrospectively evaluated the prognostic effect of PD1, PD-L1, and CD45RO in tongue and larynx squamous cell carcinomas. METHODS: FFPE tissue blocks of 63 larynx and 40 tongue squamous cell carcinoma samples were selected, cut into 3 µm sections, and immunohistochemically stained for PD1, PD-L1, and CD45RO. The slides were evaluated by an expert pathologist, and results were analysed using Chi-square, univariate, and multivariable Cox regression methods. RESULTS: TC-PD-L1 expression (P = 0.001) and its expression intensity (P = 0.002) were significantly correlated with a higher percentage of PD-1 + tumor infiltrating lymphocytes. In univariate survival analysis, TC-PD-L1 and its expression intensity had a significant impact on both DFS (HR: 0.203; P = 0.003 and HR: 0.320; P = 0.005) and OS (HR: 0.147; P = 0.002 and HR: 0.322; P = 0.005). Based on the multivariate analysis, PD1 (DFS: HR: 3.202; P = 0.011, OS: HR: 2.671; P = 0.027) and TC-PD-L1 (DFS: HR: 0.174; P = 0.006, OS: HR: 0.189; P = 0.009) were found to be independent prognostic markers. In the second part, scoring systems were defined based on the expression status of PD1 and PD-L1. Patients with higher scores were expected to have longer DFS and OS. In multivariate analysis, the PD1/TC-PD-L1 (DFS: P = 0.001, OS: P = 0.003) scoring systems showed superior prognostic effects. Interestingly, at the highest levels of this score, none of the patients experienced recurrence or cancer-caused death. CONCLUSION: Collectively, this study suggests negative prognostic behaviour for TC-PD-L1 protein and introduces the PD-1/TC-PD-L1 scoring system as a strong prognostic marker in OS and DFS prediction of tongue and larynx HNSCC patients.

Expansion of tumor-infiltrating lymphocytes in non-small cell lung cancer: Clinical potential and efficacy in EGFR mutation subsets.

Our study aimed to expand tumor-infiltrating lymphocytes (TILs) from primary non-small cell lung cancers (NSCLCs) and evaluate their reactivity against tumor cells. We expanded TILs from 103 primary NSCLCs using histopathological analysis, flow cytometry, IFN-γ release assays, cell-mediated cytotoxicity assays, and in vivo efficacy tests. TIL expansion was observed in all cases, regardless of EGFR mutation status. There was also an increase in the median CD4+/CD8+ ratio during expansion. In post-rapid expansion protocol (REP) TILs, 13 out of 16 cases, including all three cases with EGFR mutations, exhibited a two-fold or greater increase in IFN-γ secretion. The cytotoxicity assay revealed enhanced tumor cell death in three of the seven cases, two of which had EGFR mutations. In vivo functional testing in a patient-derived xenograft model showed a reduction in tumor volume. The anti-tumor activity of post-REP TILs underscores their potential as a therapeutic option for advanced NSCLC, irrespective of mutation status.

Spatial Distribution and Densities of CD103+ and FoxP3+ Tumor Infiltrating Lymphocytes by Digital Analysis for Outcome Prediction in Breast Cancer.

BACKGROUND: The evaluation of tumor-infiltrating lymphocytes (TILs) for breast cancer prognosis is now established. However, the clinical value for their spatial distributions of specific immune subsets, namely CD103+ tissue-resident memory T cells FoxP3+ regulatory T ells, have not been thoroughly examined. METHOD: Representative whole sections of breast cancers were subjected to CD103 and FoxP3 double staining. Their density, ratio, and spatial features were analyzed in tumor area and tumor-stromal interface. Their associations with clinicopathological parameters and patient's prognosis were analyzed. RESULTS: CD103 TILs were closer to tumor nests than FoxP3 TILs in the tumor-stromal interface. Their densities were associated with high-grade disease, TNBC, and stromal TILs. High stromal FoxP3 (sFoxP3) TILs and close proximity of sCD103 TILs to tumor were independently associated with better survival at multivariate analysis. Subgroup analysis showed the high FoxP3 TILs density associated better survival was seen in HER2-OE and TNBC subtypes while the proximity of CD103 TILs to tumor nests associated better survival was seen in luminal cancers. CONCLUSION: The prognostic impact of CD103 and FoxP3 TILs in breast cancer depends on their spatial localization. High sFoxP3 TIL density and the lower distance of CD103 TILs from the tumor nests had independent favorable prognostic values.

Presence, Subtypes, and Prognostic Significance of Tertiary Lymphoid Structures in Urothelial Carcinoma of the Bladder.

OBJECTIVE: To evaluate the presence and subtypes of tertiary lymphatic structures (TLSs) in urothelial carcinoma of the bladder (UCB) and to analyze their associated clinicopathological characteristics and prognostic significance. METHODS: The study enrolled 580 patients with surgically treated UCB, including 313 non-muscle invasive bladder cancer (NMIBC) and 267 muscle-invasive bladder cancer (MIBC). The presence and subtypes of TLSs were identified by immunohistochemistry (CD20, CD3, Bcl-6, and CD21). TLSs were classified into non-GC (nGC) TLS and GC TLS subtypes based on germinal center (GC) formation. Disease-free survival (DFS) was used as an endpoint outcome to evaluate the prognostic significance of TLS and its subtypes in UCB. RESULTS: TLSs were more common in MIBC than in NMIBC (67.8% vs 48.2%, P < .001), and the tumor-infiltrating lymphocyte (TIL) mean density was significantly higher in MIBC than in NMIBC (24.0% vs 17.5%, P < .001). Moreover, a positive correlation was found between TLS presence and GC structure formation and TIL infiltration in UCB. Endpoint events occurred in 191 patients. Compared to patients with endpoint events, patients without disease progression exhibited higher TIL density and more TLSs (P < .05). Kaplan-Meier curves showed that TLS was associated with better DFS in NMIBC (P = .041) and MIBC (P = .049). However, the Cox multivariate analysis did not demonstrate the prognostic significance of TLS. CONCLUSIONS: TLS is heterogeneous in UCB, and that TLS and GC structures are related to TIL density and prognostic events. However, TLS as a prognostic indicator remains unclear, warranting further investigation.

Tumor microenvironment characteristics association with clinical outcome in patients with resected intestinal-type gastric cancer.

BACKGROUND: Tumor microenvironment (TME) characteristics including tumor stroma ratio (TSR), tumor budding (TB), and tumor-infiltrating lymphocytes (TILs) were examined in resected gastric cancer. These TME features have been shown to indicate metastatic potential in colon cancer, and intestinal-type gastric cancer (IGC) has pathological similarities with that malignancy. METHODS: TSR, TB, and TILs were quantified in routine histological sections from 493 patients with IGC who underwent radical resection at 2 university hospitals in China from 2010 to 2016. TME variables were dichotomized as follows: TSR (50%), TILs (median), TB per international guidelines (4 buds/0.785mm2), and platelet-lymphocyte ratio (PLR) per survival ROC. Association of TME features with patient clinicopathological characteristics, time-to-recurrence (TTR), and cancer-specific-survival (CSS) were examined using univariate and multivariate analysis, including a relative contribution analysis by Cox regression. RESULTS: Patients whose tumors showed high TSR or high TB or low TILs were each significantly associated with increased T and N stage, higher histological grade, and poorer TTR and CSS at 5 years. Only TSR and N stage were independently associated with TTR and CSS after adjustment for covariates. PLR was only independently associated with TTR after adjustment for covariates. Among the variables examined, only TSR was significantly associated with both TTR (HR 1.72, 95% CI, 1.14-2.60, P = .01) and CSS (HR 1.62, 95% CI, 1.05-2.51, P = .03) multivariately. Relative contribution to TTR revealed that the top 3 contributors were N stage (45.1%), TSR (22.5%), and PLR (12.9%), while the top 3 contributors to CSS were N stage (59.9%), TSR (14.7%), and PLR (10.9%). CONCLUSIONS: Among the examined TME features, TSR was the most robust for prognostication and was significantly associated with both TTR and CSS. Furthermore, the relative contribution of TSR to patient TTR and CSS was second only to nodal status.

scRNA+TCR+BCR-seq revealed the proportions and gene expression patterns of dual receptor T and B lymphocytes in NPC and NLH.

While the relationship between single receptor lymphocytes and cancer has been deeply researched, the origin and biological roles of dual receptor lymphocytes in tumor microenvironment (TME) remain largely unknown. And since nasopharyngeal carcinoma (NPC) is a type of cancer closely associated with immune infiltration, studying the TME of NPC holds particular significance. Utilizing single-cell RNA sequencing combined with T cell receptor (TCR) and B cell receptor (BCR) sequencing (scRNA + TCR + BCR-seq), we analyzed data from 7 patients with NPC and 3 patients with nasopharyngeal lymphatic hyperplasia (NLH). In our research, it was firstly found that the presence of dual receptor lymphocytes in both the TME of NPC and the inflammatory environment of NLH. We also confirmed their clonal expansion, suggesting their potential involvement in the immune response. Subsequently, we further discovered the lineage and the pairing characteristics. It was found that the dual receptor lymphocytes in NPC and NLH mainly originate from memory cells, and the predominant pairing type for dual TCR was ß+α1+α2 and dual BCR was heavy+κ+λ. By further analyzing their gene expression, we compared the function of dual receptor cells with single receptor cells in the context of both NPC and NLH. This groundbreaking research has enhanced our comprehension of the features of dual-receptor cells and has contributed to a better understanding of the TME in NPC. By comparing with NLH, it illuminates part of the alterations in the process of malignant transformation in NPC. These findings present the potential to acquire improved diagnostic markers and treatment modalities.

Distinct profiles of proliferating CD8+/TCF1+ T cells and CD163+/PD-L1+ macrophages predict risk of relapse differently among treatment-naïve breast cancer subtypes.

Immunophenotypic analysis of breast cancer microenvironment is gaining attraction as a clinical tool improving breast cancer patient stratification. The aim of this study is to evaluate proliferating CD8 + including CD8 + TCF1 + Τ cells along with PD-L1 expressing tissue-associated macrophages among different breast cancer subtypes. A well-characterized cohort of 791 treatment-naïve breast cancer patients was included. The analysis demonstrated a distinct expression pattern among breast cancer subtypes characterized by increased CD8 + , CD163 + and CD163 + PD-L1 + cells along with high PD-L1 status and decreased fraction of CD8 + Ki67 + T cells in triple negative (TNBC) and HER2 + compared to luminal tumors. Kaplan-Meier and Cox univariate survival analysis revealed that breast cancer patients with high CD8 + , CD8 + Ki67 + , CD8 + TCF1 + cells, PD-L1 score and CD163 + PD-L1 + cells are likely to have a prolonged relapse free survival, while patients with high CD163 + cells have a worse prognosis. A differential impact of high CD8 + , CD8 + Ki67 + , CD8 + TCF1 + T cells, CD163 + PD-L1 + macrophages and PD-L1 status on prognosis was identified among the various breast cancer subtypes since only TNBC patients experience an improved prognosis compared to patients with luminal A tumors. Conversely, high infiltration by CD163 + cells is associated with worse prognosis only in patients with luminal A but not in TNBC tumors. Multivariate Cox regression analysis in TNBC patients revealed that increased CD8 + [hazard ratio (HR) = 0.542; 95% confidence interval (CI) 0.309-0.950; p = 0.032), CD8 + TCF1 + (HR = 0.280; 95% CI 0.101-0.779; p = 0.015), CD163 + PD-L1 + (HR: 0.312; 95% CI 0.112-0.870; p = 0.026) cells along with PD-L1 status employing two different scoring methods (HR: 0.362; 95% CI 0.162-0.812; p = 0.014 and HR: 0.395; 95% CI 0.176-0.884; p = 0.024) were independently linked with a lower relapse rate. Multivariate analysis in Luminal type A patients revealed that increased CD163 + was independently associated with a higher relapse rate (HR = 2.360; 95% CI 1.077-5.170; p = 0.032). This study demonstrates that the evaluation of the functional status of CD8 + T cells in combination with the analysis of immunosuppressive elements could provide clinically relevant information in different breast cancer subtypes.

Identification and validation of tumor-specific T cell receptors from tumor infiltrating lymphocytes using tumor organoid co-cultures.

T cell receptor-engineered T cells (TCR-Ts) therapy is promising for cancer immunotherapy. Most studies have focused on identifying tumor-specific T cell receptors (TCRs) through predicted tumor neoantigens. However, current algorithms for predicting tumor neoantigens are unreliable and many neoantigens are derived from non-coding regions. Thus, the technological platform for identifying tumor-specific TCRs using natural antigens expressed on tumor cells is urgently needed. In this study, tumor organoids-enriched tumor infiltrating lymphocytes (oeT) were obtained by repeatedly stimulation of autologous patient-derived organoids (PDO) in vitro. The oeT cells specifically responded to autologous tumor PDO by detecting CD137 expression and the secretion of IFN-γ using enzyme-linked immunospot assay. The measurement of oeT cell-mediated killing of three-dimensional organoids was conducted using a caspase3/7 flow cytometry assay kit. Subsequently, tumor-specific T cells were isolated based on CD137 expression and their TCRs were identified through single-cell RT-PCR analysis. The specificity cytotoxicity of TCRs were confirmed by transferring to primary peripheral blood T cells. The co-culture system proved highly effective in generating CD8+ tumor-specific oeT cells. These oeT cells effectively induced IFN-γ secretion and exhibited specificity in killing autologous tumor organoids, while not eliciting a cytotoxic response against normal organoids. The analysis conducted by TCRs revealed a significant expansion of T cells within a specific subset of TCRs. Subsequently, the TCRs were cloned and transferred to peripheral blood T cells generation engineered TCR-Ts, which adequately recognized and killed tumor cell in a patient-specific manner. The co-culture system provided an approach to generate tumor-specific TCRs from tumor-infiltrating lymphocytes of patients with colorectal cancer, and tumor-specific TCRs can potentially be used for personalized TCR-T therapy.