Generation of Tumor-Reactive T Cells by Co-culture of Peripheral Blood Lymphocytes and Tumor Organoids.

Cancer immunotherapies have shown substantial clinical activity for a subset of patients with epithelial cancers. Still, technological platforms to study cancer T-cell interactions for individual patients and understand determinants of responsiveness are presently lacking. Here, we establish and validate a platform to induce and analyze tumor-specific T cell responses to epithelial cancers in a personalized manner. We demonstrate that co-cultures of autologous tumor organoids and peripheral blood lymphocytes can be used to enrich tumor-reactive T cells from peripheral blood of patients with mismatch repair-deficient colorectal cancer and non-small-cell lung cancer. Furthermore, we demonstrate that these T cells can be used to assess the efficiency of killing of matched tumor organoids. This platform provides an unbiased strategy for the isolation of tumor-reactive T cells and provides a means by which to assess the sensitivity of tumor cells to T cell-mediated attack at the level of the individual patient.

Rapid and efficient generation of antigen-specific isogenic T cells from cryopreserved blood samples.

Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9)-mediated gene editing has been leveraged for the modification of human and mouse T cells. However, limited experience is available on the application of CRISPR/Cas9 electroporation in cryopreserved T cells collected during clinical trials. To address this, we aimed to optimize a CRISPR/Cas9-mediated gene editing protocol compatible with peripheral blood mononuclear cells (PBMCs) samples routinely produced during clinical trials. PBMCs from healthy donors were used to generate knockout T-cell models for interferon-γ, Cbl proto-oncogene B (CBLB), Fas cell surface death receptor (Fas) and T-cell receptor (TCRαß) genes. The effect of CRISPR/Cas9-mediated gene editing on T cells was evaluated using apoptosis assays, cytokine bead arrays and ex vivo and in vitro stimulation assays. Our results demonstrate that CRISPR/Cas9-mediated gene editing of ex vivo T cells is efficient and does not overtly affect T-cell viability. Cytokine release and T-cell proliferation were not affected in gene-edited T cells. Interestingly, memory T cells were more susceptible to CRISPR/Cas9 gene editing than naïve T cells. Ex vivo and in vitro stimulation with antigens resulted in equivalent antigen-specific T-cell responses in gene-edited and untouched control cells, making CRISPR/Cas9-mediated gene editing compatible with clinical antigen-specific T-cell activation and expansion assays. Here, we report an optimized protocol for rapid, viable and highly efficient genetic modification in ex vivo human antigen-specific T cells, for subsequent functional evaluation and/or expansion. Our platform extends CRISPR/Cas9-mediated gene editing for use in gold-standard clinically used immune-monitoring pipelines and serves as a starting point for development of analogous approaches, such as those including transcriptional activators and/or epigenetic modifiers.

Association of homozygous variants of STING1 with outcome in human cervical cancer.

DNA-sensing receptor Cyclic GMP-AMP Synthase (cGAS) and its downstream signaling effector STimulator of INterferon Genes (STING) have gained significant interest in the field of tumor immunology, as a dysfunctional cGAS-STING pathway is associated with poor prognosis and worse response to immunotherapy. However, studies so far have not taken into account the polymorphic nature of the STING-encoding STING1 gene. We hypothesized that the presence of allelic variance in STING1 would cause variation between individuals as to their susceptibility to cancer development, cancer progression, and potential response to (immuno)therapy. To start to address this, we defined the genetic landscapes of STING1 in cervical scrapings and investigated their corresponding clinical characteristics across a unique cohort of cervical cancer patients and compared them with independent control cohorts. Although we did not observe an enrichment of particular STING1 allelic variants in cervical cancer patients, we did find that the occurrence of homozygous variants HAQ/HAQ and R232H/R232H of STING1 were associated with both younger age of diagnosis and higher recurrence rate. These findings were accompanied by worse survival, despite comparable mRNA and protein levels of STING and numbers of infiltrated CD8+ T cells. Our findings suggest that patients with HAQ/HAQ and R232H/R232H genotypes may have a dysfunctional cGAS-STING pathway that fails to promote efficient anticancer immunity. Interestingly, the occurrence of these genotypes coincided with homozygous presence of the V48V variant, which was found to be individually associated with worse outcome. Therefore, we propose V48V to be further evaluated as a novel prognostic marker for cervical cancer.

Transcriptional Activity and Stability of CD39+CD103+CD8+ T Cells in Human High-Grade Endometrial Cancer.

Tumor-infiltrating CD8+ T cells (TIL) are of the utmost importance in anti-tumor immunity. CD103 defines tumor-resident memory T cells (TRM cells) associated with improved survival and response to immune checkpoint blockade (ICB) across human tumors. Co-expression of CD39 and CD103 marks tumor-specific TRM with enhanced cytolytic potential, suggesting that CD39+CD103+ TRM could be a suitable biomarker for immunotherapy. However, little is known about the transcriptional activity of TRM cells in situ. We analyzed CD39+CD103+ TRM cells sorted from human high-grade endometrial cancers (n = 3) using mRNA sequencing. Cells remained untreated or were incubated with PMA/ionomycin (activation), actinomycin D (a platinum-like chemotherapeutic that inhibits transcription), or a combination of the two. Resting CD39+CD103+ TRM cells were transcriptionally active and expressed a characteristic TRM signature. Activated CD39+CD103+ TRM cells differentially expressed PLEK, TWNK, and FOS, and cytokine genes IFNG, TNF, IL2, CSF2 (GM-CSF), and IL21. Findings were confirmed using qPCR and cytokine production was validated by flow cytometry of cytotoxic TIL. We studied transcript stability and found that PMA-responsive genes and mitochondrial genes were particularly stable. In conclusion, CD39+CD103+ TRM cells are transcriptionally active TRM cells with a polyfunctional, reactivation-responsive repertoire. Secondly, we hypothesize that differential regulation of transcript stability potentiates rapid responses upon TRM reactivation in tumors.

Diverse and heritable lineage imprinting of early haematopoietic progenitors.

Haematopoietic stem cells (HSCs) and their subsequent progenitors produce blood cells, but the precise nature and kinetics of this production is a contentious issue. In one model, lymphoid and myeloid production branch after the lymphoid-primed multipotent progenitor (LMPP), with both branches subsequently producing dendritic cells. However, this model is based mainly on in vitro clonal assays and population-based tracking in vivo, which could miss in vivo single-cell complexity. Here we avoid these issues by using a new quantitative version of 'cellular barcoding' to trace the in vivo fate of hundreds of LMPPs and HSCs at the single-cell level. These data demonstrate that LMPPs are highly heterogeneous in the cell types that they produce, separating into combinations of lymphoid-, myeloid- and dendritic-cell-biased producers. Conversely, although we observe a known lineage bias of some HSCs, most cellular output is derived from a small number of HSCs that each generates all cell types. Crucially, in vivo analysis of the output of sibling cells derived from single LMPPs shows that they often share a similar fate, suggesting that the fate of these progenitors was imprinted. Furthermore, as this imprinting is also observed for dendritic-cell-biased LMPPs, dendritic cells may be considered a distinct lineage on the basis of separate ancestry. These data suggest a 'graded commitment' model of haematopoiesis, in which heritable and diverse lineage imprinting occurs earlier than previously thought.

Antigen-specific TIL therapy for melanoma: A flexible platform for personalized cancer immunotherapy.

Tumor infiltrating lymphocyte (TIL) therapy has shown objective clinical response rates of 50% in stage IV melanoma patients in a number of clinical trials. Nevertheless, the majority of patients progress either directly upon therapy or after an initial period of tumor control. Recent data have shown that most TIL products that are used for therapy contain only low frequencies of T cells reactive against known melanoma-associated epitopes. Because of this, the development of a technology to create T-cell products that are enriched for reactivity against defined melanoma-associated antigens would seem valuable, both to evaluate the tumoricidal potential of T cells directed against different antigen classes and to potentially increase response rates. Here, we developed and validated a conditional MHC streptamer-based platform for the creation of TIL products with defined antigen reactivities. We have used this platform to successfully enrich both high-frequency (≥1%) and low-frequency (<1%) tumor-specific CD8(+) T-cell populations, and thereby created T-cell products with enhanced tumor recognition potential. Collectively, these data demonstrate that selection of antigen-specific T-cell populations can be used to create defined T-cell products for clinical use. This strategy thus forms a highly flexible platform for the development of antigen-specific cell products for personalized cancer immunotherapy.

Reproducibility of Illumina platform deep sequencing errors allows accurate determination of DNA barcodes in cells.

BACKGROUND: Next generation sequencing (NGS) of amplified DNA is a powerful tool to describe genetic heterogeneity within cell populations that can both be used to investigate the clonal structure of cell populations and to perform genetic lineage tracing. For applications in which both abundant and rare sequences are biologically relevant, the relatively high error rate of NGS techniques complicates data analysis, as it is difficult to distinguish rare true sequences from spurious sequences that are generated by PCR or sequencing errors. This issue, for instance, applies to cellular barcoding strategies that aim to follow the amount and type of offspring of single cells, by supplying these with unique heritable DNA tags. RESULTS: Here, we use genetic barcoding data from the Illumina HiSeq platform to show that straightforward read threshold-based filtering of data is typically insufficient to filter out spurious barcodes. Importantly, we demonstrate that specific sequencing errors occur at an approximately constant rate across different samples that are sequenced in parallel. We exploit this observation by developing a novel approach to filter out spurious sequences. CONCLUSIONS: Application of our new method demonstrates its value in the identification of true sequences amongst spurious sequences in biological data sets.

Expression of CD39 Identifies Activated Intratumoral CD8+ T Cells in Mismatch Repair Deficient Endometrial Cancer.

Identification of human cancer-reactive CD8+ T cells is crucial for the stratification of patients for immunotherapy and determination of immune-therapeutic effects. To date, these T cells have been identified mainly based on cell surface expression of programmed cell death protein 1 (PD-1) or co-expression of CD103 and CD39. A small subset of CD103- CD39+ CD8+ T cells is also present in tumors, but little is known about these T cells. Here, we report that CD103- CD39+ CD8+ T cells from mismatch repair-deficient endometrial tumors are activated and characterized predominantly by expression of TNFRSF9. In vitro, transforming growth factor-beta (TGF-ß) drives the disappearance of this subset, likely through the conversion of CD103- CD39+ cells to a CD103+ phenotype. On the transcriptomic level, T cell activation and induction of CD39 was associated with a number of tissue residence and TGF-ß responsive transcription factors. Altogether, our data suggest CD39+ CD103- CD8+ tumor-infiltrating T cells are recently activated and likely rapidly differentiate towards tissue residence upon exposure to TGF-ß in the tumor micro-environment, explaining their relative paucity in human tumors.

CD20 positive CD8 T cells are a unique and transcriptionally-distinct subset of T cells with distinct transmigration properties.

The presence of T cells that are dimly positive for the B cell marker CD20 is well-established in autoimmunity and correlates with disease severity in various diseases. Further, we previously identified that the level of CD20-positive T cells was three-fourfold elevated in ascites fluid of ovarian carcinoma patients, together suggesting a role in both autoimmunity and cancer. In this respect, treatment of autoimmune patients with the CD20-targeting antibody Rituximab has also been shown to target and deplete CD20-positive T cells, previously identified as IFN-gamma producing, low proliferative, CD8 cytotoxic T cells with an effector memory (EM) differentiation state. However, the exact phenotype and relevance of CD20-positive T cells remains unclear. Here, we set out to identify the transcriptomic profile of CD20-positive T cells using RNA sequencing. Further, to gain insight into potential functional properties of CD20 expression in T cells, CD20 was ectopically expressed on healthy human T cells and phenotypic, functional, migratory and adhesive properties were determined in vitro and in vivo. Together, these assays revealed a reduced transmigration and an enhanced adhesive profile combined with an enhanced activation status for CD20-positive T cells.

Tumor organoid-T-cell coculture systems.

T cells are key players in cancer immunotherapy, but strategies to expand tumor-reactive cells and study their interactions with tumor cells at the level of an individual patient are limited. Here we describe the generation and functional assessment of tumor-reactive T cells based on cocultures of tumor organoids and autologous peripheral blood lymphocytes. The procedure consists of an initial coculture of 2 weeks, in which tumor-reactive T cells are first expanded in the presence of (IFNγ-stimulated) autologous tumor cells. Subsequently, T cells are evaluated for their capacity to carry out effector functions (IFNγ secretion and degranulation) after recognition of tumor cells, and their capacity to kill tumor organoids. This strategy is unique in its use of peripheral blood as a source of tumor-reactive T cells in an antigen-agnostic manner. In 2 weeks, tumor-reactive CD8+ T-cell populations can be obtained from ~33-50% of samples from patients with non-small-cell lung cancer (NSCLC) and microsatellite-instable colorectal cancer (CRC). This enables the establishment of ex vivo test systems for T-cell-based immunotherapy at the level of the individual patient.

Tumor infiltrating lymphocytes (TIL) therapy in metastatic melanoma: boosting of neoantigen-specific T cell reactivity and long-term follow-up.

Treatment of metastatic melanoma with autologous tumor infiltrating lymphocytes (TILs) is currently applied in several centers. Robust and remarkably consistent overall response rates, of around 50% of treated patients, have been observed across hospitals, including a substantial fraction of durable, complete responses. PURPOSE: Execute a phase I/II feasibility study with TIL therapy in metastatic melanoma at the Netherlands Cancer Institute, with the goal to assess feasibility and potential value of a randomized phase III trial. EXPERIMENTAL: Ten patients were treated with TIL therapy. Infusion products and peripheral blood samples were phenotypically characterized and neoantigen reactivity was assessed. Here, we present long-term clinical outcome and translational data on neoantigen reactivity of the T cell products. RESULTS: Five out of 10 patients, who were all anti-PD-1 naïve at time of treatment, showed an objective clinical response, including two patients with a complete response that are both ongoing for more than 7 years. Immune monitoring demonstrated that neoantigen-specific T cells were detectable in TIL infusion products from three out of three patients analyzed. For six out of the nine neoantigen-specific T cell responses detected in these TIL products, T cell response magnitude increased significantly in the peripheral blood compartment after therapy, and neoantigen-specific T cells were detectable for up to 3 years after TIL infusion. CONCLUSION: The clinical results from this study confirm the robustness of TIL therapy in metastatic melanoma and the potential role of neoantigen-specific T cell reactivity. In addition, the data from this study supported the rationale to initiate an ongoing multicenter phase III TIL trial.

Targeting of cancer neoantigens with donor-derived T cell receptor repertoires.

Accumulating evidence suggests that clinically efficacious cancer immunotherapies are driven by T cell reactivity against DNA mutation-derived neoantigens. However, among the large number of predicted neoantigens, only a minority is recognized by autologous patient T cells, and strategies to broaden neoantigen-specific T cell responses are therefore attractive. We found that naïve T cell repertoires of healthy blood donors provide a source of neoantigen-specific T cells, responding to 11 of 57 predicted human leukocyte antigen (HLA)-A*02:01-binding epitopes from three patients. Many of the T cell reactivities involved epitopes that in vivo were neglected by patient autologous tumor-infiltrating lymphocytes. Finally, T cells redirected with T cell receptors identified from donor-derived T cells efficiently recognized patient-derived melanoma cells harboring the relevant mutations, providing a rationale for the use of such "outsourced" immune responses in cancer immunotherapy.

Tattoo Delivery of a Semliki Forest Virus-Based Vaccine Encoding Human Papillomavirus E6 and E7.

The skin is an attractive organ for immunization because of the presence of antigen-presenting cells. Intradermal delivery via tattooing has demonstrated superior vaccine immunogenicity of DNA vaccines in comparison to conventional delivery methods. In this study, we explored the efficacy of tattoo injection of a tumor vaccine based on recombinant Semliki Forest virus replicon particles (rSFV) targeting human papillomavirus (HPV). Tattoo injection of rSFV particles resulted in antigen expression in both the skin and draining lymph nodes. In comparison with intramuscular injection, the overall antigen expression determined at the site of administration and draining lymph nodes was 10-fold lower upon tattoo injection. Delivery of SFV particles encoding the E6 and E7 antigens of human papillomavirus type 16 (SFVeE6,7) via tattooing resulted in HPV-specific cytotoxic T cells and in vivo therapeutic antitumor response. Strikingly, despite the observed lower overall transgene expression, SFVeE6,7 delivered via tattoo injection resulted in higher or equal levels of immune responses as compared to intramuscular injection. The intrinsic immunogenic potential of tattooing provides a benefit for immunotherapy based on an alphavirus.

Manufacture of gene-modified human T-cells with a memory stem/central memory phenotype.

Advances in genetic engineering have made it possible to generate human T-cell products that carry desired functionalities, such as the ability to recognize cancer cells. The currently used strategies for the generation of gene-modified T-cell products lead to highly differentiated cells within the infusion product, and on the basis of data obtained in preclinical models, this is likely to impact the efficacy of these products. We set out to develop a good manufacturing practice (GMP) protocol that yields T-cell receptor (TCR) gene-modified T-cells with more favorable properties for clinical application. Here, we show the robust clinical-scale production of human peripheral blood T-cells with an early memory phenotype that express a MART-1-specific TCR. By combining selection and stimulation using anti-CD3/CD28 beads for retroviral transduction, followed by expansion in the presence of IL-7 and IL-15, production of a well-defined clinical-scale TCR gene-modified T-cell product could be achieved. A major fraction of the T-cells generated in this fashion were shown to coexpress CD62L and CD45RA, and express CD27 and CD28, indicating a central memory or memory stemlike phenotype. Furthermore, these cells produced IFNγ, TNFα, and IL-2 and displayed cytolytic activity against target cells expressing the relevant antigen. The T-cell products manufactured by this robust and validated GMP production process are now undergoing testing in a phase I/IIa clinical trial in HLA-A*02:01 MART-1-positive advanced stage melanoma patients. To our knowledge, this is the first clinical trial protocol in which the combination of IL-7 and IL-15 has been applied for the generation of gene-modified T-cell products.

Heterogeneous differentiation patterns of individual CD8+ T cells.

Upon infection, antigen-specific CD8(+) T lymphocyte responses display a highly reproducible pattern of expansion and contraction that is thought to reflect a uniform behavior of individual cells. We tracked the progeny of individual mouse CD8(+) T cells by in vivo lineage tracing and demonstrated that, even for T cells bearing identical T cell receptors, both clonal expansion and differentiation patterns are heterogeneous. As a consequence, individual naïve T lymphocytes contributed differentially to short- and long-term protection, as revealed by participation of their progeny during primary versus recall infections. The discordance in fate of individual naïve T cells argues against asymmetric division as a singular driver of CD8(+) T cell heterogeneity and demonstrates that reproducibility of CD8(+) T cell responses is achieved through population averaging.

TIL therapy broadens the tumor-reactive CD8(+) T cell compartment in melanoma patients.

There is strong evidence that both adoptive T cell transfer and T cell checkpoint blockade can lead to regression of human melanoma. However, little data are available on the effect of these cancer therapies on the tumor-reactive T cell compartment. To address this issue we have profiled therapy-induced T cell reactivity against a panel of 145 melanoma-associated CD8(+) T cell epitopes. Using this approach, we demonstrate that individual tumor-infiltrating lymphocyte cell products from melanoma patients contain unique patterns of reactivity against shared melanoma-associated antigens, and that the combined magnitude of these responses is surprisingly low. Importantly, TIL therapy increases the breadth of the tumor-reactive T cell compartment in vivo, and T cell reactivity observed post-therapy can almost in full be explained by the reactivity observed within the matched cell product. These results establish the value of high-throughput monitoring for the analysis of immuno-active therapeutics and suggest that the clinical efficacy of TIL therapy can be enhanced by the preparation of more defined tumor-reactive T cell products.