Main

The success of tumor infiltrating lymphocyte (TIL) therapy trials in metastatic melanoma shows that TILs contain a fraction of tumor-reactive T cells that can be harnessed for adoptive cell therapy1. This success is more limited in non-melanoma cancer types2 where the baseline fraction of experimentally verifiable, tumor-reactive CD8+ T cells is low—often not exceeding 0.5% (ref. 3). While the fraction of tumor-reactive T cells can be enriched before reinfusion via cell expansion, this process can exhaust the T cells, compromising their tumor-killing efficacy4 and leading to clonal depletion5. In contrast, personalized transgenic T cell therapies seek to identify and reinfuse defined tumor-reactive T cell receptors (TCRs), either in patient-autologous T cells6 or in induced pluripotent stem cell-derived, hypoimmunogenic (allogeneic) T cells7. While this generates a highly efficacious product, identifying tumor-reactive TCRs is a ‘needle in a haystack’ problem8. Current techniques place emphasis on tumor antigens, using mutanome analysis to determine the most likely immunogenic neoepitopes to be screened experimentally against TCRs recovered from TILs9. This is a technically challenging and time-consuming endeavor: only a fraction of predicted neoepitopes represent physiologically relevant, naturally processed T cell epitopes. Furthermore, while substantial focus has been placed on tumor-specific, single-nucleotide variant (SNV)-derived neoantigens as the source of TCR epitopes, this neglects antigens generated through diverse mechanisms that are only recently beginning to be understood. These include complex mutations such as frame shifts, gene fusions and aberrant gene splicing, as well as novel targets arising though transposable element activation10, cell stress-induced tryptophan bumps11, aberrant posttranslational modifications12, unannotated open reading frames13 and even from intracellular pathogens14. Together, these ensure that a tumor-focused, antigen-centric approach is both slow and inefficient in identifying suitable tumor-reactive TCRs for use in personalized therapies, thus raising costs and limiting clinical application.

We hypothesized that the identification of tumor-reactive TCRs could be accelerated by developing a TCR-centric, antigen-agnostic approach: ascertaining TCR sequence and tumor reactivity directly from T cells using single-cell combined RNA + VDJ sequencing (scRNA + VDJ-seq). We have previously shown that tumor-infiltrating T cells expressing a TCR against a tumor-specific neoepitope in a vaccinated patient with glioma could be distinguished from bystander T cells on the basis of their expression of CXCL13 and CD40LG15. This observation has been extended by other groups using cluster-based differential gene expression analyses to generate multigene ‘signatures’ of tumor-reactive TILs in melanoma16,17,18, lung cancer19,20, gastrointestinal cancer21, pancreatic ductal adenocarcinoma (PDAC)22 and metastatic cancer23. The reported gene signatures are only partially overlapping, implying that there may be tumor type-specific transcriptional features in TILs.

We postulated that molecular events in the process of T cell activation upon recognition of a tumor antigen are specific for tumor antigens and independent of tumor type. While differences in published signatures of T cell activation might reflect bona fide differences (for example, as a result of distinct tumor microenvironments), they might also reflect genes playing nonessential roles in T cell activation. In addition to this, the process of validating the tumor reactivity of a TCR requires the generation of tumor models that accurately recapitulate the mutational landscape and epitope processing capacity of the tumor—a process complicated by the spatial heterogeneity of many tumors. The consequence of this is that existing datasets might be noisy due to false negative TCR testing results, in which the tumor model lacks many target epitopes found in the primary tumor. Furthermore, the cost-intensive nature of these experiments and the desire to discover therapeutically useful TCRs has meant that experiments have typically focused on validating TCR clonotypes most likely to be tumor reactive rather than unbiased TCR cloning. This bias may complicate the identification of confounding transcriptional signatures not essential for T cell activation in existing data.

We reasoned that resolving these issues would allow tumor-reactive TILs to be identified regardless of tumor type from single-cell RNA sequencing (scRNA-seq) data alone. Furthermore, by cloning TCRs in an unbiased fashion and including large amounts of negative training data, a machine learning classifier could be trained to identify tumor-reactive TCR clonotypes from scRNA + VDJ-seq data in an automated manner.

Deep screening identifies tumor-reactive TCR from TILs

In this study, we set out to identify a tumor sample from which we could sequence TILs and derive a tumor cell line that appropriately recapitulated the primary tumor to allow for high-confidence TCR tumor-reactivity testing to generate a classifier training dataset (Fig. 1a). As sequencing is destructive, adjacent tumor pieces must be used for tumor and TIL sequencing as well as tumor cell line establishment. Tumor mutational heterogeneity (which correlates with TIL heterogeneity24) leads to TILs from one tumor piece recognizing antigens absent from the tumor cell line generated from a distal piece, resulting in false negatives during TCR testing that lower the quality of the training dataset. We therefore chose to use a metastatic tumor, as monoclonal metastasis seeding events represent genetic bottlenecks that fix mutations25, maximizing the similarity between primary tumor and resultant cell line. We further hypothesized that a metastasis derived from the brain—which has a degree of immune privilege—might result in improved phenotypic separation between bystander and infiltrating tumor-reactive T cells. We identified a metastatic brain tumor from a 62-year-old male patient previously diagnosed with melanoma, which was established as a tumor cell line hereafter termed BT21. Whole-exome sequencing showed that BT21 was a suitable model of the metastatic tumor, sharing 245 of the 268 functional SNVs (Extended Data Fig. 1 and Source data), and constitutively expressing major histocompatibility class I (MHC I) complexes required for epitope presentation and TCR testing (Extended Data Fig. 2).

Fig. 1: BT21 cell line accurately models resected metastatic lesion, allowing high-confidence experimental TCR tumor-reactivity testing.
figure 1

a, An overview of the experimental and computational pipeline underlying the predicTCR classifier: TILs are sorted and subject to scRNA + VDJ-seq, while adjacent resected tumor material is used to establish the BT21 tumor cell line. TCR reactivity data are then integrated with scRNA + VDJ-seq data to train the predicTCR classifier, which is later tested on externally generated TIL datasets from diverse tumor types. b, Unsupervised clustering (UMAP plot) of scRNA-seq data of TILs (n = 5,651) recovered from brain metastasis sample, with key T cell subtypes annotated. c, The percentage frequency of the top 20 TIL TCR clonotypes and their distribution projected onto the UMAP, showing that cells of the same clonotype can occupy diverse phenotypic states. d, T cells transfected with one of the 50 most frequently occurring TIL-derived TCR clonotypes (representing 58 distinct TCR α/ß chain pairs) are cocultured with BT21 cells; the resulting levels of CD107a (as quantified by flow cytometry, gated on mTCRβ+ cells, which express the transgenic TCR as a chimera with the murine constant domain) demonstrate whether a given TCR clonotype recognizes the BT21 cell line. For details of settings per TCR reactivity threshold, see Methods. DMF5 is the HLA mismatched negative control TCR. e, BT21-reactive TCR clonotypes are more frequent than nonreactive clonotypes in the TIL population. f, BT21 reactivity testing results projected onto the UMAP plot (b).

Source data

Unsupervised clustering of scRNA-seq of TIL-derived T cells (n = 5,651, hereafter referred to as TILs) showed the presence of distinct clusters expressing known markers of T cell activation including CXCL13, GZMK and GNLY (Fig. 1b). Single-cell VDJ sequencing (scVDJ-seq) of TILs showed the presence of expanded TCR clonotypes, with one clonotype representing over 5% of all clones—a signal indicative of tumor reactivity due to local T cell expansion (Fig. 1c). Additionally, TCR clonotypes found in the scRNA + VDJ of TILs could also be identified in the RNA-seq data derived from a distinct piece of tumor tissue, suggesting that the source tumor was relatively homogeneous in terms of T cell infiltration and presumably the underlying mutational landscape (Supplementary Table 1).

We cloned the most frequently occurring α/ß TCR chain pairs (n = 58) from the TIL population (representing 50 distinct TCR clonotypes as some T cells express two productive α chains). TCRs were transfected into expanded healthy donor peripheral blood mononuclear cells (PBMCs) and screened for reactivity against the BT21 cell line using a flow cytometry-mediated readout of T cell activation (CD107a+) corrected for per TCR background tonic signaling. A conservative threshold was set to determine TCR reactivity (Methods and Extended Data Fig. 3a,b). We found 34/50 TCRs to be tumor reactive (Fig. 1d and Source data), and showed that there was no significant difference in transgenic TCR expression between reactive and nonreactive TCRs (Extended Data Fig. 3c). Tumor-reactive TCR clonotypes were significantly more expanded in the TIL population than nonreactive clonotypes (Fig. 1e) and individual cells expressing tumor reactive TCRs could occupy various states (Fig. 1b,c,f).

Development of predicTCR50 classifier from scRNA + VDJ data

Using the TCR reactivity dataset we established for BT21, we set out to build a machine learning classifier that could accurately and robustly predict tumor reactivity of TIL-derived TCRs based on scRNA + VDJ-seq data using the strategy illustrated in Fig. 2. We first converted the 50 experimentally determined tumor reactivities into a binary label for each TIL cell expressing a tested TCR clonotype and used the corresponding gene expression matrix for those cells as input to train machine learning frameworks. The predictive performance of several machine learning frameworks was evaluated using the area under the receiver operating characteristic (ROC) curve (AUC) metric that ranges between 1 (perfectly predictive), 0.5 (no discrimination capacity between groups) and 0 (reciprocating classes). When making a classifier, a threshold must be set to discriminate between reactive and nonreactive states; the AUC metric assesses the best possible performance of a model on a given dataset by varying the threshold value. This preliminary comparison found eXtreme Gradient Boost (XGBoost)26 to be the most suitable framework (Extended Data Fig. 4). While XGBoost performs particularly well due to its ability to update subsequent decision trees during boosting, it has additional advantages for analysis as it effectively implements within-tree parallelization and is able to handle dropout data commonly found in scRNA datasets. Importantly, XGBoost also incorporates regularization to prevent overfitting, which otherwise limits the ability to generalize a model to new datasets.

Fig. 2: PredicTCR50 classifier training strategy.
figure 2

ScRNA data from healthy donors, as well as scRNA + VDJ and experimentally derived tumor-reactivity data for the 50 most frequent TCR TIL clonotypes from sample BT21, were used to train an intermediate model using XGBoost. Due to the sparse nature of scRNA data, we optimized this intermediate model by first performing Bayesian optimization to tune hyperparameters with stratified k-fold cross-validation. Subsequently we identified the top features (that is, genes) in this intermediate model using explainable AI SHAP, and then trained a simpler model using only these features to prevent overfitting to the training data. This simpler model was retuned as before and then applied to the remaining BT21 TIL data. Per-cell reactivity probabilities calculated by the classifier were averaged for each TCR clonotype, and the Fisher–Jenk natural break was used to determine the appropriate minimum threshold for calling TCRs as tumor reactive.

We added scRNA data from ten healthy donor PBMC samples generated by three independent groups; this produced a maximally diverse negative control dataset for training. Altogether, a total of 112,960 cells were used for training, of which 1,461 cells were TILs from BT21; the imbalanced nature of the training data required careful optimization of data weighting (Methods). XGBoost hyperparameters were tuned using stratified k-fold cross-validation with Bayesian optimization, using 70% of the TCRs for training and 30% for testing. To reduce the complexity of our model—important to prevent overfitting that would limit the performance of the classifier on new samples—we identified the key features (that is, genes) determining model performance using explainable artificial intelligence (AI) SHapley Additive exPlanations (SHAP)27. We then repeated hyperparameter optimization using only these features.

The probability of tumor reactivity was calculated for each individual T cell using the model, and a mean score then calculated for each TCR clonotype using the data from scVDJ-seq (as TILs expressing a given TCR clonotype may be found occupying various phenotypic states from naive to exhausted; Fig. 1b,c). Finally, the minimum reactivity score required for a TCR clonotype to be called as being tumor reactive was calculated using Fisher–Jenk break optimization, a deterministic statistical analysis that can set sample-specific thresholds. We named the resulting classifier ‘predicTCR50’.

PredicTCR50 prediction performance in brain metastasis

We used predicTCR50 to generate tumor-reactivity predictions for all TILs recovered from the BT21 metastasis, with the per-clonotype reactive score clearly separating TCR clonotypes into a bimodal distribution corresponding to reactive and nonreactive TCRs (Fig. 3a). We tested these predictions by cloning and experimentally validating an additional 29 α/ß TCR chain pairs (representing 22 clonotypes; Fig. 3b), finding that predicTCR50 accurately predicted tumor reactivity for 20 out of 22 TCRs (AUC of 0.92 and accuracy of 0.91; Fig. 3c and Table 1). Our CD107a+ threshold for tumor reactivity captured T cells with a broad range of activated phenotypes, with CD8+ BT21-reactive TCRs killing significantly more BT21 cells in bulk culture xCELLigence assays (Extended Data Fig. 5). We were able to recapitulate these results at single-cell resolution by tracking hundreds of individual transgenic effector T cells using miniaturized microwell coculture assays using the Cellply VivaCyte platform (Extended Data Fig. 6). We then implemented the previously published gene signature-based approaches that generate per-cell TCR tumor-reactivity predictions19,20,23 and used the same clonotype thresholding procedure as for predicTCR50 to distinguish tumor-reactive from nonreactive TCR clonotypes. We found that predicTCR50 performed considerably better than the signature-based approaches at predicting reactivity in our 22 TCR set: NeoTCR8 (AUC of 0.87 and accuracy of 0.50), Hanada and Caushi (both AUC of 0.77 and accuracy of 0.72) and Meng TR30 (AUC of 0.85 and accuracy of 0.77) as presented in Table 1 (detailed per-clonotype predictions, Uniform Manifold Approximation and Projection (UMAP) plots and ROC curves shown in Extended Data Fig. 7b–e). This was not unexpected given that the signature approaches were derived from other tumor types, while predicTCR50 was trained on BT21 data.

Fig. 3: PredicTCR accurately predicts tumor-reactive TCRs in diverse tumor types.
figure 3

a, A UMAP plot as in Fig. 1 overlaid with predicTCR50 per-cell tumor-reactivity predictions. b, An additional 22 TCR clonotypes (29 distinct TCR α/ß chain pairs) were tested for reactivity against the BT21 cell line. c, The performance of predicTCR50 in prospective prediction of tumor-reactive TCR in BT21 patient. dg, The performance of predicTCR in predicting TCR tumor reactivity in published scRNA + VDJ datasets with TCR reactivity data available: seven PDAC samples from Meng et al.22 (d), one colon metastasis23 (e), two NSCLC19 (f) and three gastrointestinal cancers21 (g). The metrics were calculated by clonotype, with the number of TCR clonotypes for each sample and the AUC value listed. The overall performance was assessed using all available TCRs per cancer modality. Additional metrics and details of the sequencing technology and reactivity testing method used for each sample are listed in Table 3. h, PredicTCR reactivity predictions for PDAC sample TIPC418 from Meng et al.22 who tested eight TCRs and found none to react to the TIPC418-derived tumor cell line (blue dots, dot size scaled to number of TIL TCR clonotypes). PredicTCR analysis predicted seven of these eight TCRs to be nonreactive (reactivity scores below the Fisher–Jenk natural break threshold, dashed line in plot). Seven additional TCR clonotypes (red dots) predicted to be tumor reactive were cloned for prospective validation of predicTCR. i,j, Flow cytometry analysis of T cells expressing predicted TIPC418-reactive TCRs cocultured with TIPC418 cells (top) or irrelevant MeWo control cells (bottom) confirmed all seven TCRs to be reactive as assessed by CD107a (i) and TNFα (j). k, The relative frequency and absolute number of recovered TILs for the TCR clonotypes tested in hj.

Source data

Table 1 Performance of tumor-reactivity prediction methods on BT21

Benchmarking predicTCR50 false positive rate

Given that 34/50 of the TCRs in the training set and 13/22 of the TCRs in our validation set were tumor reactive, we questioned whether our predicTCR50 classifier might have a bias toward calling TCRs as tumor reactive. Published TCR reactivity datasets (such as those used to derive the signature-based approach) typically present more data for tumor-reactive than nonreactive TCRs; this imbalance means that a classifier that calls many TCRs to be tumor reactive would have an apparently high performance. We therefore evaluated the false positive rate of our classifier by analyzing scRNA data from PBMCs of patients with coronavirus disease 2019 (COVID-19). Severe COVID-19 disease is associated with an enrichment of proliferating and effector memory Tem populations28, and previous studies have shown that virus-reactive T cells have a transcriptional signature similar to—but distinct from—tumor-reactive T cells. We found that predicTCR50 did not classify any T cells from patients with COVID-19 as tumor reactive (Table 2 and Extended Data Fig. 8), suggesting that predicTCR50 is highly specific to tumor-reactive T cells and has a low false positive rate. In contrast, gene signature-based approaches such as NeoTCR8 typically called 1–2% of PBMCs as tumor reactive in a majority of patients with COVID-19, even those recovered from infections with mild symptoms where fewer T cells would be expected to express the virus-reactive signature.

Table 2 PredicTCR does not falsely detect tumor-reactive T cells in PBMC samples from patients with COVID-19 before and after infection

PredicTCR performance generalizes to diverse tumor types

Having shown that our training method did not generate a classifier with a high false positive rate, we created the final version of predicTCR by retraining on all 72 BT21 derived TCRs (1,679 cells) and healthy donor data (111,499 cells). We set out to compare the performance of predicTCR with that of signature approaches using only externally generated data. Given the aforementioned imbalance in validation data, we primarily used the geometric mean (G-mean) of sensitivity (true positive rate) and specificity (true negative rate) to benchmark model performance. We first applied predicTCR to nine PDAC tumors from which tumor cell lines, TIL scRNA + VDJ data and TCR reactivity testing for 118 clonotypes were available22. Despite not being trained on PDAC data, we found that predicTCR could accurately predict experimentally determined tumor reactivity as shown in Fig. 3d and detailed in Table 3 (accuracy of 0.88, G-mean of 0.88 and AUC of 0.88). This suggested that predicTCR detects core transcriptional features of tumor-mediated T cell activation that are independent of tumor type. These scores are notably higher than those achieved when applying gene signature approaches including NeoTCR8 (accuracy of 0.47, G-mean of 0.03 and AUC of 0.65; Supplementary Table 2), Hanada (accuracy of 0.77, G-mean of 0.76 and AUC of 0.76; Supplementary Table 3), Caushi (accuracy of 0.54, G-mean of 0.13 and AUC of 0.51; Supplementary Table 4) and the TR30 signature from Meng et al. (accuracy of 0.81, G-mean of 0.81 and AUC of 0.88; Supplementary Table 5), highlighting the increased predictive value of the machine learning-derived classifier.

Table 3 Summary of predicTCR TCR tumor-reactivity predictions in diverse cancer types

We next extended our analysis to include additional publicly available TIL scRNA + VDJ datasets from additional tumor types. From Lowery et al., we analyzed a single colorectal metastatic cancer patient (SR4323) for whom both reactive and nonreactive TCRs were available, showing predicTCR accuracy with a G-mean of 0.76 (accuracy of 0.83 and AUC of 0.96; Fig. 3e and Table 3). By comparison, NeoTCR8 performed perfectly on its training dataset with a G-mean of 1.00 (accuracy of 1.00 and AUC of 1.00), whereas the Hanada et al., Caushi et al. and Meng et al. TR30 gene signature-based approaches performed with respective G-means of 0.53 (accuracy of 0.72 and AUC of 0.64), 0.00 (accuracy of 0.61 and AUC of 0.50) and 0.53 (accuracy of 0.72 and AUC of 0.84), suggesting that gene signature-based approaches fail to generalize beyond the tumor type in which they were derived (Supplementary Tables 25).

We analyzed three non-small cell lung cancer (NSCLC) samples from Caushi et al. for which TCRs were cloned and tested. Since only ten TCRs were directly tested in these samples, we also included TCRs shown to be neoepitope-reactive based on mutation-associated neoantigen functional expansion or virus-reactive based on viral antigen functional expansion. PredicTCR once again performed well, with a G-mean of 0.87 (accuracy of 0.87 and AUC of 0.94; Fig. 3f and Table 3), better than the Caushi et al. signature derived from these samples, with which we observed a G-mean of 0.75 (accuracy of 0.74 and AUC of 0.83; Supplementary Table 4). The Hanada et al. signature was also derived from NSCLC samples, and as expected, it performed similarly to the Caushi et al. signature with a G-mean of 0.76 (accuracy of 0.81 and AUC of 0.86; Supplementary Table 3). NeoTCR8, on the other hand, was not predictive with a G-mean of 0.00 (accuracy of 0.58 and AUC of 0.50; Supplementary Table 2), while surprisingly the TR30 signature derived from PDAC samples performed well with a G-mean of 0.79 (accuracy of 0.77 and AUC of 0.98).

Finally, we analyzed five gastrointestinal cancer samples generated by Zheng et al.21 using Smart-seq2 (ref. 29), which contain an order of magnitude fewer cells (mean 328 cells per sample) than other external datasets generated using the 10x Genomics platform19,20,23. Notably, this dataset contained testing data for both CD4 and CD8 T cells; however, as published signature-based prediction approaches focused on CD8 cells, we restricted our comparisons to only the CD8 T cell data. For three samples, predicTCR performed with high accuracy (Fig. 3g and Table 3), while for two samples accuracy was reduced, leading to an overall G-mean of 0.78 (accuracy of 0.60 and AUC of 0.74). All other gene signature approaches were not predictive, with G-means of 0.00 (accuracy of 0.91 and AUC of 0.50; Supplementary Table 2), 0.41 (accuracy of 0.24 and AUC of 0.54; Supplementary Table 3), 0.16 (accuracy of 0.11 and AUC of 0.57; Supplementary Table 4) and 0.41 (accuracy of 0.24 and AUC of 0.52; Supplementary Table 5) for NeoTCR8, Hanada, Caushi and Meng TR30 gene signatures, respectively. These results suggest that predicTCR is applicable to datasets with few cells (probably to include tumor biopsies that can be more easily obtained than resection material), and sequenced at lower cost due to the reduced cell number.

Having demonstrated the generalizability of predicTCR, we set out to experimentally validate a number of TCRs predicted to be tumor reactive in a different tumor type. Meng et al. processed a PDAC sample (TIPC418) and tested 12 TCR clonotypes, eight of which were found to be nonreactive and four of which showed weak reactivity22. PredicTCR analysis identified many other TCR clonotypes with a high chance of being tumor reactive (Fig. 3h). From these, we selected seven new TCR clonotypes expressed in multiple TILs (Source data) and confirmed that all seven showed reactivity against the TIPC418 PDAC line as assessed by flow cytometry-based quantification of CD107a and TNFα but not against a negative control MeWo cell line (Fig. 3i,j). In this sample, many of the nonreactive TCRs were present at higher frequencies in the TIL population than the reactive clonotypes (Fig. 3k); we found that the two most frequent clonotypes shared a CDR3 α sequence that has been reported to bind to a cytomegalovirus-derived epitope in VDJdb30, confirming the utility of predicTCR in identifying TCRs for personalized cell therapies.

Discussion

Here, we present predicTCR, the first automated classifier of tumor TCR reactivity capable of highly accurate identification of tumor-reactive TCR clonotypes in TILs derived from diverse cancer types through the use of machine learning models combined with deterministic thresholding. We show that through careful sample choice, generation of a large, high-confidence TCR reactivity dataset and inclusion of extensive negative training data, an accurate classifier can be generated. In contrast to previous approaches using differential gene expression to elucidate a gene signature specific to one tumor type, predicTCR enables rapid, antigen-agnostic identification of tumor-reactive TCRs in diverse tumor types—the first step in manufacturing personalized TCR-transgenic T cell cancer therapies.

The majority of signature-based approaches rely on gene set enrichment analysis of clustered tumor-reactive T cells, and thus identify genes that are upregulated in the CXCL13 expressing cluster that we have previously shown to contain tumor-reactive infiltrating T cells15. However, CXCL13 expression does not always define a discrete population of T cells (see the example in Extended Data Fig. 9), as cell clustering is highly dependent on upstream processing methods such as normalization31, the type of clustering algorithm used and the number of cells in a particular dataset32. Furthermore, tumor-reactive TCRs may be expressed in T cells of diverse phenotypes (including memory and exhausted populations): here, cluster-based approaches struggle to interpret genes that are expressed across clusters but which have context-specific predictive value that can be discriminated by machine learning. Finally, clustering approaches require manual verification and annotation to achieve optimal results, making automation difficult. This particularly affects small datasets such as those generated with Smart-seq2, for which cluster-based gene signatures did not detect reactive T cell clusters in as many as six out of ten patients (Zheng et al.21).

We interrogated the predicTCR classifier using explainable AI SHAP to determine the key genes marking tumor reactivity in T cells. While the known reactivity marker CXCL13 contributed the most to our classifier for prediction (Extended Data Fig. 10), of the two next best genes, AC243829.4 was only identified by Caushi et al., while LINC02099 was completely absent from signature approaches. AC243829.4 has recently been reported to correlate with the presence of immune cells in the tumor microenvironment in clear cell renal cell carcinoma, and is associated with positive patient prognosis33, possibly by regulating the expression of the inflammatory cytokine CCL3 (ref. 34). Notably, the relationship between expression of LINC02099 and tumor reactivity is not linear, which we believe is the result of LINC02099 being identified as the hub of a large long noncoding RNA–messenger RNA (mRNA) regulatory network in a breast cancer study35, giving rise to complex interaction effects that can be best determined by machine learning approaches.

Given the high cost of manufacturing personalized TCR-mediated cell therapies under GMP or GMP-like conditions, only a limited number of TCRs can be manufactured per patient, so it will be important to avoid manufacturing nontumor-reactive TCRs (that is, false positive predictions). As predicTCR generates per-cell reactivity predictions, predicted reactive TCR clonotypes can be ranked by their mean reactivity score: for BT21 picking TCR clonotypes with reactivity scores above the 95th percentile threshold would exclude the one false positive predicTCR prediction we obtained by using a binary reactivity threshold (Extended Data Fig. 7). Among the TCR clonotypes exceeding a given threshold, the most frequent TCR clonotypes can be prioritized as having a more reliable reactivity score, as well as showing evidence of antigen-driven expansion. Finally, complementary analysis of the CDR3 repertoire can assist in picking between TCRs of similar frequency and score, such as identifying clusters of similar CDR3 sequences that are statistically unlikely to occur in naive repertoires using tools such as ALICE (antigen-specific lymphocyte identification by clustering of expanded sequences)36. We illustrate a cluster of TCRs in the BT21 TIL repertoire that have convergently recombined the tumor-reactive CDR3 ß sequence ‘CASSLGGASYEQYF’ in Supplementary Table 6. Of less importance in a translational context is the single false negative reactivity prediction made by predicTCR in the BT21 test set. We speculate that this might reflect a bona fide tumor-reactive TCR which could not be validated using the BT21 cell line, either due to transcriptional changes occurring to the cell line during adaptation to cell culture conditions resulting in downregulation of the TCR’s target antigen or due to the BT21 cell line lacking SNVs found in the original tumor. We note that in general, the performance of predicTCR is better on TCR tumor reactivity datasets generated using a tumor cell line as the T cell target. Tumor cell lines recapitulate the diversity of potential TCR target antigens found in the original tumor, including tumor-associated antigens, posttranslationally modified antigens and neoepitopes derived from cryptic splicing or the dark proteome, some of which are hard to capture using tandem minigene (TMG) assays. It is therefore possible that the higher false positive prediction rate exhibited by predicTCR when analyzing external samples generated using TMG assays to validate TCR reactivity actually reflects a higher false negative TCR reactivity testing rate in the source assays.

In general, predicTCR predicted more tumor-reactive TCR clonotypes for each sample than could be practicably manufactured for a personalized cell therapy. We found this to be the case even for PDAC samples, which are generally considered to be ‘cold tumors’ with a low tumor mutation burden and limited T cell infiltration, which may therefore be refractory to conventional TIL therapies2. PredicTCR predictions can be refined with accessory analyses, such as computational prediction of TCR avidity that has been shown to enrich for neoantigen-specific TCRs over tumor-associated antigen-specific TCRs37. Optimally, a minimal panel of computationally predicted tumor-reactive TCRs will advance to experimental resolution of the target epitope using sensitive, high dynamic range reporters of T cell activation38. Such analyses might be further informed by computational reconstruction of tumor heterogeneity to identify clonal or near-clonal tumor mutations39; TCRs reactive to these targets are most likely to result in tumor clearance. Combining these new computational and experimental tools will allow for the creation of a validated patient-derived cell therapy product targeting diverse, tumor-specific, clonal antigens at lower cost than current screening. However, for aggressive cancers in which patient survival is short, the rapid sample-to-vein turnaround enabled by predicTCR would allow for the creation of a personalized cell therapy product in an entirely antigen-agnostic fashion. Although the TCRs contained in such a product would target unknown antigens, given that autologous TCRs have undergone thymic selection, they pose little risk to patients, and targeting subclonal (that is nonoptimal) tumor antigens may yet offer patients clinical benefit by nucleating an immune cascade and epitope spreading effects. Furthermore, resolving the target epitope of a TCR from TCR sequence alone is a rapidly advancing field40 and we believe that by pairing tumor mutanome data with predicTCR reactivity predictions, datasets with dramatically reduced numbers of possible TCR–epitope interactions can be generated, serving to train and test TCR–epitope prediction tools, which will themselves allow for the training of TCR reactivity classifiers with ever higher predictive accuracy. As the costs of TCR synthesis fall and validated scRNA + VDJ-seq datasets become more widely available, it will become possible to generate increasingly large training datasets, ensuring that future classifiers can identify tumor-reactive TCRs (or specialized subsets thereof) with even greater accuracy.

In conclusion, we believe that accurate machine learning classifiers such as predicTCR will accelerate the realization of personalized T cell-mediated transgenic cell therapies by reducing overall sample-to-vein turnaround times and increasing the likelihood of therapy delivery before tumor progression, while reducing the costs that currently limit implementation.

Methods

Sample and patient consent

Patient BT21, a 62-year-old male previously diagnosed with melanoma, was treated for a brain metastasis at the University Hospital Mannheim following written consent. The patient was not financially compensated for participation. The study was approved by the institutional review board (Ethikkommission 2019-643N).

Processing of tumor samples for sequencing

Freshly resected brain tumor tissue was obtained from the University Hospital in Mannheim. The patient gave informed written consent before sample collection. Tissue was transported on ice in phosphate-buffered saline (PBS) (Sigma-Aldrich) and processed within 3 h of resection by dissection into into small pieces (2 × 2 × 2 mm). Individual tumor pieces were snap frozen and stored at −80 °C before extracting DNA and RNA for sequencing. The whole-exome library was prepared using SureSelect Human All Exon V7 (5191-4028, Agilent) and the RNA sequencing library was prepared using Ultra Low Input RNA-Seq from TakaraBio. Both were sequenced using NovaSeq 6000 (2× 100 bp). DNA isolated from PBMCs from patient BT21 was included as the whole-exome reference sample.

The remaining tumor pieces were gently mashed through a 100 µm cell strainer using the back side of a syringe plunger to generate a single-cell suspension. To generate a tumor cell line, a portion of the single-cell suspension was spun down (350g, 5 min, room temperature) and resuspended in Dulbecco’s modified Eagle medium/F12 (Gibco) supplemented with 1× penicillin–streptomycin (Sigma), 1× B27 supplement (Thermo Fisher), 20 ng ml−1 epidermal growth factor (236-EG, R&D Systems) and 20 ng ml−1 fibroblast growth factor (13256-029, Thermo Fisher). Cells were placed in a 37 °C CO2 incubator where they started to grow as spheroids. Cells were subsequently transferred into Roswell Park Memorial Institute (RPMI)-1640 media (Sigma) supplemented with penicillin–streptomycin and 10% fetal bovine serum (FBS), whereupon they grew as a monolayer. Cells were split with accutase (A1110501, Thermo Fisher) when appropriate during establishment of the robustly growing tumor cell line.

The remaining single-cell suspension was filtered through a 70 µm cell strainer, myelin was removed using myelin removal beads II (130-096-433, Miltenyi) and LS columns (130-042-401, Miltenyi) according to the manufacturer’s protocol, and aliquots of the single-cell suspension were cryopreserved as described for PBMCs. Thawed aliquots were used for fluorescence-activated cell sorting (FACS)-based enrichment of T cells (CD3+ and CD45+) and prepared for sequencing using Chromium Single Cell V(D)J Reagent kit v1.1 chemistry (PN-1000006, PN-1000020, PN-1000005 and PN-120262, 10X Genomics) according to the manufacturer’s protocol. The constructed scVDJ library and scGEX libraries were sequenced using the NovaSeq 6000 platform (Illumina).

Exome sequence variant calling

Variant calling was performed by the German Cancer Research Center Omics Data Core Facility using previously described pipelines41. Briefly: exome sequencing was performed on DNA extracted from PBMCs, tumor and the tumor cell line. SNVs were called relative to the human genome reference sequence GRCh37, and tumor and cell line SNVs determined by subtracting germline SNVs present in the PBMC sample using the One Touch Pipeline42.

In silico HLA typing from bulk RNA-seq data

For in silico human leukocyte antigen (HLA) typing on paired fastq files from bulk RNA-seq analysis, arcasHLA43 was used to perform in silico HLA typing on paired fastq files from bulk RNA-seq analysis.

Recovery of TCR sequences from bulk RNA-seq data

We used TRUST4 to reconstruct unpaired α and ß TCR chain sequences from within the bulk RNA-seq data as described by Song et al.44.

Generation of TCR in vitro-transcribed mRNA constructs

Cell Ranger-derived TCR clonotype data were processed in R using tidyverse functions45. VDJ regions of TCRs were ordered as synthetic DNA fragments from Twist Biosciences and cloned in 96-well format as chimeric TCRs, using murine TRAC or TRBC constant region sequences that had been further modified to include an additional disulfide bond to improve stability and avoid mismatches with the endogenous human TCR after transduction into human T cells46,47. As negative controls, we cloned two TCRs targeting HLA-A*02:01 restricted epitopes of MART1 (DMF5 TCR: CDR3α CAVNFGGGKLIF and CDR3β CASSLSFGTEAFF) or influenza (CDR3α CAVSESPFGNEKLTF and CDR3β CASSSTGLPYGYTF). For in vitro transcription, RNA-mediated expression TCR constructs were PCR amplified using a primer to add a T7 promoter, and the resulting PCR product used as a template for the T7 mScript Standard mRNA Production System (CELLSCRIPT C-MSC11610). mRNA was m7G capped and enzymatically polyadenylated following the manufacturer’s instructions. For TCR killing assays, TCR constructs were subcloned into S/MAR nanovectors using classical molecular biology techniques as previously described48.

Isolation and expansion of healthy donor PBMCs

PBMCs from healthy donors were isolated from heparinized blood. In short, 15.5 ml of Ficoll Paque Plus Media (Cytiva) was loaded per Leucosep tube (Greiner Bio-One) and spun down. After adding 3 ml of PBS (Sigma), up to 25 ml of blood was loaded on top and a density-gradient centrifugation was performed at 800g (acceleration 4 and deceleration 3). After collection of the interphase, PBMCs were washed twice with PBS and frozen in a controlled rate freezing device at −80 °C in 50% freezing medium A (60% X-Vivo 20 and 40% fetal calf serum) and 50% medium B (80% fetal calf serum and 20% dimethylsulfoxide). Cells were stored in liquid nitrogen at −140 °C until further analysis.

The rapid expansion protocol was used to expand T cells. PBMCs from three independent donors were irradiated at 40 Gy using a Gammacell 1000 (AECL) irradiation device to serve as feeder cells. Then, 1 × 107 cells from each donor were pooled together, cells were spun down (400g, 10 min, room temperature) and resuspended in rapid expansion protocol media (X-Vivo15 (Lonza, BE02-060Q), 2% human AB serum (H4522-100ML, Sigma-Aldrich), 2.5 µg ml−1 Fungizone (15290-018, Gibco), 20 µg ml−1 gentamicin (2475.1, Roth), 100 IU ml−1 penicillin and 100 µg ml−1 streptomycin (15140122, Life Technologies)). Next, 150,000 PBMCs were plated into a standing T25 flask and 666 ng of OKT-3 antibody (Life Technologies, 16-0037-85) was added to the culture and the flask was topped up to a total volume of 20 ml. The next day, 5 ml of X-Vivo15 supplemented with 2% AB serum containing 7,500 IU interleukin-2 (IL-2) was added to the culture. Three days later, 12.5 ml of medium was removed and replaced with 12.5 ml of X-Vivo15 supplemented with 2% AB serum containing 600 IU ml−1 IL-2.

Melan A expression

Melan A expression was confirmed using anti-Melan A-FITC (cat. no. sc-20032, clone A103, Santa Cruz Technology), diluted at 1:10.

TCR reactivity screening via flow cytometry

TCR-encoding RNA was electroporated into expanded healthy donor PBMCs using the Lonza 4D-Nucleofector (program EO-115, solution P3 supplemented according to the manufacturer’s recommendations), which were plated into 48-well plates containing TexMACS media (130-097-196, Miltenyi) supplemented with 2% human AB serum. At 18–24 h after electroporation, cells were collected and 50 IU ml−1 benzonase (YCP1200-50KU, Speed BioSystems) was added to avoid cell clumping. TCR expression levels were measured via flow cytometry with markers including fixable viability dye (AF700, BD), CD3 (clone HIT3A, BV510, BD) and mTCRb (clone H57-597, PE, Biolegend).

To assess TCR reactivity, a total of 150,000 T cells and 75,000 cells of the patient-autologous tumor cell line were cocultured in U-bottom 96-well plates in a total volume of 200 µ. Wells with only T cells, or T cells and TransAct beads (130-111-160, Miltenyi) were used as negative and positive controls, respectively. Then, 5 µl of CD107a FACS antibody (REF 561343, BD) was added per well. After 1 h of coculture, GolgiPlug and GolgiStop (555029 and 554724, BD) were added to reach a 1:1,000 dilution, and after four additional hours of coculture cells were used for flow cytometry analysis. Markers included fixable viability dye (AF700, 1:1,000 dilution, eBioscience), CD3 (clone HIT3A, BV510, 1:20 dilution, BD), mTCRb (clone H57-597, 1:50 dilution, PE, Biolegend) and TNFa (clone MAb11, BV711, 1:10 dilution, Biolegend). Samples were acquired on a FACS Lyric device and flow cytometry data were analyzed using FlowJo software, v10.6.2 (FlowJo LLC).

TCRs were classified as reactive or nonreactive based on flow cytometry data acquired after coculture. The percentage of CD107a positive cells (%CD107a) was quantified by gating on viable CD3+ mTCRβ+ singlets. TCRs were included in the analysis if the mTCRβ expression was >2%.

The %CD107a signal per TCR after coculture with the cell line (‘TCR versus cell line’) or after running the coculture assay without stimulation (‘TCR, unstimulated’) was corrected for background by calculating

$$\begin{array}{l}( \mathrm\% {\mathrm{CD}}107{{\mathrm{a}}}_\mathrm{TCRvscellline}- \mathrm\% {\mathrm{CD}}107{{\mathrm{a}}}_\mathrm{TCR,unstimulated})-\\( \mathrm\% {\mathrm{CD}}107{{\mathrm{a}}}_\mathrm{Mockvscellline}- \mathrm\% {\mathrm{CD}}107{{\mathrm{a}}}_\mathrm{Mock,unstimulated})\end{array}$$

where mock refers to expanded T cells electroporated without TCR-encoding RNA. TCRs were classified as reactive if the background corrected %CD107a signal per TCR was larger than 2× the standard deviation of the %CD107a+ signal measured in all samples without stimulation (1× s.d. of 0.34%). Where a TCR clonotype expressed two α chains, data are presented for the α chain resulting in the %CD107a expression (that is, the functional pair).

TCR reactivity screening via xCELLigence real-time killing assays

Primary human CD3+ cells were isolated from healthy donor volunteers using the Pan T cell isolation kit from Miltenyi Biotec according to the manufacturer’s instructions. The isolated T cells were then activated for 3 days using the human T Cell TransAct kit (Miltenyi Biotec) according to the manufacturer’s instructions and cultured in TexMACS medium from Miltenyi Biotec supplemented with IL-7 and IL-15, both at a final concentration of 10 ng ml−1, at a concentration of 1 × 106 cells ml−1. 3 days post activation 2 × 106 cells were washed and resuspended in 20 μl of primary P3 solution (Lonza), mixed with 2 μg of S/MAR DNA nanovectors and pulsed with the FI-115 pulsing code using the Lonza 4D-Nucleofector.

Primary human T cells were collected, washed two times and resuspended in FACS buffer (PBS containing 1% of FBS). TCR expression was detected by flow cytometry and T cells were stained with a PE-conjugated antibody (clone H57-597, PE, Biolegend) for 30 min on ice in the dark. Dead cells were excluded by 4,6-diamidino-2-phenylindole gating and alive TCR+ cells were gated. Data analysis was performed using FlowJo software.

A real-time killing assay using the xCELLigence was performed. Briefly, BT21 tumor cells were seeded on a 96-well plate (3 × 104 cells per well) and incubated for 24 h. Transgenic T cells were added at an effector–target cell ratio of 2:1 and co-incubated at 37 °C in RPMI 10% medium for 24 h. Cell growth was then monitored for 24 h.

TCR reactivity screening via cell-mediated cytotoxicity

Analysis of transgenic TCR cell cytotoxicity at microfluidic scale was carried out on the VivaCyte platform (Cellply) loaded with a CC-Array microfluidic device based on a modified version of the open-microwell technology49. The CC-Array contains 16 lanes, each lane comprising 1,200 microwells where effector and target cells can interact. Lower microfluidic channels under the microwell array of the CC-Array device were initially preloaded with 6% gelatin methacryloyl hydrogel (900622, Sigma-Aldrich) in PBS and the gel was polymerized with an ultraviolet lamp. BT21 target cells were prestained with CellTracker Blue CMAC Dye (C2110, Thermo Fisher, Invitrogen). Transgenic T cells and BT21 target cells were resuspended in 100% FBS (10270106, Thermo Fisher, Gibco) and loaded on the upper channels of the CC-Array device, resulting in the formation of cocultures on the bottom part of the microwell at the interface between the liquid and the underlying gelatin methacryloyl layer. Each lane was loaded with T cells expressing a single TCR. After cell delivery, a solution of RPMI-1640 (R0883, Sigma-Aldrich) and propidium iodide (P3566, Thermo Fisher) was then delivered into the microchannels and the microfluidic design allowed to rapidly exchange media in the microwells without displacing the cells. The CC-Array device was maintained at 37 °C, 5% CO2 and >90% relative humidity in the VivaCyte instrument for the duration of the assay and fluorescence images were acquired every 2 h for 12 h.

An automated analysis of the images was carried out by the VivaCyte software featuring a pretrained deep learning method50 to detect target cell cytoplasm. Nine hundred microwells were imaged per microchannel by acquiring 20 subarrays per microchannel. Cell viability was quantified as the frequency of cells stained with CMAC and not stained with propidium iodide.

ScRNA-seq analysis

Fastq files from sequenced TIL samples were processed using 10X Genomics’ Cellranger v6.1.2 (ref. 51) and count matrices are imported into R v4.1 (ref. 52). Briefly, SoupX53 was used to removed background noise and miQC54 used to remove poor quality or degraded cells (that can be identified as having an unusually high mitochondrial gene expression). Cells with an ‘RNA count’ <1,200 and ‘Feature count’ <500 were excluded from further analysis.

Healthy PBMC datasets

PBMC datasets enriched with T cells from healthy donors were obtained as follows: a single healthy donor PBMC sample from 10X Genomics55, two donors from Szabo et al.56 and seven donors from Gao et al.57. In total, data from 111,499 T cells were obtained.

PredicTCR classifier training

All scRNA count data from both internally and externally generated datasets were normalized using the ‘sctransform’ method as implemented in Seurat v4 (ref. 58), resulting in a gene–cell matrix of Pearson residuals that was used as the model input. TCR reactivity was converted to a binary value from the CD107a flow cytometric quantification as described above; all healthy donor PBMCs were assumed to be nonreactive. The model was trained using scRNA + VDJ-seq data from healthy donors (111,499 cells) plus data from experimentally validated BT21 derived TCRs for predicTCR50 (1,461 cells) or predicTCR (1,679 cells) as appropriate. Data were imported in Python (v3.9.16) using pandas (v2.0.2) for preprocessing before training with xgboost (v1.7.4). Due to the scRNA data having many dropouts, we performed hyperparameter tuning before feature selection. The XGBoost hyperparameters ‘colsample_bytree’, ‘gamma’, ‘learning_rate’, ‘max_delta_step’, ‘max_depth’, ‘min_child_weight’, ‘n_estimators’, ‘alpha’, ‘lambda’, ‘scale_pos_weight’ and ‘subsample’ were tuned by Bayesian optimization using scikit-optimize (v0.9.0)59 with ten stratified k-fold cross-validations to generate an intermediate classifier model. Due to the imbalanced nature of the training dataset, particular attention was put on optimizing data weighting (‘scale_pos_weight’). We used 70% of the data as training data, and the remaining 30% as testing dataset for hyperparameter training. To prevent overfitting to the BT21 training data, we simplified the intermediate classifier using SHAP27 to identify the key genes contributing to the model. The final predicTCR classifier was then trained on the top 100 SHAP features and hyperparameters were again optimized as before.

Prediction of tumor-reactive T cells using predicTCR

External datasets used to validate predicTCR were downloaded and the raw data preprocessed as described above. The prediction probability for each cell was averaged for each clonotype and the subsequent prediction probability for each clonotype was used to calculate the AUC using pROC. The threshold used to classify TCR reactivity was determined using Fisher–Jenk natural break optimization as implemented in jenkspy. The confusion matrix and accuracy of the resulting prediction were then calculated using caret (v6.0-94), and G-mean (the square root of sensitivity and specificity) was calculated using the output of caret.

Prediction of tumor-reactive T cells using the NeoTCR8 gene signature

Predictions using the NeoTCR8 gene signature were performed as described in Lowery et al. Briefly, the raw gene count matrix was imported into R and scGSEA (using GSVA package, v1.46.0) was performed using the signature gene list (NeoTCR8) obtained from Lowery et al.23. Cluster(s) that correspond to 0.95 percentile expression were designated as reactive. A reactive score was calculated using the ratio of predicted reactive cell to the total number of cells for each clonotype. The AUC was then calculated based on this probability score using pROC. To make direct comparisons with the performance of predicTCR, we applied the same Fisher–Jenk optimization to determine the threshold for distinguishing between reactive and nonreactive TCR clonotypes on the basis of the reactive score.

Prediction of tumor-reactive T cells using the Hanada et al. gene signature

Signature analysis using the Hanada et al. gene signature was performed as described in Hanada et al. Briefly, the raw gene count matrix was imported into R and the score was calculated by adding the genes that contributed positively to the signature and minus the genes that contributed negatively to the signature. Cells that were positive for the signature were called as (neoantigen) reactive. A reactive score was calculated and a minimum threshold for tumor reactivity was determined using Fisher–Jenk optimization as described above.

Prediction of tumor-reactive T cells using the Caushi et al. gene signature

Signature analysis using the Caushi et al. gene signature was performed similarly to Caushi et al. Briefly, the raw gene count was imported into R and analyzed using Seurat. Seurat was used to normalize the raw count data, then using ‘AddModuleScore’, a signature score was calculated using the mutation-associated neoantigen functional expansion genes. Cells that were positive for the signature were called as reactive. A reactive score was calculated and a minimum threshold for tumor reactivity was determined using Fisher–Jenk optimization as described above.

Prediction of tumor-reactive T cells using the Meng et al. TR30 gene signature

Signature analysis using Meng TR30 gene signature was performed as described Meng et al.22. Briefly, the raw gene count was imported into R and analyzed using Seurat. Seurat was used to normalize the raw count data; then the TR30 signature was computed using the UCell package (v2.2)60. The mean of the TR30 signature score was then calculated for each TCR clonotype and termed the Meng TR30 score. The minimum threshold for tumor reactivity was similarly determined using Fisher–Jenk optimization.

Material availability

The use of the primary tumor cell lines specified in this manuscript is restricted by patient informed consent and institutional review board approval to this study.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.